Insect viruses and their uses in protecting plants

Abstract

The present invention relates to an isolated small RNA virus capable of infecting insect species including Heliothis species, and to the nucleotide sequences and proteins encoded thereby. The invention contemplates uses of the virus in controlling insect attack in plants.

Description

FIELD OF THE INVENTION

The present invention relates to insect viruses useful in control of insect attack on plants. It particularly relates to biological insecticides, especially those comprised of insect viruses. In particular applications, the invention also provides recombinant viruses and transgenic plants.

BACKGROUND OF THE INVENTION

There is increasing awareness of the desirability of insect pest control by biological agents. Considerable effort in recent years has been devoted to the identification and exploitation of DNA viruses with large genomes, especially the baculoviruses. These viruses generally require extensive genetic manipulation to become effective insecticides, and the first such modified viruses are only now being evaluated.

In contrast, very little effort has been devoted to the study and use of small viruses with RNA genomes.

Four main groups of small RNA viruses have been isolated from insects. These include members of the Picornaviridae, the Nodaviridae, the Tetraviridae and the unclassified viruses. Descriptions of these groups can be found in the Atlas of Invertebrate Viruses (eds J. R. Adams and J. R. Bonami) (CRC Press, Boca Raton, 1991) and Viruses of Invertebrates (ed. E. Kurstak) (Marcel Dekker, New York, 1991). These disclosures relating to these viruses concern their pathology and biology, not their use in biological control.

Further information regarding small RNA viruses of insects an be found in P. D. Scotti et al (1981). “The biology and ecology of strains of an insect small RNA virus complex” Advances in Virus Research 26, 117-143. This review describes the insect picornaviruses cricket paralysis virus and Drosophila C virus (diameters estimated at 27-30 nm with one RNA component of 7.5-8.5 kb). N. F. Moore & T. W. Tinsley (1982). The small RNA viruses of insects. Brief review Archives of Virology 72, 229-245. This review included viruses of the following families:

Nodaviridae (diameter 29-30 nm, 2 RNA components totalling 4.5 kb)

Picornaviridae (diameter 27-30 nm, one RNA component of 7.5-8.5 kb)

Nudaurelia β family (now called Tetraviridae) (diameter around 35 nm, either one RNA of 5.5 kb or two totalling 8 kb)

N. F. Moore, B. Reavy & L. A. King (1985) General characteristics, gene organisation and expression of small RNA viruses of insects. Journal of general Virology 66, 647-659. This reference defines small RNA viruses of insects as being those less than 40 nm in diameter. The review covers Picornaviridae, Nodaviridae and the Nudaurelia β family (now called Tetraviridae).

D. Hendry, V. Hodgson, R Clark and J Newman (1985) Small RNA viruses co-infecting the pine emperor moth ( Nudaurelia cytherea capensis ). Journal of general Virology 66, 627-632 described viruses with mean diameters of 40 nm and 38 nm and one or two RNA components up to 5.5 kb in length.

Most recently, the term insect small RNA viruses has been used by one of the present inventors to cover three main recognised toxic groups: the Picornaviridae, the Tetraviridae and the Nodaviridae (P. Scotti & P. Christian (1994) The promises and potential problems of using small RNA insect viruses for insect control. Sains Malaysiana 23, 9-18).

These references illustrate a long standing usage of the term in this field of the term “small RNA virus” for viruses with certain characteristics as listed above. Another important characteristic of these virus groups is that they are not occluded, in contrast to many large viruses like the cytoplasmic polyhydrosis (RNA) viruses or the DNA baculoviruses, granulosis viruses and entomopox viruses. The term would also be applied to viruses not members of the three families listed above, as long as they satisfied the definition of being up to 40 nm in size. There are reports of such unclassified viruses (eg in Hendry et al. 1985). Moreover, the taxonomic status of some members of the Tetraviridae still requires clarification and it might even be possible for this family to be split, with Ha SV and other members with two RNA components in their genome being separated from those with only one component, like the type member Nudaurelia β virus, which has not yet been sequenced. The above definition of “small RNA virus” would still cover all members of such virus families.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided an isolated small RNA virus wherein the virus is up to 40 nm in size, is not occluded and infects insect species including Heliothis species.

In one particular embodiment, the present invention provides an isolated preparation of Heliothis armigera stunt virus referred to as “ Ha SV” herein.

In a further aspect of the present invention there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence hybridizable with RNA 1 (SEQ ID No: 39) or RNA 2 (SEQ ID No: 47) described herein under low stringency conditions.

In still a further aspect the invention provides a vector comprising a nucleic acid molecule, the sequence of which is hybridizable with RNA 1 (SEQ ID No: 39) or RNA 2 (SEQ ID No: 47) as described herein. These vectors include expression and transfer vectors for use in animals including insect, plant and bacterial cells.

In a further aspect the invention provides an isolated protein or polypeptide preparation of the proteins or polypeptides derivable from the isolated virus of the present invention. The invention also extends to antibodies specific for the protein and polypeptide preparations.

In a yet further aspect the invention provides a recombinant insect virus vector incorporating all or a part of the isolated virus of the present invention.

In a still further aspect of the present invention there is provided a method of controlling insect attack in a plant comprising genetically manipulating said plant so that it is capable of producing Ha SV or mutants, derivatives or variants thereof or an insecticidally effective portion of Ha SV, mutants, variants or derivatives thereof such that insects feeding on the plants are deleteriously effected. The present invention also provides a transgenic plant so manipulated.

In another aspect of the present invention there is provided a preparation of Ha SV or a mutant variant or derivative thereof, or an insecticidally effective portion of Ha SV, mutant, variant or derivative thereof, suitable for application to plants, wherein the preparation is capable of imparting an insect protective effect.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 a is a restriction map of RNA 1 (SEQ ID No. 39) clones.

FIG. 1 b is a restriction map of RNA 2 (SEQ ID No. 47) clones.

FIG. 2 is the complete sequence of RNA 1 (SEQ ID No. 39) and of major encoded polypeptide.

FIG. 3 a is the complete sequence of RNA 2 (SEQ ID No. 47) in the authentic version, and its encoded polypeptides.

FIG. 3 b is the sequence of RNA 2 variant (a 5C version) (SEQ ID No.51) and its major encoded polypeptide(s).

FIG. 4 A and FIG. 4B are bioassay data showing Ha SV induced stunting of larvae.

FIG. 5 is a map of Vector plasmid pT7T2b and pT7T2c.

FIG. 6 is a schematic representation of the proteins encoded by RNA 1 (SEQ ID No. 39) and RNA 2 (SEQ ID No. 47).

FIG. 7 is a schematic representation of the proteins expressed by RNA 2 (SEQ ID No. 47) in bacteria DNA fragments encoding P17 (SEQ Id No. 48), P71 (SEQ ID No. 50), P64, P7 and the fusion protein P70 (SEQ ID No. 52) were synthesized by PCR. The flanking NdeI and BamHI sites used in cloning are indicated. (Note that P17 is followed by BgIII site, whose cohesive ends are compatible with those of BamHI).

FIG. 8 illustrates the 3′-terminal secondary structure of Ha SV RNAs. The tRNA-like structures at the 3′ ends of RNAs 1 and 2 (SEQ ID Nos. 39 & 47) are shown. Residues in bold are common to both sequences.

FIG. 9 Expression strategies for Ha SV cDNAs in insect cells. The upper part of the figure shows the genome organization of RNAs 1 and 2 (SEQ ID Nos. 39 & 47). The lower part shows insertion of cDNAs corresponding to these RNAs into a plasmid vector, between the heat shock protein (HSP70) promoter of Drosophila and a suitable polyadenylation (pA) signal. The HSP promoter was obtained by PCR using suitable primers, with a BaMHI site inserted by PCR immediately upstream of the start of transcription, giving the following sequence: GGATCCACAGnnn (SEQ ID No. 1), where the underlined residue is the transcription start site for either RNA. The cDNAs are termined by Cla1 sites, allowing direct linkage to ribozyme sequences as described in the text.

FIG. 10 Ribozymes to yield correct 3′ ends. The sequences of ribozymes inserted as short cDNA fragments into Ha SV cDNA clones are shown. The ribozyme fragments were assembled and cloned as described in the text. Designed self-cleavage points are indicated by bold arrows.

FIGS. 11A , 11 B, and 11 C, Immunoblots to map epitopes on Ha SV. A. Detected with Ha SV antiserum. Lane 1 : pTP70delSP; lane 2 : pTP70; lane 3 : pTP17; lane 4 : control; lane 5 : pTP70delN; lane 6 : pTP70; lane 7 : pTP71; lane 8 : Ha SV virions; lane 9 ; molecular weight markers. B. Detected with Ha SV antiserum. Lane 1 : pTP70delN; lane 2 : pTP70delSPN; lane 3 : pTP70. C. Detected with an antiserum to the Bt toxin (CryIA(c)), lane 1 : pTP70; lane 2 : Ha SV virions; lane 3 : control extract.

FIG. 12 New field isolates of Ha SV. The genomic organization of RNA 2 is shown at the top of the Figure. PCR using appropriate primers with BamHI restriction sites and in some cases altered context sequences of the AUG initiating translation of the P17 (SEQ ID No. 48) or P71 (SEQ ID No. 50) genes were used to make fragments for cloning into the BamHI sites of the expression vectors. Constructs 17E71 (SEQ ID No. 35) and P71 (SEQ ID No. 50) have altered context sequences of the AUG initiating translation of the P17 (SEQ ID No. 48) and P71 (SEQ ID No. 50) genes respectively; these alterations correspond to the context derived from the JHE gene (see text). All context sequences are given on the right of the figure. R2 is a clone of the complete RNA sequence as a BamHI fragment in the vector.

FIG. 13 Maps of the expression constructs in baculovirus vectors.

FIG. 14 a to f Various strategies utilizing the present invention.

FIG. 15 Expression of RNAs 1 and 2 (SEQ ID Nos. 39 & 47) from baculovirus vectors. The full-length cDNA clone of Ha SV RNA 1 or 2 (SEQ ID Nos. 39 & 47) was inserted as a BamHI fragment into the baculoexpression vectors. PCR was used to add BamHI sites immediately adjacent to the 5′ and 3′ termini of the RNA 1 sequence; sequences of the primers are given in the text. Constructs R1RZ and R2RZ carry cis-acting ribozymes immediately adjacent to the 3′ end of the sequence of RNA 1 and 2 (SEQ ID Nos. 39 & 47) respectively.

FIG. 16 Expression strategies for Ha SV cDNAs in plant cells. The upper part of the Figure shows the genome organization of RNAs 1 and 2 (SEQ ID Nos. 39 & 47). The lower part shows insertion of cDNAs corresponding to these RNAs into a plasmid vector, between 35S promoter of cauliflower mosaic virus and the polyadenylation (pA) signal on plasmid pDH51 (Pietrzak et al, 1986). The cDNAs were obtained by PCR using suitable primers, with a BaMHI site inserted by PCR immediately upstream of the start of each cDNA. The cDNAs are terminated by ClaI sites, allowing direct linkage to ribozyme sequences as described in the text.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A first aspect of the invention contemplates use of small RNA viruses for biological control of insects. In particular, in accordance with the first aspect of this invention there is provided an isolated small RNA virus, particularly H. armigera stunt virus or mutants, variants or derivatives thereof capable of infecting insects, in particular the insect species such as Helicoverpa armigera . The small RNA virus isolate of the instant invention is insecticidal and in particular stunts the growth of insect larvae, for example Helicoverpa armigera larvae and inhibits or prevents development into the adult stage.

The small RNA viruses of the instant invention have insecticidal, anti-feeding, gut-binding or any synergistic property or other activity useful for insect control.

In particular, Helicoverpa armigera stunt virus ( Ha SV) particles are isometric and approximately 36 nm in diameter with a buoyant density on CsCl gradients of 1.36 g/ml. The virus is composed of two major capsid proteins of approximately 64 and 7 KDa in size as determined on SDS-PAGE. The Ha SV genome is much later than the largest known nodavirus (another class of RNA viruses) and comprises two ss (+) RNA molecules of approximately 5.3 and 2.4 kb. The genome appears to lack a blockage of unknown structure at the 3′ termini that is found in Nodaviridae. The Ha SV genome however shares a capped structure and non-polyadenylation with Nodaviridae. Ha SV differs significantly from Nodaviridae and Nudaurelia w virus in terms of its immunological properties. In particular the large capsid protein has different antigenic determinants. Other properties of Ha SV are described in the Examples.

The host range of Ha SV includes Lepidopterans such as from the subfamily Heliothinae. Species known to be hosts are Helicoverpa ( Heliothis ) armigera, H. punctigera, H. zea, Heliothis virescens and other such noctuides as Spodoptera exigua. H. armigera which is known by the common names corn ear worm, cotton ball worm, tomato grub and tobacco bud worm is a pest of economic significance in most countries. H. punctigera , the native bud worm, is a pests of the great economic significance in Australia. Members of the Heliothinae, which include Helicoverpa and Heliothis, and especially H. armigera are among the most important and widespread pests in the world. In the U.S. Heliothis virescens and Helicoverpa zea are particularly important pests.

The first aspect of the invention provides an isolated small RNA virus capable of infecting insects including Heliothis species. In a particularly preferred form the invention relates to mutants, variants and derivatives of Ha SV. The terms “mutant”, “variant and “derivative” include all naturally occurring and artificially created viruses or viral components which differ from the Ha SV isolate as herein described in nucleotide content or sequence, amino acid content or sequence, immunological reactivity, non-glycosylation or glycosylation pattern and/or infectivity but generally retain insecticidal activity. Specifically the terms “mutant”, “variant” and “derivative” of Ha SV covers small RNA viruses which have one or more functional characteristic of Ha SV described herein. Examples of mutants, variants or derivatives of Ha SV include small RNA viruses that have different nucleic or amino acid sequences from Ha SV but retain one of more functional features of Ha SV. These may include strains with genetically silent substitutions, strains carrying replication and encapsidation sequences and signals that are functionally related to Ha SV, or strains that carry functionally related protein domains.

In a preferred aspect the invention relates to mutants, variants or derivatives 2 of Ha SV which encode replication or encapsidation sequences, structures or signals with 60%, preferably 70%, more preferably 80%, still more preferably 90% and even more preferably 95% nucleotide sequence identity to the nucleotide sequences Ha SV.

In another preferred aspect the invention relates to mutants, variants or derivatives of Ha SV which encode proteins with at least 50%, preferably 60%, preferably 70%, more preferably 80%, still more preferably 90% and even more preferably 95% amino acid sequence identity to proteins or polypeptides of Ha SV.

In another preferred aspect the invention relates to mutants, variants or derivatives of Ha SV with 50%, more preferably 60%, still more preferably 70%, more preferably 80%, still more preferably 90 or 95% nucleotide sequence identity to the following biologically active domains encoded by the Ha SV genome:

RNA 1 (SEQ ID No: 39)

amino acid residues 401 to 600 or the other domains in the replicase

RNA 2 (SEQ ID No: 47) (in the capsid protein)

amino acid residues 273 to 435

amino acid residues 50 to 272

amino acid residues 436 to the COOH terminus

Preferably the viral isolate of the present invention is biologically pure which means a preparation of the virus comprising at least 20% relative to other components as determined by weight, viral activity or any other convenient means. More preferably the isolates are 50% pure, still more preferably it is 60%, even more preferably it is 70% pure, still more preferably it is 80% pure and even more preferably it is 90% or more, pure.

In a second aspect the present invention relates to a nucleotide sequence or sequences hybridizable with those of Ha SV. The term nucleotide sequence used herein includes RNA, DNA, cDNA and nucleotide sequences complementary thereto. Such nucleotide sequences also include single or double stranded nucleic acid molecules and linear and covalently closed circular molecules. The nucleic acid sequences may be the same as the Ha SV sequences as herein described or may contain single or multiple nucleotide substitutions and/or deletions and/or additions thereto. The term nucleotide sequence also includes sequences with sufficient homology to hybridize with the nucleotide sequence under low, preferably medium and most preferably high stringency conditions (Sambrook J, Fritsch, E. F. & Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratories Press) and to nucleotide sequences encoding functionally equivalent sequences. In still a more preferred embodiment the invention comprises the nucleotide sequences of genome components 1 and 2 (SEQ ID Nos: 39 and 47) as represented by FIGS. 1 and 2 hereinafter or parts thereof, or mutants, variants, or derivatives thereof The terms “mutants”, “variants” or “derivatives” of nucleotide genome components 1 and 2 (SEQ ID Nos: 39 and 47) has the same meaning, when applied to nucleotide sequences as that given above and includes parts of genome components 1 and 2 (SEQ ID Nos: 39 and 47).

The second aspect of the invention also relates to nucleotide signals, sequences or structures which enable the nucleic acid on which they are present to be replicated by Ha SV replicase. Furthermore the invention relates to the nucleotide signals, sequences or structures which enable nucleic acids on which they are present to be encapsidated.

In a particularly preferred embodiment of the second aspect, the invention comprises nucleotide sequences which are mutants of the capsid gene having the following sequences:

ATG GGC GAT GCC GGC GTC GCGT TCA CAG (SEQ ID No: 2)

ATG GAG GAT GCT GGA GTG GCG TCA CAG (SEQ ID No: 3)

ATG AGC GAG GCC GGC GTC GCG TCA CAG (SEQ ID No: 4)

In a preferred aspect the invention relates to nucleotide sequences of Ha SV encoding insecticidal activity including the capsid protein gene and P17 (SEQ ID No: 48) and mutants, variants and derivatives thereof.

In another preferred aspect the invention comprises nucleotide sequences including the following ribozyme oligonucleotides:

5′CCATCGATGCCGGACTGGTATCCCAGGGGG (called “HVR1Cla” herein) (SEQ ID No: 5)

5′ CCATCGATGCCGGACTGGTATCCCGAGGGAC (called “5′HVR2Cla” herein) (SEQ ID No: 6)

5′ CCATCGATGATCCAGCCTCCTCGCGGCGCCGGATGGGCA (called “RZHDV1” herein) (SEQ ID No: 7)

5′ GCTCTAGATCCATTCGCCATCCGAAGATGCCCATCCGGC (called “RZHDV2” herein) (SEQ ID No: 8)

5′ CCATCGATTTATGCCGAGAAGGTAACCAGAGAAACACAC (called “RZHC 1” herein) (SEQ ID No: 9)

5′ GCTCTAGACCAGGTAATATACCACAACGTGTGTTTCTCT (called “RZHC2” herein) (SEQ ID No: 10)

Ribozyme sequences are useful for obtaining translation, replication and encapsidation of the transcript. It is therefore desirable to cleave the transcript downstream of its tRNA-like structure or poly A tail prior to translation, replication or encapsidation of the transcript.

The present invention also further extends to oligonucleotide primers for the above sequences, antisense sequences and nucleotide probes for the above sequences and homologues and analogues of said primers, antisense sequences and probes. Such primers and probes are useful in the identification, isolation and/or cloning of genes encoding insecticidally effective proteins or proteins required for viral activity, from Ha SV or another virus (whether related or unrelated) carrying a similar gene or similar RNA sequence. They are also useful in screening for Ha SV or other viruses in the field or in identifying Ha SV or other viruses in insects, especially in order to identify related viruses capable of causing pathogenecity similar to Ha SV.

Any pair of oligonucleotide primers derived from either RNA 1 or RNA 2 (SEQ ID Nos: 39 and 47) and located between ca 300 and 1500 bp apart can be used as primers. The following pairs of primer sequences exemplify particularly preferred embodiments of the present invention: Specifically for RNA 1 (SEQ ID No: 39):

1. HVR1B5′ (SEQ ID No: 38) (described below) and the primer complementary to nucleotides 1192-1212 of FIG. 1 .

2. The primer corresponding to nucleotides 4084 and 4100 of FIG. 13 and the primer HVR 13 p (SEQ ID No: 12) described below

Specifically for RNA 2 (SEQ ID No: 47):

1. The primer corresponding to nucleotides 459 to 476 of FIG. 2 and the primer complementary to nucleotides 1653 to 1669 of FIG. 2 (this would include the central variable domain)

2. R2cdha5 and the primer complementary to nucleotides 1156 to 1172 of FIG. 2

3. The primer corresponding to nucleotides 1178 to 1194 and the primer complementary to nucleotides 2072 to 2091 (of FIG. 2 ).

Other combinations giving shorter fragments are also possible.

Further preferred primers include:

5′ GGGGGGAATTCATTTAGGTGACACTATA G TTCTGCCTCCCCGGAC (called “HvR1SP5p” herein) (SEQ ID No: 11)

5′ GGGGGGATCCT G GTATCCCAGGGGGGC (called “HvR13p” herein) (SEQ ID No: 12)

5′ CCGGAAGCTT G TTTTTCTTTCTTTACCA (called “Hr2cdna5” herein) (SEQ ID No: 13)

5′ GGGGGATCCGATGGTATCCCGAGGGACGC TCAGCAGGTGGCATAGG (called “HvR23p” herein) (SEQ ID No: 14)

AAATAATTTTGTTACTTTAGAAGGAGATATACAT ATGAGCGAGCGAGCACA C (called “HVPET65N” herein) (SEQ ID No: 15)

AAATAATTTTGTTTAACCTTAAGAAGGAGATCTACAT ATGCTGGAGTGGCG TCAC (called “HVPET63N” herein) (SEQ ID No: 16)

GGAGATCTACAT ATGGGAGATGCTGGAGTG (called “HVPET64N” herein) (SEQ ID No: 17)

GTAGCGAACGTCGAGAA (called “HVRNA2F3” herein) (SEQ ID No: 18)

GGGGGATCCTC AGTTGTCAGTGGCGGGGTAG (called “HVP65C” herein) (SEQ ID No: 19)

GGGGATCC CTAATTGGCACGAGCGGCGC (called “HVP6C2” herein) (SEQ ID No: 20)

AATTACATATGGCGGCCGCCGTTTCTGCC (called “HVP6MA” herein) (SEQ ID No: 21)

AATTACATATGTTCGCGGCCGCCGTTTCT (called “HVP6MF” herein) (SEQ ID No: 22)

The invention also relates to vectors encoding the nucleotide sequence described above and to host cells including the same. Preferably these vectors are capable of expression in animal, plant or bacterial cell or are capable of transferring the sequences of the present invention to the genome of other organisms such as plants. More preferably they are capable of expression in insect and crop plant cells.

In a preferred aspect the invention relates to the vectors pDHVR1, pDHVR1RZ, pDHVR2, pDHVR2RZ, p17V71, p17E71, pPH, pV71, p17V64, p17E64, pP64, pV64, pBacHVR1, pBacHVR1RZ, pBacHUR2, pBacHVR2RZ, pHSPR1, pHSPR1RZ, pHSPR2, pHSPR2RZ, pSR1(E3)A, pSR1(E3)B, pSR2A, pSR2B, pSX2P70, pSXR2P70, pSRP2B, pBHVR1B, pBHVR2B, pT7T2P64, pSR2P70, pT7T2P65, pT7T2P70, pT7T2-P71, pBSKSE3, pBSR15, pBSR25p, pSR25, phr236P70, phr235P65, pGemP63N, pGemP64N, pGemP65N, pP64N, pP65H, pTP6MA, pTP6MF, pTP17, pTP17delBB, pP656 or p70G as described hereinafter.

In a third aspect the invention relates to polypeptides or proteins encoded by Ha SV and to homologues and analogues thereof. This aspect of the invention also relates to derivatives and variants of the polypeptides and proteins of Ha SV. Such derivatives and variants include substitutions and/or deletions of one or more amino acids, and amino and carboxy terminal fusions with other polypeptides or proteins. In a preferred aspect the invention relates to the proteins P7, P16, P17 (SEQ ID No: 48), P64, P70 (SEQ ID No: 52), P71 (SEQ ID No: 50), P11a (SEQ ID No: 42), P11b (SEQ ID No: 44), P14 (SEQ ID No: 46) and P187 (SEQ ID No: 40) described herein and to homologues and analogues thereof, including fusion proteins particularly of P71 (SEQ ID No: 50) such as P70 (SEQ ID No: 52) described herein. In a most preferred aspect the invention relates to polypeptides or proteins from Ha SV which have insecticidal activity themselves or provide target specificity for insecticidal agents. In particular the invention relates to polypeptides or fragments thereof with insect gut binding specificity, particularly to the variable domains thereof as herein described. In addition, homologues and analogues with said insecticidal activity of the polypeptides and proteins are also included within the scope of the invention. In addition the invention also relates to antibodies (such as monoclonal or polyclonal antibodies or chimeric antibodies including phage antibodies produced in bacteria) specific for said polypeptide and protein sequences. Such antibodies are useful in detecting Ha SV and related viruses or the protein products thereof.

In a fourth aspect the invention provides an infectious, recombinant insect virus including a vector, an expressible nucleic acid sequence comprising all of, or a portion of the Ha SV genome, including an insecticidally effective portion of the genome and optionally, material derived from another insect virus species or isolate(s).

Insect virus vectors suitable for the invention according to this aspect, include baculoviruses, entomopoxviruses and cytoplasmic polyhedrosis viruses. Most preferably, the insect virus vector is selected from the group comprising the baculovirus genera of nuclear polyhedrosis viruses (NPV's) and granulosis viruses (GV's). In this aspect of the invention the vector acts as a carrier for the Ha SV genes encoding insectidical activity. The recombinant insect virus vector may be grown by either established procedures Shieh, (1989), Vlak (in press) or any other suitable procedure and the virus disseminated as needed. The insect virus vectors may be those described in copending International application No. PCT/AU92/00413.

The nucleic acid sequence or sequences incorporated into the recombinant vector may be a cDNA, DNA or DNA sequence and may comprise the genome or portion thereof of a DNA or RNA of Ha SV or another species. The term “material derived from another insect virus species or isolate” includes any nucleic acid sequence, or protein sequence or parts thereof which are useful in exerting an insecticidal effect when incorporated in the recombinant vector of the invention. Suitable nucleic acid sequences for incorporation into the recombinant vector include insecticidally effective agents such as a neurotoxin from the mite Pyemotes tritici (Tomalski, M. D. & Miller, L. K. Nature 352, 82-85 (1991) a toxin component of the venom of the North African scorpion Androctonus australia Maeda, S. et al. Virology 184-777-780 (1991) Stewart, L. M. D. et al., Nature 352, 85-88 (1991), Conotoxins from the venom of Conus spp. (Olivera B. M. et al., Science 249, 257-263 (1990); Woodward S. R. et al., EMBO J. 9, 1015-1020 (1990); Olivera B. M. et al., Eur. J. Biochem. 202, 589-595 (1991).

The exogenous nucleic acid sequence may be operably placed into the insect virus vector between a viral or cellular promoter and a polyadenylation signal. Upon infection of an insect cell, the vector virus will cause the production of either infectious virus genomic RNA or infectious encapsidated viral particles.

The promoters may be constitutively expressed or inducible. These include tissue specific promoters, temperature sensitive promoters or promoters which are activated when the insect feeds on a metabolite in the plant that it is desired to protect.

Recombinant insect virus vectors according to the present invention may include nucleic acid sequences comprising all or an infectious or insecticidally effective portion of genome the Ha SV and optionally another insect virus species or isolate.

In a particularly preferred embodiment of the present invention there is provided assembled capsids comprising one or more of the capsid proteins of the present invention, or derivatives or variants thereof as contemplated or described herein. These assembled virus capsids are useful as vectors for insecticidal agents. As such the assembled viral capsids may be used to administer insecticidal agents such as various nucleotide sequences with insecticidal activity or various toxins to an insect. Nucleotide sequences in the form of RNA or DNA which can be used include those of the Ha SV genome or other insect viruses. Toxins which can be used advantageously include those which are active intracellularly and may also include neurotoxins with an appropriate transportation mechanism to reach the insect neurones.

The efficacy or insecticidal activity of infectious genomic RNA or viral particles produced by insect cells infected with insect vectors according to this aspect of the invention, may be enhanced as described below. Moreover the virus vector itself may include within a non-essential region(s), one or more nucleic acid sequences encoding substances that are deleterious to insects such as the insecticidally effective agents described above. Alternatively an extra genome component may be added to the Ha SV genome either by insertion into one of the Ha SV genes or by adding it to the ends of the genome.

In a particularly preferred embodiment there is provided a recombinant baculovirus vector comprising Ha SV or part thereof having insecticidal properties.

Other modifications which may be made to the infectious recombinant insect virus according to the fourth aspect include:

i) splitting the exogenous Ha SV nucleic acid molecules comprising the genome and cloning the fragments into the insect vector so that they cannot rejoin. One component, preferably the virus RNA replicase, could be expressed from a separately-transcribed fragment, the transcripts of which would not be replicated by the replicase they encode. The remainder of the genome (having insecticidal activity or encoding the capsid protein or a separate toxin m-RNA) could be encoded by, for example, a second separately-transcribed fragment, the transcripts of which are capable of being amplified by the replicase. Consequently, whilst the transcripts from the second or other fragment would effect their insecticidal activity upon the infected insect cell, they would not be able to infect another insect cell, (even if encapsidated) because the replicase or replicase-encoding transcripts would be absent;

This modification would allow an inherent biological containment to be built into the insecticidal vectors, which, when used in conjunction with the use of non-persistent DNA virus vectors such as those described in the above mentioned copending application, would allow a new level of environmental safety greatly extending earlier approaches based on baculovirus vectors.

ii) Manipulation of encapsidation signals or sequences essential for replicase binding or production of sub-genomic mRNA's including expression of exogeneous insect control factors as RNAs dependent on the virus for replication. This involves determination of RNA sequences and signals important for replication and encapsidation of virus RNAs, such as by analysis of replication of deletion mutants carrying reporter genes in appropriate cells, followed by studies on the transmission of the reporter gene to larvae by feeding of virus. These deletion mutants can be used to carry genes for insect control factors/toxins to larvae after replacing the reporter gene by a suitable toxin gene such as shown in FIG. 12 ;

iii) using an insect promoter responsive to virus infection and, for example, placing copies of the viral replicase gene under the control of two promoters, one which is constitutive or expressed at early stages of vector infection, and the other being a cellular promoter turned on by the ensuing RNA viral infection. This system would then make more copies of the replicase mRNA available as the amount of its template increased. Such a promoter may be isolated using techniques analogous to enhancer trapping, that is, transforming insect cells with a suitable reporter gene and looking for induction of the reporter upon virus infection of a population of transformed cells.

In a fifth aspect the invention relates to a method of controlling insect attack in plants by genetically manipulating plants to express Ha SV or parts thereof which can confer insecticidal activity optionally in combination with other insecticidally effective agents. Such plants are referred to as transgenic plants.

The term “express” should be understood as referring to the process of transcribing the genome or portion thereof into RNA or, alternatively, the process of transcribing the genome or portion thereof into RNA and then, in turn, translating the RNA into a protein or peptide.

In a sixth aspect the invention relates to the transgenic plants per se as described above. Transgenic plants according to the invention may be prepared for example by introducing a DNA construct including a cDNA or DNA fragment encoding all or a desired infectious portion of Ha SV, into the genome of a plant. The cDNA or DNA fragment may, preferably, be operably placed between a plant promoter and a polyadenylation signal. Promoters may cause constitutive or inducible expression of the sequences under their control. Furthermore they may be specific to certain tissues, such as the leaves of a plant where insect attack occurs but not to other parts of the plant such as that used for food. The inducible promoters may be induced by stimuli such as disturbance of wind or insect movement on the plant's tissues, or may be specifically turned on by insect damage to plant tissues. Heat may also be a stimulus for promoter induction such as in spring where temperatures increase and likelihood of insect attack also increases. Other stimuli such as spraying by a chemical (for instances a harmless chemical) may induce the promoter.

The cDNA or DNA fragment may encode all or a desired infectious portion of the wild-type, recombinant or otherwise mutated Ha SV. For example, deletion mutants could be used which lack segments of the viral genome which are non-essential for replication or perhaps pathogenicity.

The nucleotide sequences of the invention can be inserted into a plant genome by already established techniques, for example by an Agrobacterium transfer system or by electroporation.

Plants which may be used in this aspect of the invention include plants of both economic and scientific interest. Such plants may be those in general which need protection against the insect pests discussed herein and in particular include tomato, potato, corn, cotton, field pea and tobacco.

To enhance the efficacy of infectious genomic RNA or viral particles expressed by transgenic plants according to the invention, the DNA construct introduced into the plants' genome may be engineered to include one or more exogenous nucleic acid sequences encoding substances that are deleterious to insects. Such substances include, for example, Bacillus thuringiensis d-toxin, insect neurohormones, insecticidal compounds form wasp or scorpion venom or of heterologous origin, or factors designed to attack and kill infected cells in such a way so as to cause pathogenesis in the infected tissue (for example, a ribozyme targeted against an essential cellular function).

DNA constructs may also be provided which include:

i) mechanisms for regulating pathogen expression (for example, mechanisms which restrict the expression of ribozymes to the insect cells) by tying for example, expression to abundant virus replication, production of minus-strand RNA or sub-genomic mRNA's; and/or

ii) mechanisms similar to, or analogous to, those described in copending International patent application number PCT/AU92/00413 so as to achieve a limited-spread system (such as control of replication).

Transgenic plants according to the present invention may also be capable of expressing all or an infectious or insecticidal portion of genomes from Ha SV and one or more species or isolates of insect viruses.

In a seventh aspect of the invention Ha SV, or insecticidally effective parts thereof, or the infectious recombinant virus vectors of the fourth aspect of the present invention may be applied directly to the plant to control insect attack. Ha SV or the recombinant virus vectors may be produced either in whole or in part in either whole insects or in culture cells of insects or in bacteria or in yeast or in some other expression system. Ha SV or the recombinant virus forms may be applied in a crude form, semi purified or purified form optionally in admixture with agriculturally acceptable carrier to the crop in need of protection. Ha SV may also be applied as a facilitator of infection where existing insect populations already infected with another agent, such as one or more other viruses whereby Ha SV is able to act synergistically to bring about an insecticidal effect. Alternatively Ha SV and another agent such as one or more viruses may be applied together to plants to control insects feeding thereon.

A deposit of Ha SV No. 18.4 was made on Aug. 5, 1992 at the Australian Government Analytical Laboratories. The deposit was given accession No. N92/35575.

EXAMPLE 1

Taxanomic, Physiochemical and Biochemical Characterisation of an Insect Virus: Ha SV

Materials and Methods

A Animals and Virus Production

H. Armigara larvae were raised as described in Teakle R. E. and Jensen J. M. (1985) Heliothis punctiger in Singh P and Moore R. F. (eds) Handbook of Insect Rearing Vol 2., Elsevier, Amsterdam pp 313-322. Larvae were infected for virus production by feeding five day old larvae on 10 mg pieces of diet to which 0.064 OD 260 units of Ha SV had been applied. After 24 hours the larvae were then transferred to covered 12-well plates (BioScientific, Sydney, Australia) that contained sufficient diet and grown for eight days after which they were collected and frozen at −80° C. until further processed. Frozen larvae were weighed to 100 g, placed into 200 ml of 50 mM Tris buffer (pH 7.4), homogenized, and filtered through four layers of muslin. This homogenate was centrifuged in a Sorvall SS-34 rotor at 10,000×g for 30 minutes whereupon the supernatant was transferred to fresh tubes and recentrifuged in Beckman SW-28 rotor at 100K×g for 3 hours. The resultant band was collected and repelleted in 50 mM pH 7.2 Tris buffer in a Beckman SW-28 tube by centrifugation at 100K×g for 3 hours. The pelleted virus was resuspended overnight in 1 ml of buffer at 4° C. then layered onto a discontinuous CsCl gradient containing equal volumes of 60% and 30% CsCl (w/v) in a Beckman SW-41 tube and centrifuged at 12 h at 200×g. The resultant pellet was suspended in 100 ml of buffer and frozen for further use.

B Particle Characterization

Staining with acridine orange was as described in Mayor H. D. and Hill N. O. (1961) Virology 14: p264. Buoyant density was estimated in CsCl gradients according to Scotti P. D., Longworth J. F., Plus N, Crozier G. and Reignanum C. (1981) Advances in Virus Research 26: 117-143.

C Immunological Procedure

Rabbit anti-sera to Ha SV was produced by standard immunological procedures. Rabbit antisera to the Nudaurelia o virus in addition to the virus itself was provided by Don Hendry (Rhodes University, Grahamstown, South Africa). Rabbit antisera to the Nudaurelia b virus was supplied by the late Carl Reinganum (Plant Research Institute, Burnley, Vic, Australia). The immunological relationship to the Nudaurelia w virus was determined by the standard reciprocal double diffusion technique. Immunoblotting was performed according to Towbin H., Staeheln T. and Gordon J. (1979) PNAS. Antibodies monospecific for the major 65 kDa capsid protein were prepared by incubating polyclonal antisera with sections of nitrocellulose blotted with the 65 kDa protein. After extensive washing in Tris buffered saline, the bound antibodies were eluted in 50 mM citric buffer, pH 8.0 after a 5 minute incubation.

D Protein Characterization

Polyacrylamide gel electrophoresis in the presence of SDS followed the procedure of Laemmli UK 1970 Nature 227: 680-685 and was done with 12.5% gels unless otherwise noted with low and high molecular weight standards from BioRad. Staining was done with a colloidal preparation of Coomassie Blue G-250 (Gradipore Ltd, Pyrmont, New South Wales, Australia). Determination of the M r of the smallest protein was done with a 16% gel and standards of 3.4 kDa, 12.5 kDa and 21.5 kDa (Boehringer Mannheim). Glycosylation of the viral proteins was determined by a general glycan staining procedure with reagents supplied by Boehringer Mannheim; the positive control was fetuin. N-termini of proteins were sequenced using procedures described by Matsudairia (1989) Purification of Proteins and Peptides by SDS-PAGE in A Practical Guide to Protein and Peptide Purification for Microsequencing ed Matsudaira P. T. Academic Press, San Diego pp 52-72 on an Applied Biosystems 477A gas phase sequencer.

E Nucleic Acid Characterization

RNA was removed from capsids by twice vortexing a virus suspension with equal volumes of neutralized phenol then with phenol/chloroform (50:50). RNA was then precipitated from the aqueous phase in the presence of 300 mM sodium acetate and 2.5 volumes of ethanol. Digestions of the Ha SV nucleic acid with RNAse A and DNAse I (Boehringer Mannheim) were done with pBSSK(−) phagemid ssDNA and dsDNA (Stratagene) and RNA controls (BRL). Denaturing agarose gel electrophoresis in the presence of formaldehyde was performed according to Sambrook et al (1989). The state of polyandenylation of the viral RNA was determined by two methods. The first method was to compare the binding of identical amounts (20 mg) of viral RNA and poly(A)-selected RNA from Helicoverpa virescens to a 1 ml slurry of 5 mg of oligo-d(T) cellulose (Pharmacia) in a binding buffer consisting of 20 mM Tris pH 7.8, 500 mM NaCl, 1 mM EDTA and 0.04% SDS. The second method was to observe specific priming of viral RNA and viral RNA polyadenylated with poly(A) polymerase (Pharmacia) with d(T) 16 A/C/G primers in RNA sequencing reactions using reverse transcriptase (U.S. Biochemical) and a protocol provided by the supplier. The 5′ cap structure of the genomic RNA and Ha SV was determined by observing the ability of polynucleotide kinase to phosphorylate viral RNA with and without preincubation with tobacco acid pyrophosphatase and alkaline phosphatase (Promega) under conditions described by the supplier.

F In vitro Translation of Ha SV RNA

In vitro translation of Ha SV RNA was performed with lysates of both rabbit reticulocytes and wheat germ (Promega) as directed by the supplier. Reactions were conducted in 10 ml volumes with 1.0 mg of RNA in the presence of five u Ci 35 S-methionine. The labelled proteins were resolved on 10% and 14% SDS-PAGE gels as described above then visualised by autoradiography of the dried gels. The two viral RNAs were separated by a “freeze and squeeze” method after resolution on nondenaturing low-melting-point agarose gels in TAE (Sambrook, et al. 1989). Briefly, agarose slices containing the RNA were melted at 65° C. in a volume of TAE buffer equal to six times the agarose volume. The solution was allowed to gel on ice before freezing at −80° C. for 30 minutes. The frozen solution was thawed on ice then centrifuged at 14,500×g for 10 minutes after which the supernatant was withdrawn and precipitated by the addition of ethanol.

G Bioassay of Virus-Induced Pathogensis

Known amounts of virus isolate, as shown in FIGS. 4 A-B, were fed to larvae at the growth stages indicated by admixture to standard diet. At the time points shown, the larvae were weighed and the mean and SD calculated. Growth of infected larvae was compared to those of uninfected control populations from the same hatching batch in every experiment.

Results

i) Characteristics and Taxonomy of Ha SV

The virus particles are isometric and are approximately 36-38 nm in diameter. They are composed of two major capsid proteins, of 65 kDa and 6 kD is size. The virions contain two single-stranded (+) RNA species of 5.3 kb and 2.4 kb length. The virus bears a similarity in these respects to the Nudaurelia w virus, which has been tentatively regarded as a member of the Tetraviridae; these two viruses differ however, in the above respects from other viruses in this group and are likely to form a new virus family, sharing chiefly their capsid structure (T=4) with the Tetraviridae.

ii) Particle Characterization and Serology

The buoyant density of Ha SV was calculated to be 1.296 g/ml in CsCl at pH 7.2. The A 260 /A 280 ratio of Ha SV viral particles was 1.22 indicating a nucleic acid content of approximately 7% (Gibbs and Harrison, (1976) Plant Virology: The Principles London: Edward Arnold. Reciprocal immuno-double diffusion comparisons between Ha SV and the Nudaurelia w virus showed no serological relationship. The more sensitive technique of immunoblotting also showed a complete lack of any antigenic relationship. In addition, Ha SV did not react with antisera to the Nudaurelia b virus in a immuno-diffusion test or when immunoblotted. However, no Nudaurelia b virus was available as a positive control in these latter two immunological experiments. When Ha SV was stained with acridine orange then irradiated with 310 nm UV light, the particles fluoresced red which indicated a single stranded genome.

iii) Protein Characterization

Examination of the capsid proteins of Ha SV with polyacrylamide gel electrophoresis in the presence of SDS showed variable results depending on the quantity of protein present. At low protein loadings, two proteins in major abundance were evident that had M r 's of 65,000 and 6,000 along with a protein in minor abundance with M r of 72,000 (data not shown). When more protein was present on the gels, however, at least 12 more distinct bands with M r 's ranging between 15,000 and 62,000 became evident. Probing the resolved and blotted proteins with antibodies monospecific for the major 65 kDa capsid protein showed all but two of the proteins shared common antigens with the major 65 kDa protein. The major 6 kDa capsid protein and a minor band migrating at M r =16,000 failed to react with both the monospecific antibodies and untreated antisera.

The capsid proteins were shown to be non-glycosylated as they failed to react with a hydrazine analog after oxidation with periodic acid. The N-terminus of the 65 kDa protein appeared to be blocked in some manner as two efforts to conduct an Edman degradation failed. After the second attempt, the sample was treated with n-chlorosuccinimide and shown to be in a quantity normally adequate for sequencing. The N-terminus of the 6 kDa protein, however, was not blocked as an unambiguous 16-residue sequence was readily obtained. The sequence of the N-terminus of the 6 kDa capsid protein and those of a cyanogen bromide cleaved fragment of the 65 kDa protein are as follows:

6 kDa protein:

PheAlaAlaAlaValSerAlaPheAlaAlaAsnMetLeuSerSerValLeuLysSer (SEQ ID No: 23)

65 kDa protein:

ProThrLeuValAspGlnGlyPheTrpIleGlyGlyGlnTyrAlaLeuThrProThrSer (SEQ ID No: 24)

Detailed sequence analysis of the RNA genome carried out in Example 3 showed that RNA 1 (SEQ ID No: 39) encodes a protein of molecular weight 186,980 hereinafter referred to as P187 (SEQ ID No: 40) and RNA 2 (SEQ ID No: 47) encodes proteins with molecular weight 16,522 (called P17 (SEQ ID No: 48)) and 70,670 (called P71 (SEQ ID No: 50)). P71 (SEQ ID No: 50) is processed into two proteins of molecular weight 63,378 (called P64) and 7,309 (called P7).

iv) Nucleic Acid Characterization

The extracted nucleic acid from Ha SV was readily hydrolysed by RNAse A but not by DNAse I. Denaturing agarose gel electrophoresis of the extracted RNA genome of Ha SV indicated two strands that migrated at 5.5 kb and 2.4 kb. The RNA strands were shown not to have extensive regions of polyadenylation as only 24% of the viral RNA bound to the oligo-d(T) cellulose matrix as opposed to 82% of poly(A)-selected RNA. Further evidence for the non-polyadenylation of the viral genome was provided by the observation that the oligo primer, d(T) 16 G, gave a clear sequencing ladder using reverse transcriptase only after in vitro polyadenylation of the viral strands with poly(A)-polymerase.

The demonstration that the strands could be modified with poly(A)-polymerase also showed the lack of any 3′ modification. The 5′ termini of the viral strands were shown to be capped, most likely with m 7 G(5′)ppp(5′)G, as they could not be labelled with polynucleotide kinase unless pretreated with tobacco acid pyrophosphatase and alkaline phosphatase.

v) In vitro Translation

In vitro translation of the viral RNA yielded different results in the two translation systems used (data not shown). The 5.5 kb RNA translated very poorly in the reticulocyte system whereas it produced in the wheatgerm system more than 20 proteins ranging in size from M r =195,000 to M r =12,000. The 2.4 kb viral RNA strand yielded a major protein with an M r =24,000 in both systems in addition to a minor protein at M r =70 kDa. A time course of the translation reaction with the 5.5 kb RNA strand showed all labelled proteins were produced at similar rates indicating that the smaller products did not arise through processing of the larger ones. However when a time course experiment was done with translation of the smaller 2.4 kb RNA strand, the 24 kDa protein appeared before the 70 kDa protein.

vi) Presence of Another Form of Ha SV

Frequently, during purification of Ha SV virions, a minor band appeared in varying amounts on the CsCl gradient that had a buoyant density of 1.3 g/ml. On four occasions, when particles from this minor band were used to infect H. armigera larvae that were then processed as before for purification of Ha SV virions, the Ha SV band with a density of 1.296 g/ml was again recovered in vast excess to a varying minor amount of the more dense band. No virions of either type were recovered from uninfected control larvae. Proteins extracted from the more dense particles appeared identical to those from the less dense particles when examined by SDS-PAGE and immunoblotting with antibodies specific for the 65 kDa capsid protein of Ha SV. Extraction and examination of the RNA genome with denaturing agarose gel electrophoresis also showed the same 5.5 and 2.4 kb bands. When particles from the more dense band were examined by electron microscopy as before, they appeared to have a larger diameter 45 nm but otherwise highly similar to the 38 nm particles.

The molar ratio of the two RNA strands was determined by quantitative densitometry of fluorograms of the resolved strands. The ratio derived from an average of four measurements of various loadings on denaturing gels proved to be 1.7:1 (5.5 kb strand:2.4 kb strand) which is somewhat lower than the expected ratio of 2.3:1 for equimolar amounts of each strand.

The genome of Ha SV has major differences that make it distinct from those of the nodaviruses, the only other group of bipartite small RNA viruses pathogenic to animals. Although Ha SV shares the characteristic of a bipartite genome with the only animal viruses having such a divided genome, the nodaviridae, it differs in virtually every other aspect from this group. Both segments of its genome are considerably larger than the corresponding nodaviral RNAs (Hendry D. A., (1991) Nodaviridae of Invertebrates in (ed. E. Kurstak) Viruses of Invertebrates. Marcel Dekker, New York, pp. 227-276). However, the division of genetic labour is similar with the larger component carrying the replicase gene and the smaller one encoding the capsid proteins. Direct comparison of the sequences shows little homology between these viruses, at either RNA or protein level. The Nodaviruses, have the already mentioned unusual 3′blockage (probably a protein), whereas the Ha SV RNAs terminate in a distinctive secondary structure resembling a tRNA.

vii) Bioassays of Virus Isolates on Larvae

The original constructs made to express the capsid proteins (precursor and processed forms) in E. coli for bioassay started at the first AUG (nts 284 to 286). Production of full-length, immuno-reactive protein from these was due to these clones being the 5C sequence version with the extra C residue. Bioassays of these proteins have been difficult due to problems with obtaining suitable Heliothis larvae for the tests.

Purified native Ha SV was used to conduct bioassays in non-noctuid insect species. The native Ha SV was orally administered, the larvae scored for symptoms of infection and growth was measured. Dot blotting for Ha SV RNA was also conducted. Based on these experiments native Ha SV does not appear to infect the following larvae.

	Species	Order	Family
	Galleria mellonella	Lepidoptera	Pyradidae
	Tineola bissellia	Lepidoptera	Tineidae
	Epiphyas postvittana	Lepidoptera	Tortricidae
	Lucilia cuprina	Diptera	Calliphoridae
	Dacus tyronii	Diptera	Tephritidae
	Antitrogus parvulus	Coleoptera	Scarabaediae
	Lepidiota picticollis	Coleoptera	Scarabaediae
	Sericesthis germinata	Coleoptera	Scarabaediae

The above experiment conducted with the larvae of Spodoptera exigua and S. litura showed that native Ha SV infects these species but not to the same degree as seen in Heliothis armigera.

EXAMPLE 2

Other Virus Isolates

Materials and Methods

A Virus isolation

Apparently infected (viz diseased) larvae of Helicoverpa sp were collected in February 1993 at Mullaley (NSW), Narrabri (NSW) and Toowoomba (QLD) (Australia). Referring to FIG. 10 the samples in wells 2A-2D were from parasitised H. armigera larvae collected from sorghum at Mullaley; the sample in 6C was collected from sunflower at Toowoomba; the sample in 7D was collected from cotton at the Narrabri Research Station. The latter two larvae may have been either H. armigera or H. punctigera , which are both easily infected with Ha SV.

B Virus RNA Extraction

Larvae collected were ground up and RNA extracted. RNA extraction and purification were as per Example 1.

C Dot-Blot Northern Hybridization

Extracts of viral RNA was analysed by Northern dot-blot hybridisation using a probe made from cloned Ha SV sequences derived from 3′-terminal 1000 units of RNA 1 and RNA 2 by random priming in a Boehringer Mannheim kit according to the supplier's instructions were employed. RNA extracts were transferred to Zeta-Probe (BioRad) for probing. Hybridization under high stringency washing conditions were as specified by BioRad. Hybridizations were carried out in the following solution:

1 mM EDTA, 500 mM HaH 2 PO 4 , pH 7.2, 7% SDS, at 65° C. in a rotating Hybaid hybridization chamber. After completion of hybridization and removal of the solution containing the probe, the filters were washed twice in 1 mM EDTA, 40 mM HaH 2 PO 4 pH 7.2, 5% SDS, at 65° C. (1 h each), followed by 2 washes in 1 mM EDTA, 40 mM HaH 2 PO 4 , pH 7.2 1% SDS, at 65° C. (1 h each), before autoradiography.

Results

Referring to FIG. 10 , samples 9A, 9B, 10A, 10B and 10C contain Ha SV infected positive control lab-raised larvae; 9C-H contain healthy ( Ha SV-free) negative control lab-raised larvae; All other wells (beginning 1-8) contain extract from field-collected larvae. Numbers 2A-D, 6C and 7D gave positive signals indicating that these isolates are either the same as Ha SV or derivatives or variants thereof Election microscopy employing (−) staining confirmed that the samples which gave positive signals contained abundant icosohedral virus particles of approximately 36 mm in size.

The presence of Ha SV in larvae which had tested positive in the Northern hybridization dot-blot was confirmed by Western blotting of crude extracts from such infected larvae, using the polyclonal antibody to the Ha SV capsid protein. For routine screening of such extracts in order to identify further isolates of Ha SV or to confirm the presence of the virus, use of a monoclonal antibody or its equivalent is preferable, in order to achieve (i) higher sensitivity of detection and (ii) greater specificity of detection.

EXAMPLE 3

Identification, Isolation and Characterisation of Insect Virus Genes

Materials and Methods

A Animals and Virus Production

H. armigera larvae were raised as described in Example 1.

B Protein Characterization

Was conducted as described in Example 1.

C Nucleic Acid Characterization

Was conducted as in Example 1.

D Fractionation of Virus RNA

The two viral RNAs were separated by a “freeze and squeeze” method after resolution on nondenaturing low melting point agarose gels in TAE (Sambrook, et al, 1989). Briefly, agarose slices containing the RNA were melted at 65° C. in a volume of TAE buffer equal to six times the agarose volume. The solution was allowed to gel on ice before freezing it at −80° C. for 30 minutes. The frozen solution was thawed on ice then centrifuged at 14,500 g for 10 minutes after which the supernatent was withdrawn and precipitated by the addition of ethanol.

E In vitro Translation of Ha SV RNA

Was as in Example 1.

F cDNA Synthesis and Cloning of Virus Genome

The virus RNAs were reverse transcribed into cDNA using the Superscript RTase (a modified form of the Moloney murine leukaemia virus (MMLV) RTase, produced by Life Technologies Inc). Oligo(dT) was used as a primer on RNA which had been polyadenylated in vitro. After size selection of DNA fragments over 1 kbp in length, the cDNA was then blunt-end ligated using T4 DNA ligase (Boehringer Mannheim or Promega, under conditions described by the suppliers) into vector pBSSK(−) (Stratagene) which had been cut with EcoRV and dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim). E. coli strain JM109 or JPA101 were electroporated with the ligation mixture and white colonies selected on colour-indicator plates Sambrook et al, 1989.

For some clones of RNA2 (SEQ ID No: 47), cDNA was synthesised using the RTase of AMV (Promega) and a specific primer complementary to nucleotide sequence 2285-2301 of RNA 2 (SEQ ID No: 47). The same buffer and conditions were used for the Superscript RTase (above). The AMV RTase was found not to make cDNA form a primer annealing to the terminal 18 nucleotide sequence (see below), nor to be able to reach the 5′-end of the RNA with the primer here described.

G Sequencing of DNA and RNA

The cDNA clones were separated as single-stranded or double-stranded DNA, using the deaza-dGTP and deaza-dITP nucleotide analogues (Pharmacia) in the deaza T7 sequencing kit as recommended by this supplier. Synthetic oligonucleotides were used as primers. The 5′ terminal sequences of the two RNAs were determined using reverse transcriptase to sequence the RNA template directly, from specific oligonucleotide primers located about 200 nucleotides downstream from the termini. Such RNA sequencing was performed using the reverse transcriptase sequencing kit from Promega, under the conditions described by the manufacturer.

The sequence of the 20 or so nucleotides at the 5′ terminus of each RNA was checked using direct RNase digestion of 5′-labelled RNA under conditions designed to confer sequence-specificity. Direct RNA sequence using RNases was performed with the RNase sequencing kit from U.S. Biochemicals, following the protocols provided by the manufacturer. This also confirmed that the sequence of the most abundant RNA is consistent with that of the RNA analysed using the specific primer and RTase.

All transcription of plasmids linearized as described were performed as recommended by the suppliers of SP6 RNA polymerase, in the presence of 1 mM cap analogue, 0.2 mM GTP, and 0.5 mM of the other NTPs.

H Subcloning and Expression

PCR Amplification

The polymerase chain reaction (PCR) was used to obtain sequences covering virus genes in a form suitable for cloning into expression vectors. The reaction was performed with Taq DNA polymerase (Promega) as described by the supplier, in a rapid cycling thermal sequencer manufactured by Corbett Research (Sydney, Australia). A typical reaction involved 1 cycle of 1 min at 90° C., 25 cycles of 95° C. (10 sec), 50° C. (20 sec), 72° C. (1.5 min), followed by one cycle of 72° C. for 5 min. Templates were generally cDNA or cDNA clones derived from Ha SV RNAs, made as described below. Primers were as described below for the relevant constructs.

Upon termination of the PCR reaction, the product's ends were made blunt by treatment with E. coli DNA polymerase I (Klenow fragment) at ambient temperature for 15 minutes. After heating at 65° C. for 10 minutes, the reaction was cooled on ice and the reaction mix made 1 mM in ATP. The product then 5′-phosphorylated using 5 units of T4 polynucleotide kinase at 37° C. for 30 minutes. After heating at 65° C. for 10 minutes, the product was run on a 1% low-melting agarose gel and purified as described for RNA in section E above.

ligations: Vectors and restriction fragments cut with the enzymes described were run on 1% low-melting-point agarose gels and excised as slices. These slices were then melted at 65° C. for 5 minutes, before cooling to 37° C. Fragment and vectors were then ligated in 10 ul total volume at 14° C. overnight using T4DNA ligase (BRL, Boehringer Mannheim or Promega), in the buffers supplied by the manufacturers.

Expression: Expression plasmids containing viral genes (e.g. for the capsid protein) were transformed into E. coli strain BL21 (DE3) or HMS 174 (DE3) (supplied by Novagen). After growth as specified by the supplier, protein expression was induced by the addition of isopropyl b-D-thiogalactopyranoside (IPTG), at 0.4 nM to the growing culture for a period of 3 h. Expressed proteins were analysed by SDS-polyacrylamide gel electrophoresis of bacterial extracts (Laemmli, 1970).

Results

i) Mapping cDNA Clones of Ha SV

The template for cDNA synthesis was virus RNA which had been polyadenylated in vitro. Oligo(dT) was used as a primer for the Superscript reverse transcriptase (RTase; a modified form of the Moloney murine leukaemia virus (MMLV) RTase, produced by Life Technologies Inc). The cDNA was cloned into vector pBSSK(−) as described earlier. The larger clones were selected for further analysis by restriction mapping and Northern hybridization. All the probes tested hybridized either to RNA 1 or to RNA 2, suggesting that there are no regions of extensive sequence homology between the two RNA's. Furthermore, screening of a number of other clones excluded the theoretical possibility that either RNA band may actually contain more than one species.

ii) RNA 1 (SEQ ID No: 39) Clones

Three large RNA 1 (SEQ ID No: 39) clones (B11U, B11O and B35) obtained for the first round of clones were further analysed by restriction mapping and shown to form an overlap spanning over 3 kbp (this was later confirmed by sequencing). The second round of cloning then yielded E3 of 5.3 kbp, representing 99.7% of RNA 1 (SEQ ID No: 39). A complete restriction map of clone E3 showed it to align with that previously determined for three overlapping clones. On the basis of this alignment, the 5′ end of the insert in B11U was placed about 300 nucleotides downstream from the 5′ end of the RNA.

Once clones covering a contiguous block had been identified, the orientation 3 relative to the RNA was determined.

iii) RNA 2 (SEQ ID No: 47) Clones

Three significant cDNA clones were isolated for RNA 2 (SEQ ID No: 47) (FIG. 2 ). One, hr236, contains about 88% of RNA 2 (SEQ ID No: 47) (2470 bp total length), and runs from the 3′ end to 240 bp from the 5′ end. The other clones, hr247 and hr 249 are 3′ coterminal subgenomic fragments of 1520 bp and 760 bp, respectively. Orientation of clone hr236 was determined by strand specific hybridization. While a much stronger signal was seen with a probe for one orientation, the probe specific for the other orientation also yielded a signal, indicating that there are extensive regions of reverse complementarity within the positive strand sequence. Such sequences are likely to form extensive short and long-range secondary structure.

The clones contain the 3′ sequence of Ha SV RNA 2 (SEQ ID No: 47) as they all have the same 3′ sequence adjacent to the poly (A) stretch added in vitro before cDNA priming. The remaining 5′ sequence of RNA 2 has been obtained by direct RNA sequencing using two reverse transcriptases as described above.

iv) Sequencing of Virus Genome

The clones mapped in section (i) were selected for further analysis by sequencing.

The cDNA clones were completely sequenced as single-stranded DNA in both orientations, using the deaza-dGTP and deaza-dITP nucleotide analogues (Pharmacia) and synthetic oligonucleotides as primers.

v) Sequence of Genome Component 1 (SEQ ID No: 39) (see FIG. 1 )

The 5310 nucleotides of RNA 1 (SEQ ID No: 39) encode a protein of molecular weight 187,000 which is regarded as the RNA-dependent RNA polymerase (replicase) in view of its amino acid sequence similarity in certain limited regions to replicases of other RNA viruses. The apparent molecular weight of this protein upon in vitro translation of virus RNA and SDS-PAGE is 195,000.

Sequence analysis of RNA 1 (SEQ ID No: 39) was concentrated on clone E3 which extends from the 3′ end of RNA 1 to 18 nucleotides form the 5′ end (FIG. 1 ). The complete sequence has been confirmed by sequencing in both directions. An ORF of 1750 amino acids and spanning virtually the complete RNA (5310 nucleotides in length) has been detected. This ORF begins with the first AUG on the sequence at position 34 and terminates at nucleotide 5290 and is thought to encode the RNA-dependent RNA polymerase (replicase)(referred to as P187 (SEQ ID No: 40) in FIG. 1 ) required for virus replication, since it contains the Gly-Asp-Asp conserved triplet and surrounding sequences identified in these enzymes, which are usually large (over 100 kDa), in addition to further homology with the polymerase encoded by tobacco mosaic virus and other plus-stranded RNA viruses.

Referring to FIG. 1 the sequence is presented as the upper strand of the cDNA sequence. This strand is therefore in the same sense as the viral (positive-sense) RNA. The sequence of the protein encoded by the major open reading frame, encoding the putative RNA-dependent RNA replicase, is shown, as are those of the small open reading frames at the 3′ end, corresponding to the proteins P11a (SEQ ID No: 42), P11b (SEQ ID No: 44) and P14 (SEQ ID No: 46).

Clone E3 was inserted downstream of the SP6 promoter for in vitro transcription. As mentioned above, the transcript of this clone can be translated in the wheat germ system to yield the 195 kDa protein observed upon translation of fractionated RNA 1 (SEQ ID No: 39) from the virus. The latter yields more lower molecular weight products, presumably due to being contaminated with nicked and degraded RNA. The products derived from the in vitro transcript can therefore be regarded as defining the coding capacity of the complete RNA 1 (SEQ ID No: 39) of Ha SV.

vi) Sequence of Genome Component 2 (see FIG. 2 )

The 2470 nucleotides encode a protein of molecular weight 71,000 which contains the peptide sequences corresponding to those determined from the two virus capsid proteins. This protein is therefore the precursor of these capsid proteins. The protein is a major product of in vitro translation of this RNA obtained either from virus particles or by in vitro transcription of a full-length cDNA clone; in addition, another major translation product of apparent molecular weight 24,000 is obtained. This protein is derived from a molecular weight 17,000 reading frame overlappling the slab of the capsid protein gene.

Clones hr236 and hr247 were completely sequenced as the first step in RNA 2 sequencing. These sequences were then extensively compared to that obtained by direct RNA sequencing using AMV reverse transcriptase.

Comparison of the cloned sequence with that by direct RNA sequencing showed both clones lacked 50 nucleotide present in the RNA (at around nucleotide 1500). The sequence of this stretch was obtained by direct RNA sequencing using the AMV RTase. The MMLV “Superscript” RTase, which was used to make all the cDNA clones, was found to simply by-pass this region in sequencing reactions. These 50 nucleotides contain a very stable GC-rich hairpin flanked by a 6 bp direct repeat, and the MMLV RTase skips from the first repeat to the second.

The sequence of RNA 2 (SEQ ID No: 47) was then completed using plasmids pSR2A and pSR2P70 constructed as described below. The plasmids contain a segment of cDNA derived for the AMV RTase, as well as the sequence corresponding to the 5′ 240 nucleotides of RNA 2 (SEQ ID No: 47) which are not present on phr236 (FIG. 2 ). The sequence of RNA in FIG. 2 is presented as the upper strand of the cDNA sequence. This strand is therefore in the same sense as the viral (positive-sense) RNA. The sequences of the proteins encoded by the major open reading frames, encoding the capsid protein precursor P71 (SEQ ID No: 50), and P17 (SEQ ID No: 48).

The sequence of RNA 2 (SEQ ID No: 47) encodes a major ORF running from a methionine initiation codon at nucleotides 366 to 368 to a termination codon at nucleotides 2307 to 2309. This protein encoded by this ORF has a theoretical molecular weight of 71,000 (SEQ ID No: 50). This initiation codon is in a good context (AGGatgG), suggesting that it will be well recognized by scanning ribosomes. The size of the product is close to that of the residual putative precursor protein identified in purified virus, and to the size of the in vitro translation product obtained from RNA 2 (SEQ ID No: 47).

The approach adopted to identify the gene encoding the capsid protein was to obtain amino acid sequence information from the two abundant capsid proteins and then locate these on the protein encoded by the sequence of the virus RNA's. CNBr cleaved products of the capsid protein were therefore sequenced. These fragments gave a clear and unambiguous sequence shown in Example 1. These sequences determined were then located on the large ORF of RNA 2 (SEQ ID No: 47). ( FIG. 2 )

In the case of the small capsid protein, the clear and unambiguous sequence, obtained is located near the carboxy terminus of the major ORF on RNA 2 (SEQ ID No: 47). Starting at the point corresponding to the amino-terminal residue of the sequence determined for the 6 kDa protein, and continuing to the carboxy-terminus of the complete reading frame, the protein encoded by the sequence 7.2 kDa and has a hydrophobic N-terminal region and an arginine rich (basic) C-terminal region. It is an extremely basic protein with a pI of 12.6.

The two abundant capsid proteins are derived from a single precursor, which is processed at a specific site. This is presumably immediately amino-terminal to the sequence FAAAVS . . . (SEQ ID No: 25).

RNA 2 (SEQ ID No: 47) appears to be a bicistronic mRNA (see FIGS. 2 and 5 ). The first methionine codon is encoded on the sequence of RNA at nucleotides 283 to 285. This ATG is in a poor context (TTTatgA), making it a weaker initiation codon. It initiates a reading frame of 157 amino acids, encoding a protein of molecular weight 17,000 (SEQ ID No: 48). (The second AUG [nts 366 to 368] initiates the 71 kDa (SEQ ID No: 50) precursor of the capsid protein). Since the first AUG is in a poor context, abundant expression of the capsid precursor would be expected. In fact, in vitro translation of a full length RNA 2 (SEQ ID No: 47) transcribed from a reconstructed cDNA clone yields two major protein products of relative mobility 71,000 (SEQ ID No: 50) and 24,000, similar to those already observed upon translation of viral RNA 2 (SEQ ID No: 47). The protein of Mr 24,000 appears to correspond to the 157 amino acid protein, despite the significant anomaly in apparent size. The 24,000 Mr product was also observed upon translation of an in vitro transcript covering only nucleotides 220 to 1200 of RNA 2 (SEQ ID No: 47). This region contains no open reading frame other than those already mentioned and cannot encode a protein longer than 157 amino acids.

The protein of Mr 24,000 seen upon in vitro translation appears to correspond to P17 (SEQ ID No: 48), with the anomaly in apparent size probably being due to the high content of proline (P), glutamate (E), serine (S) and threonine (T). These amino acids cause the protein run more slowly on a gel thereby giving it an apparent size of Mr 24,000.

The Mr 24,000 protein (hereinafter referred to as P17 (SEQ ID No: 48)) may have a function in modifying or manipulating the growth characteristics or cell cycle of Ha SV-infected cells. Although a protein of 16 kDa (identified in Example 1) is found in small amounts in the capsid, it does not react with antiserum against the virus particles this is unlikely to correspond to P17 (SEQ ID No: 48), since a preparation of the latter proteins migrates with a molecular weight of 24,000 on SDS gels.

Sequence analysis of the Region from nucleotide 500 to 600 of RNA 2 (SEQ ID No: 47) showed that it has the sequence shown in FIG. 2 , as do the plasmids pSR2A, pSR2P70, pSR2B and pSXR2P70. However, plasmids pT7T72P65 and pT7T2P70 have an extra C residue at nucleotide 570. The RNA sequence from which they are derived from is shown in FIG. 2 (the “5C” version). In this sequence the first ATG (nucleotides 283 to 285) is in the same reading frame as most of the capsid protein gene. The resultant fusion protein is called “P70” (SEQ ID No: 52) and its carboxyterminal-truncated version (a variant of the native P64) is “P65”. In view of these clones it was considered important to resolve whether any virus RNA carrying the extra C residue was present in the viral RNA population first isolated for investigation.

Direct sequencing of the virus RNA using reverse transcriptase confirmed that the 4C version lacking the extra residue was the abundant form of the RNA. In order to exclude the possibility of a small amount of the RNA having the extra residue, a sensitive PCR assay was designed. This showed that the extra C residue was not present on any RNA in the viral population, and had been introduced into some clones as a PCR artefact. These clones were however retained and used in bacterial expression experiments (below) because of the high level expression obtained of the P65 and P70 (SEQ ID No: 52) fusion proteins.

vii) Comparison With the Sequence of the Nudaurelia w Capsid Gene

The sequence of most of the RNA2 of the Nudaurelia w virus has recently been published by Agrawal D. K. and Johnson J. E. (Virology 190 806-814, 1992). From the published sequence it has been determined that this sequence shows 63% homology to that of Ha SV RNA2 (SEQ ID No: 47) at the nucleotide level and 66% at the overall amino acid level. A detailed comparison of the capsid proteins of these two viruses shows the amino-terminal 45 residues to be variable, the next 220 residues to be highly conserved, the next 180 residues to be variable and the c-terminal 200 residues covering the small protein P7 to be highly conserved. A more detailed comparison is discussed below.

The published report did not find a complete reading frame corresponding to the 157 amino acid protein (P17 (SEQ ID No: 48)) gene reported above. The AUG is however present, as is a reading frame—starting upstream of the start of the capsid gene—showing considerable amino acid homology to P17 (SEQ ID No: 48) of Ha SV. In vitro translation of purified Nudaurelia w virus RNA 2 and a re-examination of the nucleotide sequencing data for this RNA may help to resolve the question of whether the Nudaurelia w virus also encodes a protein homologous to the Ha SV P17.

More interestingly, antisera against these two viruses, which are similar at a nucleotide sequence level, do not show any cross-reactivity.

viii) Construction of Full-Length Clones

RNA 1 (SEQ ID No: 39)

cDNA clone E3, described above contains all but the 5′-18 nucleotides of RNA 1 (SEQ ID No: 39) and included the complete ORF present on the sequence. The first full-length clone of RNA 1 (SEQ ID No: 39) is therefore based on E3. The 4.9 kbp XbaI-ClaI fragment from clone E3 was recloned into pBSKS(−) (Stratagene) cut with XbaI and ClaI, giving pBSKSE3.

The full-length clone of RNA 1 (SEQ ID No: 39) was completed using PCR. The primer defining the 5′ end of the RNA carried an EcoRI site, the promoter for the SP6 RNA polymerase and a sequence corresponding to the 5′ 17 nucleotides of RNA 1, as shown in FIG. 1 . The sequence of this primer was:

HvR1SP5p: 5′-GGGGGGAATTCATTTAGGTGACACTATA G TTCTGCCTCCCCGGAC (SEQ ID No: 11)(The G which initiates transcription is underlined)

Using an oligonucleotide complementary to nucleotides 1192-1212, a PCR product of 1240 bp was efficiently made. The template was cDNA synthesised using the MMLV RTase and the same oligonucleotide complementary to nucleotides 1192-1212 was the primer. Upon termination of the PCR reaction, the product's ends were made blunt and then 5′-phophorylated as described below. The purified PCR fragment was then cleaved with restriction endonuclease XbaI and the 450 bp subfragment corresponding to the 5′ end of RNA 1 (SEQ ID No: 39) cloned into the plasmid pBSSK(−)(Stragene) cut with EcoRV and XbaI, to give pBSR15.

To assemble the full-length of RNA 1 (SEQ ID No: 39), pBSKSE3 (above) was cut with XbaI and ScaI giving fragments of 1.2 kbp and 6.8 kbp. pBSR15 was cut with the same enzymes, giving fragments of 2 and 1.8 kbp. Ligation of the 6.8 kbp fragment for pBSKSE3 and the 1.8 kbp fragment for mpBSR15 yielded pSR1(E3)A. Upon linearization at ClaI and in vitro transcription with the SP6 RNA polymerase, and RNA corresponding to RNA 1 (SEQ ID No: 39), and terminating in a poly(A) stretch of about 50 nucleotides, is obtained.

Since the natural RNA 1 (SEQ ID No: 39) does not have a poly (A) tail, an alternative plasmid was constructed which carries a BamHI restriction site immediately downstream of the 3′end of RNA 1 (SEQ ID No: 39). Again this terminal fragment was made using PCR as above. The sequence of the primer was as follows:

HvR13p: 5′-GGGGGGATCCT G GTATCCCAGGGGCGC (SEQ ID No: 12) (the nucleotide complementary to that which was determined as the 3′ one, based on its adjacency to the poly(A) stretch, is underlined; RNA terminating at the BamHI site will have the sequence GCGCCCCCUGGGAUACCaggauc (SEQ ID No: 26)).

The template was clone E3 and an oligonucleotide corresponding to nucleotides 4084-4100 was the other primer. The 1220 bp product was blunt-ended, kinased and gel-purified as described above, before cleavage with HindIII. The resulting 420 bp subfragment corresponding to the 3′ end of RNA 1 (SEQ ID No: 39) cloned into plasmid pSR1(E3)A cut with ClaI, end-filled with Klenow and then cut with HindIII. The resulting plasmid is pSR1(E3)B. Upon linearization at BamHI and in vitro transcription with the SP6 RNA polymerase, and RNA corresponding to RNA 1 (SEQ ID No: 39), and terminating as described immediately above is obtained.

ix) RNA 2 (SEQ ID No: 47)

In constructing the full-length cDNA clone to enable in vitro transcription of this RNA hr236 described above was used as a basis. Two separate PCR products, one corresponding to the 5′ portion of RNA 2 (SEQ ID No: 47), which is missing from this clone altogether, and another covering the region where clone hr236 lacks the hairpin-forming sequence described above, were required.

The primer defining the 5′ end of the RNA carried a HindIII site and a sequence corresponding to the 5′ 18 nucleotides of RNA 2 (SEQ ID No: 47), as shown in FIG. 2 . The sequence of this primer was:

Hr2cdna5: 5′-CCGGAAGCTT G TTTTTCTTTCTTTACCA (SEQ ID No: 13) (The nucleotide underlined corresponds to that identified as the first nucleotide of RNA 2. (SEQ ID No: 47)).

Using an oligonucleotide complementary to nucleotides 1653-1669, a PCR product of 1.67 kbp was made. The template was cDNA synthesised using the MMLV RTase and an oligonucleotide complementary to the 18 nucleotides at the 3′ end of RNA 2 (SEQ ID No: 47) as the primer. Upon termination of the PCR reaction, the product was blunt-ended, kinased and gel-purified as described above, before cleavage with PstI. The resulting 1.3 kbp subfragment corresponding to the 5′ half of RNA 2 (SEQ ID No: 47) was cloned into plasmid pBSSK(−) (Stragene) cut with EcoRV and PstI, giving plasmid pBSR25p. In order to place this subfragment corresponding to the 5′ half of RNA 2 (SEQ ID No: 47) downstream of the SP6 promoter for in vitro transcription, a 1.3 kbp HindIII-BamHI fragment was excised from pBSR25p and ligated into HindIII-BamHI cut pGEM-1 (Promega), giving plasmid pSR25.

The second PCR product, covering the region where clone hr236 lacks the hairpin-forming sequence described above, was synthesised using as primers oligonucleotides corresponding to nucleotide sequence 873 to 889 of RNA 2 (SEQ ID No: 47) and to the complement of nucleotide sequence 2290-2309. Upon termination of the PCR reaction, the product was blunt-ended, kinased and gel-purified as described above, before cleavage with AatII. The resulting 1.1 kbp subfragment covering the required region was cloned into plasmid phr236 cut with HindIII, end-filled with Klenow and cut with AatII, giving plasmid phr236P70.

The two segments were joined covering the first 230 nucleotides of RNA 2 (SEQ ID No: 47) together. Plasmid phr236P70 was cut at the SacI site in the vector adjacent to the 5′ end of the insert and this made blunt-ended using Klenow in the absence of dNTPs. After heat-inactivation of the Klenow, the plasmid was cut with EcoRI, yielding fragments of 4.5 kbp and 380 bp. Plasmid pSR25 was cut with NheI, blunt-ended by end-filling with Klenow and cut with EcoRI, yielding fragments of 2.8 kbp, 900 bp and 750 bp. The 4.5 kbp fragment of phr236P70 and the 900 bp fragment of pSR25 were ligated to give pSR2P70. This clone covers all of RNA 2 (SEQ ID No: 47) except for the 3′ 169 nucleotides.

To complete the full-length clone of RNA 2 (SEQ ID No: 47), it was necessary to insert a fragment covering the 3′ end. As with RNA 1 (SEQ ID No: 39), two versions were made. One, called pSR2A, used the 3′ end as present in phr236, together with the poly(A) tail present in this version. The other pSR2B, used a PCR fragment carrying a BamHI site immediately downstream of the 3′ nucleotide, as in pSR1(E3)B above. To construct pSR2A, a 350 bp NotI-ClaI fragment was excised from phr236 and cloned into pSR2P70 cut with the same endonucleases. Linearization at the unique ClaI site allows in vitro transcription of the complete RNA 2 (SEQ ID No: 47) and a poly(A) tail of about 50 nucleotides in length.

To make pSR2B, an appropriate PCR product was made using as primers an oligonucleotide corresponding to nucleotide sequence 1178 to 1194 and to the 3′ terminal 18 nucleotides of RNA 2 (SEQ ID No: 47). The latter primer carried a BamHII site attached, giving it the sequence:

HvR23p:5′-GGGGGATCCGATGGTATCCCGAGGGACGC (SEQ ID No: 14)

The template used was a plasmid phr236. Upon termination of the PCR reaction, the product was blunt-ended, kinased and gel-purified as described above, before cleavage with NotI. The resulting 400 bp subfragment covering the required region was cloned into plasmid pSR2P70 cut with ClaI, end-filled with Klenow and cut with NotI, giving plasmid pSRP2B. Linearization at the unique BamHI site allows in vitro transcription of the complete RNA 2 (SEQ ID No: 47), terminating with the sequence ACCaggatc.

x) Construction of pSXR2P70

This plasmid was made to determine where p24 starts. A 2.1 kbp XhoI-BamHI fragment was cut from clone pSR2P70 and ligated into the vector pGEM-1 (Promega) which had been cut with SalI and BamHI. In vitro transcription of the resulting plasmid after linearization at the unique BamHI site yielded an RNA covering about 70 nucleotides upstream of the first ATG at nucleotides 283 to 286, plus a short sequence derived from the vector.

In vitro translation of the RNA from pSXR2P70 yielded both proteins (P70 (SEQ ID No: 52)+P24).

xi) Description of Virus-Induced Pathology

The virus induces a rapid anti-feeding effect in Helicoverpa larvae as determined by experiments with larvae the results of which are shown in FIG. 3 . FIG. 3 shows: A neonate larvae (less than 24 h old) were fed the designated concentrations of isolated virus (in particles per ml [of diet] added to solid diet). They were weighed on following days and the mean of a statistically significant number (24) of larvae shown. Where necessary, mortality was recorded for the higher concentrations. The vertical axis shows the fold-increase in weight from the hatching weight of 0.1 mg per larvae. This scale therefore also corresponds to weight in units of 0.1 mg (ie 300 is equivalent to 30 mg). B. As for A, but the larvae were 5 days old at the start of the virus feeding. The vertical scale is in mg weight.

No weight gain at all was detectable with neonates which had been fed the doses of virus over 10 8 particles per ml (virus added to diet). In addition, 100% mortality was evident after four days at the highest doses. Virus doses as low as 10 6 particles per ml (virus added to diet) still cause significant stunting. The five day old larvae showed a cessation of feeding after 48 hours and significant stunting at 4 dpi, but no mortality at comparable virus doses (FIG. 3 ). Neonates are therefore very sensitive indeed to this virus. Virus particles accumulate specifically in the midgut. This potent anti-feeding effect may be due to the capsid protein or another protein encoded by the virus, or to the effect of any combination of such proteins.

xii) Expression of Virus-Encoded Proteins in Bacteria

The Vectors

The expression system used initially was derived from the pET-11 system (Novagen). Trimmed down versions of pET-11b and c were constructed and used to compare expression of the capsid proteins. However, due to difficulties experienced with this system substantial modification of the original vectors was carried out in order to achieve much higher yields. These results are described in xiii-b) below.

The initial trimmed-down vectors discussed above were made as follows: pGEM-2 (Promega) which carries T7 promoter adjacent to a poly-linker sequence, but has no sequences corresponding to the lac operon, was cut at the unique XbaI (34) and ScaI (1651) sites, giving fragments of 1.61 and 1.25 kbp. The plasmids pET-11b and c were cut with the same enzymes, giving fragments of 4.77 and 0.91 kbp. The 1.61 kbp fragment of pGEM-2, carrying the c-terminal portion of the ampicillin-resistance gene, the origin of replication and the T7 promoter, was then ligated to the 0.91 kbp fragment of the pET vector, which carries a sequence covering the Shine-Dalgarno sequence, the ATG (in a NdeI site), the terminator for the T7 polymerase and the N-terminal portion of the ampicillin-resistance gene. The resulting plasmids of approximately 2.53 kbp, called pT7T2-b and c, therefore carry a complete T7 transcription unit, which may be used as an expression system in a manner similar to the original pET-11 plasmids, but are repressor-neutral within the cell; they neither titrate away repressor by carrying a binding site, nor do they carry the gene producing the repressor. They were found to grow very well in E. coli strains JM109 and BL21 (DE3), and to be very efficient expression vectors. The repressor present in the cells was found to be sufficient to keep the genomic T7 polymerase gene uninduced and therefore the foreign gene unexpressed in the absence of IPTG.

xiii-a) Construction of Plasmids for Expression of Capsid Proteins

In this section, all proteins expressed from segments of Ha SV RNA 2 (SEQ ID No: 47) are referred to by the size of their gene, as defined in FIG. 4 and in section vi) of this example. The following plasmids were constructed by PCR, using the abovementioned full-length clone of RNA 2 (SEQ ID No: 47), plasmid pSR2A as the template, except where mentioned otherwise.

Groups of plasmids expressed protein starting at each of the first three methionine initiation codons found on the sequence of Ha SV RNA 2 (SEQ ID No: 47). For those proteins initiating at the first methionine initiation codon found on the sequence of Ha SV RNA 2 (SEQ ID No: 47) (which initiates the P17 (SEQ ID No: 48) gene; oligonucleotide primer HVPET65N (SEQ ID No: 15)), an extra group of plasmids was made by PCR using as a template the version of the RNA 2 sequence carrying an extra C residue inserted at residue 570 (SEQ ID No: 51) (as depicted in FIG. 2 ). Expression constructs initiating at the third methionine initiation codon found on the sequence of Ha SV RNA 2 (which is located within the P17 gene; oligonucleotide primer HVPET63N (SEQ ID No: 16)) were made by PCR using as a template only the version of the RNA 2 sequence carrying an extra C residue inserted at residue 570 (SEQ ID No: 51). For these latter expression constructs, as well as those designed to initiate expression from the second methionine initiation codon found on the sequence of Ha SV RNA 2 (SEQ ID No: 47) (which initiates the P71 gene; oligonucleotide primer HVPET64N (SEQ ID No: 17)), two versions were constructed.

One version terminated at a point corresponding to the c-terminus of the processed (P64) form of the capsid protein and was made using oligonucleotide primer HVP65C (SEQ ID No: 19). The other version terminated at a point corresponding to the c-terminus of the precursor (P71 (SEQ ID No: 50)) form of the capsid protein and was made using oligonucleotide primer HVP6C2 (SEQ ID No: 20).

The sequence encoding P64 (or the precursor, P71 (SEQ ID No: 50)) was synthesised in two segments using PCR. The amino-terminal half of the gene was obtained using as primers oligonucleotides incorporating one of the three ATG possible initiation codons for the ORF, in addition to an oligonucleotide with the sequence TCAGCAGGTGGCATAGG (SEQ ID No: 27); complementary to nucleotides 1653 to 1669 of the sequence shown in FIG. 2 . The forward primers were as follows:

HVPET65N:

AAATAATTTTGTTTACTTTAGAAGGAGATATACAT ATGAGCGAGCGAGCAC AC (SEQ ID No: 15)

(the underlined sequence corresponds to nucleotides 283 to 296 of the sequence shown in FIG. 2 )

HVPET63N

AAATAATTTTGTTTAACCTTAAGAAGGAGATCTACAT ATGCTGGAGTGGCG TCAC (SEQ ID No: 16)

(the underlined sequence corresponds to nucleotides 373 to 390 of the sequence shown in FIG. 2 ; the AflII (CTTAAG) and BglII (AGATCT) sites introduced into the sequence by single nucleotide changes (shown in italics) in the oligonucleotide are shown in bold).

HVPET64N

GGAGATCTACAT ATGGGAGATGCTGGAGTG (SEQ ID No: 17)

(the underlined sequence corresponds to nucleotides 366 to 383 of the sequence shown in FIG. 2 ; the BglII site introduced into the sequence by a single nucleotide change in the oligonucleotide is shown in bold).

The PCR products obtained from each combination of one of these primers with the abovementioned one were treated with the Klenow fragment of E. coli DNA polymerase, and then with T4 polynucleotide kinase in the presence of 1 mM ATP, before purification by agarose gel electrophoresis as described above. Each product was then cleaved with AatII to yield fragments of 0.95 and 0.4 kbp, and each resulting fragment of about 0.95 kbp cloned intro vector pGEM-2 (Promega) cut with HincII and AatII, giving plasmids pGEMP63N (in which the insert commenced with oligonucleotide HVPET63N (SEQ ID No: 16)), pGEMP64N (in which the insert commenced with oligonucleotide HVPET64N (SEQ ID No: 17)) and pGemP65N (in which the insert commenced with oligonucleotide HVPET65N (SEQ ID No: 15)). The fragment covering portion of the Ha SV capsid gene was then excised with enzymes AatII and XbaI.

Two versions of plasmid pGemP65N were made, using different templates as described above. pGemP65N was derived from the sequence of the viral RNA, as in plasmid pSF2A; plasmid pGemP65Nc was derived from the sequence carrying an extra C residue, as shown in FIG. 2 (see “5C version”).

In parallel, the carboxy-terminal halves of the major capsid protein variant, whether terminating as for P64 or for P71 (SEQ ID No: 50), were also produced using PCR. An oligonucleotide primer, HVRNA2F3, with the sequence GTAGCGAACGTCGAGAA (SEQ ID No: 18) (corresponding to nucleotides 873 to 889 of the sequence shown in FIG. 2 ) was used in conjunction with each of the two primers following:

HVP65C

GGGGGATCCTC AGTTGTCAGTGGCGGGGTAG (SEQ ID No: 19)

(the underlined sequence is complementary to nucleotides 2072 to 2091 of the sequence shown in FIG. 2 ).

HVP6C2

GGGGATCC CTAATTGGCACGAGCGGCGC (SEQ ID No: 20)

(the underlined sequence is complementary to nucleotides 2290 to 2309 of the sequence shown in FIG. 2 ).

The PCR products obtained from each combination of one of these primers with the above mentioned one (HvRNA2F3 (SEQ ID No: 18)) were treated with the Klenow fragment of E.coli DNA polymerase, and then with T4 polynucleotide kinase in the presence of 1 mM ATP, before purification by agarose gel electrophoresis as described above. Each product was then cleaved with AatII to yield fragments of 0.9 kbp (in the case of HVP65C (SEQ ID No: 19)) or 1.1 kbp (in the case of HVP6C2 (SEQ ID No: 20)) and 0.4 kbp, and each resulting fragment of about 0.9 or 1.1 kbp cloned into plasmid phr236 cut with HindIII, treated with Klenow and AatII, giving plasmids phr236P65C and phr236P70 (which has already been described above), respectively. The fragment covering the c-terminus of the capsid protein gene was then excised with enzymes AatII and BamHI.

To assemble plasmids for expression in suitable strains of E. coli , the excised XbaI-AatII fragments of 0.95 kbp covering the amino-terminal half of the gene and the excised AatII-BamHI fragments of 0.9 or 1.1 kbp covering the carboxy-terminal half of the gene were simultaneously ligated into the vector pT7T2 cut with XbaI and BamHI. Initial transformation was of E. coli strain JM109. Recombinant plasmids carrying the correct insert were then transformed into strain BL21(DE3) for expression as described above.

The plasmid obtained by ligating the aminoterminal fragment commencing with oligonucleotide primer HVPET63N (SEQ ID No: 16) to the c-terminal fragment ending at oligonucleotide primer HVP65C (SEQ ID No: 19) in the epxression vector pT7T2b was called pP65G.

In the case of plasmid pP64N, containing an insert from HVPET64N (SEQ ID No: 17) to HVP65C (SEQ ID No: 19), the fragment covering the amino-terminal half of the oligonucleotide was excised by BglII and ScaI from the plasmid pGemP64N and the fragment covering the remainder of the gene was excised with ScaI and EcoRI from plasmid pT7T2-P65. These two fragments were then ligated simultaneously into pP65G which had been cut with BglII sand EcoRI.

The resulting construct carrying the complete P71 (SEQ ID No: 50) precursor gene was called pT7T2-P71 and that carrying the P64 form of the gen was called pT7T2-P64. In the case of plasmids derived from pGemP65N and pGemP65Nc, carrying inserts commencing as defined by primer HVPET65N, the expression plasmid derived from pGemP65N which is based on PCR products made using as the template the sequence of the viral RNA, as in plasmid pSR2A, was called pTP17; a truncated form of this plasmid, which expresses P17 (SEQ ID No: 48), was made by cutting at the unique BglII and BamHI sites, removing the intervening fragment (which corresponds to the c-terminal part of the insert) and religating the compatible cohesive ends, to give pTP17delBB. The expression plasmids derived from plasmid pGemP65Nc (which was derived from the sequence carrying an extra C residue, were called pT7T2-P65 (carrying an insert terminating at the primer HVP65C (SEQ ID No: 19)) and pT7T2-P70 (carrying an insert terminating at the primer HVP6C2 (SEQ ID No: 20)).

Expression of P6

Two forms of this protein, which arises through processing of the large capsid protein variant precursor P70 (SEQ ID No: 52) and therefore lacks its own initiation codon, were made. One form (protein MA) replaced the phenylalanine at the start of this protein with methionine, giving it the amino-terminal sequence MAA . . . ; the other carries an additional methionine residue, giving it the amino-terminal sequence MFAA. . . . The oligonucleotides used for PCR-amplified products covering the p6 coding sequence carried a NdeI site (bold) at the ATG codon, for direct ligation into the pET-1I vectors. The primers used were:

HVP6MA: AATTACATATGGCGGCCGCCGTTTCTGCC (SEQ ID No: 21)

HVP6MF: AATTACATATGTTCGCGGCCGCCGTTTCT (SEQ ID No: 22)

Each of these primers was used in conjunction with primer HVP6C2 (SEQ ID No: 20) to generate a PCR product of 0.2 kbp. These products were blunt-end ligated into vector pBSSK(−) which had been cut with EcoRV and dephosphorylated. The insert corresponding to the p6 gene was excised with NdeI and BamHI (using the BamHI site in the primer HVP6C2 (SEQ ID No: 20)) and ligated into the expression vector pET-1Ib, which had been cut with the same enzymes. For expression at higher levels, the insert was transferred to PT7T2 as a XbaI-BamHI fragment, yielding plasmids pTP6MA and pTP6MF.

IPTG induction of bacteria containing plasmids pTP6MA or pTP6MF were used produce p6 for bioassay.

xiii-b) Expression of Viral Genes in E. coli and Bioassay in Larvae

Expression of P64

IPTG induction of bacteria containing plasmid pT7T2-P65, which contains an insert running from the location of primer HVPET65N (SEQ ID No: 15) to that of primer HVP65C (SEQ ID No: 19), yielded a protein of molecular weight 68 000. This was 3 000 molecular weight greater than the size of the authentic coat protein, as expected. Expression of pP65G, which contains an insert running from HVPET63N (SEQ ID No: 16) to HVP65C (SEQ ID No: 19), yielded a protein of 65 000 molecular weight.

The authentic capsid protein (P64) was expressed poorly from plasmid pT7T2-P64. Recloning this insert as a NdeI-BamHI fragment back into the other form of the vector (PT7T2b) did not alter this.

Expression of P70

IPTG induction of bacteria containing plasmid pT7T2-P70, which contains an insert running from the location of primer HVPET65N (SEQ ID No: 15) to that of primer HVP6C2 (SEQ ID No: 20), yielded a protein of molecular weight 73 000. This was 3 000 molecular weight larger than the size of the precursor of the coat protein, as expected.

The authentic capsid protein precursor (P71 (SEQ ID No: 50)) was expressed poorly from plasmid pT7T2-P71. Recloning this insert as a Ndel-BamHI fragment back into the other form of the vector (pT7T2b) did not alter this.

Due to the observation mentioned in vi) above, plasmids designed to express all forms of the capsid proteins from several possible ATG's at the start of the open reading frame were constructed.

It was found that both authentic P64 and P71 (SEQ ID No: 50) were expressed poorly in bacteria. In contrast, P17 (SEQ ID No: 48) and the forms of the capsid protein commencing at the P17 ATG were expressed very well. The extra C residue present in the latter two constructs resulted in a fusion protein being made from these expression plasmid. The sequence of the fusion proteins can be derived from FIG. 2 by including an extra C at position 570. The fusion caused the first 67 residues of the Ha SV capsid protein to be replaced by the first 95 residues of P17 (SEQ ID No: 48). Good expression of the large capsid precursor and protein was achieved, but the size of these proteins were above 3 kDa larger than the authentic forms. Notwithstanding this the expression products of the vectors containing the 5C variant of RNA 2 (SEQ ID No: 51) are still useful because the resulting product, a P70 (SEQ ID No: 52) variant, is only modified at the NH 2 terminus. Since this terminus is thought to be embedded in the capsid structure and therefore not to participate in the initial interaction with the larval midgut cell, the variant is still useful.

In order to produce constructs which ensure that the expressed proteins possessed the native amino terminus, new plasmids carrying the correct sequence were then cloned into the expression vector (pT7T2). It was found these plasmids to express proteins of the correct size.

The P6 has not yet been to expressed from the new constructs. No evidence has been found for processing of P70 to yield the mature proteins in bacteria, nor upon in vitro translation of synthetic full-length RNA 2 (SEQ ID No: 47).

The P17 (SEQ ID No: 48) gene has also been cloned into the same vectors for expression and bio-assay. This protein accumulates well in bacteria upon induction, and electron microscopy analysis has shown it form spectacular honeycomb-like structures under the bacterial cell wall, completely surrounding the cell interior (results not shown). The properties of this protein including its amino acid composition and ability to form tube-like structures when expressed in bacteria suggest that it may be an homolog of a gap junction protein. The latter is involved in forming the channels linking the cytoplasms of adjacent epithelial cells in the insect gut. P17 could then play a role in enlarging or forming these channels, thereby enabling cell-to-cell movement of the virus in the insect gut, analogous to the movement or spreading proteins encoded by plant RNA viruses.

In order to ensure that the expressed proteins carried the native amino terminus the correct sequence has also been cloned into the expression vector (pT7T2). The vector had been very slightly modified to that described above to introduce two novel restriction sited (for AfIII and BgIII) flanking the Shine-Dalgarno sequence. The resulting constructs have been found to be poor producers of the capsid proteins. The complete coding regions (which have been completely checked by re-sequencing) have therefore been recloned into the more satisfactory vectors. Results using these constructs suggest that the amino-terminus of the capsid protein presents inherent difficulties in expression. These difficulties may be imposed by either the nucleotide sequence encoding the amino terminus, or the actual amino acid sequence itself To discriminate between these possibilities, two types of mutants were made in the sequence encoding the amino terminal 5 residues of the Ha SV capsid protein. These amino-terminal mutants are as follows:

HVP71GLY
CCCATATG GGC GAT GCC GGC GTC GCG TCA CAG (SEQ ID NO: 28)
Met Gly Asp Ala Gly Val Ala Ser Gln (SEQ ID NO: 29)
HVP71SER:
CCCATATG AGC GAG GCC GGC GTC GCG TCA CAG (SEQ ID NO: 30)
Met Ser Glu Ala Gly Val Ala Ser Gln (SEQ ID NO: 31)
Native HaSV seq:
ATG GGA GAT GCT GGA GTG GCG TCA CAG (SEQ ID NO: 32)
Met Gly Asp Ala Gly Val Ala Ser Gln (SEQ ID NO: 33)

EXAMPLE 4

Expression in Baculovirus Vectors and Bioassay on Larvae

Materials and Methods

A(i) Cloning of Ha SV capsid protein gene.

The capsid protein gene was amplified by PCR using the following primers:

5′ primers:

HV17V71:

5′ GGGGGATCCCGCGGATTT ATG AGCGAG (SEQ ID No: 34)

HV17E71:

5′ GGGGGATCCCGCGGAGAC ATG AGCGAGCACAC (SEQ ID No: 35)

HVP71:

5′ GGGGGATCCAGCGAC ATG AGAGATGCTGGAGTGG (SEQ ID No: 36)

HVV71:

5′ GGGGGATCCAGCGAC ATG AGAGATGCTGGAGTGG (SEQ ID No: 37)

The ATG triplets initiating P17 (SEQ ID No: 48) (in HV17V71 (SEQ ID No: 34) and HV17E71 (SEQ ID No: 35)) or P71 (SEQ ID No: 50) (in HVP71 and HVV71) are underlined)

3′ primers:

Primers HVP65C (SEQ ID No: 19) and HVP6C2 (SEQ ID No: 20), described in Example 3. Results section Xiiia, were used. These constructs were made using one of the four 5′ primers and HVP6C2 (SEQ ID No: 20). Plasmids constructed from PCR products made using one of the four 5′-primers and HVP65C (SEQ ID No: 19) are called 17V64 (made using 5′ primer 17E71 (SEQ ID No: 35)), P64 (made using 5′ primer P71 (SEQ ID No: 36)) and V64 (made using 5′ primer V71 (SEQ ID No: 37)). These plasmids allow expression of P64.

A(ii) Cloning a Full Length cDNA of Ha SV RNA 1 (SEQ ID No: 39)

For expression of an RNA transcript corresponding to full length Ha SV RNA 1 (SEQ ID No: 39), in insect cells by baculovirus infection or plasmid transfection, PCR was used to generate a fragment of cDNA linking the 5′ end of RNA 1 (SEQ ID No: 39) to a Bam HI site.

The primers were:

HVR1B5′

5′ GGGGGATCC G TTCTGCCTCCCCGGAC (SEQ ID No: 38)

(where the underlined nucleotide represents the start of natural RNA 1 (SEQ ID No: 39)), and an oligonucleotide complementary to nucleotides 1192=1212 of RNA 1 (SEQ ID No: 39).

The template was plasmid pSR1(E3)B described in Example 3 above.

A segment of the 1240 bp PCR fragment corresponding to the 5′ 320 nucleotides of RNA 1 (SEQ ID No: 39) was excised with Bam HI and ASC II and cloned into the Bam HI site of pBSSK(−)[Stratagene] together with the 5 kbp ASCII-Bam HI fragment of pSR1(E3)B, giving plasmid pBHVR1B, which carries the complete cDNA to Ha SV RNA 1 (SEQ ID No: 39), flanked by Bam HI sites.

A(iii) Cloning a Full Length CDNA of Ha SV RNA 2 (SEQ ID No: 47)

For expression of an RNA transcript corresponding to full length RNA 2 (SEQ ID No: 47) in insect cells by baculovirus infection or plasmid transfection, plasmid pB+NR2B was made by inserting a fragment carrying Hind III and Bam HI sites from the multiple cloning site of vector pBSSK(−) [Stratagene] into plasmid pSR2B described above. The resulting plasmid, called pBHVR2B, carried the cDNA corresponding to full length Ha SV RNA 2 (SEQ ID No: 47), flanked by Bam HI sites.

A(iv) Baculovirus Transfer Plasmids.

Bam HI fragments of 5.3 and 2.5 kbp corresponding to Ha SV RNA's 1 and 2 (SEQ ID Nos: 39 and 47) respectively, were excised from pBHVR1B and pBHVR2B respectively and inserted into the baculovirus transfer vectors described below, which had been linearised with Bam HI.

B. Baculovirus Expression of Protein.

Baculovirus transfer vectors and engineered AcMNPV virus were transfected into Spodoptera frugiperda ( Sf 9) cells as described by the supplier (Clontech) and as described in the following references:

Vlak, J. M. & Kens, R. J. A. (1990) in ‘Viral Vaccines”, Wiley-Liss Inc., N.Y., pp.92-128;

Kitts, P. A. et al (1990) Nucleic Acids Research 18: 5667-5672; Kitts, P. A. and Possee, R. P. (in preparation); Possee, R. D. (1986) Virus Research, 5: 43-59.

C. Western Blotting.

As in Example 1

D. Oligonucleotides

The following Ribozyme Oligonucleotides were produced according to standard methods.

HVR1Cla

5′ CCATCGATGCCGGACTGGTATCCCAGGGGG (SEQ ID No: 5)

5′ HVR2Cla

5′ CCATCGATGCCGGACTGGTATCCCGAGGGAC (SEQ ID No: 6)

RZHDV1

5′ CCATCGATGATCCAGCCTCCTCGCGGCGCCGGATGGGCA (SEQ ID No: 7)

RZHDV2

5′ GCTCTAGATCCATTCGCCATCCGAAGATGCCCATCCGGC (SEQ ID No: 8)

RZHC1

5′ CCATCGATTTATGCCGAGAAGGTAACCAGAGAAACACAC (SEQ ID No: 9)

RZHC2

5′ GCTCTAGACCAGGTAATATACCACAACGTGTGTTTCTCT (SEQ ID No: 10)

Results

A series of recombinant baculoviruses has been constructed, based on the pVL941 transfer vector (PharMingen) or pBakPak8 (Clontech) and the AcMNPV. These are designed to express the correct forms of the precursor and processed Ha SV capsid proteins (P64 and P71 (SEQ ID No: 50)) as well as the smaller capsid protein P6, and P17 (SEQ ID No: 48). In all systems where replicatable RNA encoding the nucleotide sequences of the present invention are to be used, such as eukaryotic systems, in order to get efficient replication, translation or encapsidation of the RNA it is necessary to excise structures downstream of the t-RNA like structure such as the 3′ extension or poly A tail on the RNA. In order to carry out such an excision, ribozymes or other suitable mechanisms may be employed. This self cleavage activity of the ribozyme containing transcript should proceed at such a rate that most of the transcript is transported into the cytoplasm of the cell before the regeneration of a replicatable 3′ end occurs. Such ribozyme systems are more fully explained in Examples 7 and 9. In the results presented here highly efficient production of P64 and P71 (SEQ ID No: 50) has been achieved. Electron microscopy and density gradient analysis have confirmed that empty particles (“capsoids”) are being produced in infected cells that efficiently express the P71 precursor gene. P17 (SEQ ID No: 48) placed in the context of the H. virescens juvenile hormone esterase (JHE) gene (Hanzlik T. N., et al, J. Biol. Chem. 264, 12419-25 (1989)) is produced, but not in large amounts. The latter construct results in a reduction of expression of the capsid protein from the same recombinant, presumably due to a reduction in the number of ribosomes reaching the AUG for the capsid gene.

Sf 9 cells infected with recombinant baculovirus have been shown to contain large amounts of icosahedral virus particles by electron microscopy (data not shown). These particles contained no RNA, and were empty inside. This observation shows that signals on the viral RNA required for encapsidation of RNA must be located in either the 5′ 270 nucleotides or the 3′ 170 nucleotides, or both, since these sequences were missing from the RNA transcripts made using recombinant baculovirus. Expression of Ha SV proteins was confirmed by Western blotting of total protein extracts from infected insect cells.

In addition, the pAcUW31 vector (Clontech), which carries two promoters, is being used to simultaneously express p6 and p64 as separate proteins. In order to bioassay the capsid protein produced in baculovirus infected cells, it is first necessary to purify it from the baculovirus expression vector. Preliminary attempts have made use of density gradients, based on the observation that empty virus particles (“assembled capsids”) are in fact produced in infected cells.

As outlined earlier, the Ha SV genome or portion thereof is a particularly effective insecticidal agent for insertion into baculovirus vectors. Such a vector is constructed by insertion of the complete virus genome or portion thereof (preferably the replicase gene) into the baculovirus genome as shown in FIG. 13 . Preferably the virus genome or replicase is transcribed from a promoter active constitutively in insect cells or active at early stages upon baculovirus infection. An example of such a promoter is the heat shock promoter described in Example 7. Heat shock promoters are also activated in stressed cells, for example cells stressed by baculovirus infection. An even more preferable use of such a baculovirus construct is to use the HSP promoter to drive the Ha SV replicase and another gene for a toxin (as exemplified elsewhere in the specification) where the RNA expressing the toxin gene is capable of being replicated by the Ha SV replicase. Such recombinant baculoviruses carrying the Ha SV genome or portions thereof for expression in larvae at early or other stages of the baculovirus infection cycle are particularly effective biological insecticides.

EXAMPLE 5

Effect of Ha SV Genes and Their Products on Plants

Materials and Methods

A. Electroporation of Protoplasts

Protoplasts of Nicotiana tobacum, N. plumbaginifolia and Triticum aesticum and oats were produced and electroporated with either Ha SV or Ha SV RNA as described in Matsunaga et al (1992) J. Gen. Virol. 73: 763-766.

B. Northern Blot Analysis—RNA Extraction From Protoplasts After Harvest

The protoplasts are subjected to 3 cycles of freezing and thawing, and then an equal volume of 2× extraction buffer (100 mM Tris-HCl, pH 7.5, 25 mM EDTA, 1% SDS, made in DEPC treated water) is added, followed by 1 volume of phenol (equilibrated in 10 mM Tris-HCl pH 8.0) heated to 65° C. The samples are mixed by vortexing and incubated at 65° C. for 15 min, vortexing every 5 min. After phase separation by centrifugation at room temperature for 5 min, the aqueous phase is re-extracted with phenol, re separated by centrifugation and re-extracted with chloroform/isoamyl alcohol. To the aqueous phase are then added 0.1 volume of DEPC-treated sodium acetate (pH 5.0) and 2 volumes of ethanol. The RNA is recovered by precipitation at −70° C., followed by centrifugation at 4° C. for 15 min. The samples were then analysed by agarose gel electrophoresis as described in example 1.

After blotting to Zeta-Probe membrane (BioRad), the hybridization protocols were as above for Example 2.

C. Total Protein From Ha SV-Electroporated Protoplasts

Protoplasts were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting as described in Example 1.

Results

i) Use of Complete (Replication-Competent) RNA Virus Genome in Protoplasts

a) Ha SV Replication in Protoplasts

The nodavirus FHV has previously been shown to replicate in barley protoplasts (Selling H. H., Allison, R. F. and Kaesberg, P. Proc. Natl. Acad. Sci. U.S.A. 87,434-8 (1990). To determine whether Ha SV virus RNA can replicate in plants protoplasts, when introduced by electroporation, experiments using protoplasts from Nicotiana plumbaginifoli and wheat have been conducted. (These are all species for which protoplasts are regularly available in the Division of Plant industry). Assays for replication including RNA (Northern) blots using probes derived from cloned fragments of cDNA to RNAs 1 and 2 (SEQ ID Nos: 39 and 47), and Western blots, using the antiserum to purified Ha SV particles. Initial experiments showed that both Ha SV virus and RNA electroporated into protoplasts of N. plumbaginifolia resulted in Ha SV replication as studied using and verified by northern blots and ELISA. As a positive control TMV RNA was electroporated and was replication observed.

b) Bioassays

Protoplasts into which Ha SV RNA had been introduced by electroporation were harvested after 6 or 7 days post electroporation and used in bioassays on neonate larvae by addition to normal diet. The results showed significant stunting of test larvae in comparison to control larvae (see Table 1 below). Protoplasts lacking Ha SV RNAs had no effect on the larvae, confirming the result of control experiments. This result confirms that Ha SV RNA, when expressed or replicated in plant cells, is able to cause the formation of infectious virus particles able to control insect larvae feeding on the plant material.

Northern blotting has been used to confirm that RNA electroporation into protoplasts leads to RNA replication.

TABLE 1. Results of Bioassay From a Typical Experiment With Nicotiana and Oat Protoplasts (Oat Results are Shown in Brackets) [see over]

TABLE 1
Results of Bioassay from a typical experiment with Nicotiana and oat
protoplasts (oat results are shown in brackets) [see over]
				Number
	Treatment	Number	Escapes	stunted
1.	diet only	12 (12)	2 (3)	0/10 (0/9)
2.	diet + protoplasts	12 (12)	0 (1)	0/12 (0/11)
3.	HaSV + diet	12 (12)	0 (1)	12/12 (11/11)
4.	diet + HaSV/protoplasts	12 (n.d.)	0 (n.d.)	12/12 (n.d.)
5.	diet + RNA/protoplasts	12 (12)	0 (0)	11/12 (10*/12)
*HaSv replication in the larvae was confirmed except for two larvae which were dead. The letters “n.d.” mean the experiment was not done.

The above results demonstrate assembly of Ha SV particles from electroporated RNA in protoplasts of both moncot and dicot plant species.

c) Plasmids to Test Replication of Cloned and Engineered Forms of Ha SV

(1) Plasmids allowing in vitro transcription of Ha SV RNAs 1 and 2 (SEQ ID Nos: 39 and 47) for electroporation into protoplasts have already been described above. (2) Plasmids for transient expression of individual Ha SV RNAs (1 or 2) (SEQ ID Nos: 39 and 47) in protoplasts. Full-length cDNAs for the two viral RNAs have been inserted into expression plasmids pDH51 (with the CaMV 35 S promoter. Pietrzak M., et al (9186) Nucl. Acids Res. 14, 5857-68) for dicots and pActI.cas (with the rice actin promoter) for monocots (McElroy et al (1990) The Plant Cell 2: 163-171). As with the vectors for expression in insect cells, these expression plasmids are being modified to include a cis-acting ribozyme for generation of authentic ends. The non-ribozyme plasmids gave no virus replication.

ii) Expression of Capsid Protein in Plants

In view of the present inventors' observation that empty particles (“assembled capsids”) are being produced in baculovirus-infected cells that efficiently express the P71 precursor gene, expression of the coding region for the capsid protein in tobacco plants was investigated. The vector chosen for this purpose is based on pDH51 which carries the CaMV 35S promoter and polyadenylation signal. If necessary for improved expression, this vector can be modified by the addition of a translation enhancer sequence from e.g. TMV. Although certain groups have constructed transgenic plants expressing the capsid proteins of plant viruses, there has been only one recent report of assembly of empty capsids in such plants (Bertioli et al.,(1991) J. gen. Virol. 72: 1801-9). Bertioli et al point out that the protein-protein interactions in most icosohedral plant RNA viruses may be too weak to allow assembly of such capsids. In addition to the present inventors' observation of empty Ha SV capsids, it has been found these capsids are very tough, showing great resilience to e.g. repeated cycles of freezing and thawing, so that it is expected to see assembly of empty Ha SV capsids (“assembled capsids”) in transgenic plants.

Construction of Capsid Protein Expression Plasmid

Vector used was pDH51; linearised with BamHI and phosphatased.

Insert was PCR product made using following 2 primers:

CAPPLANT:

5′ GG GGATCC ACA ATG GGA GAT GCT GGA GTC-3′ (BamHI)

(i.e. A BamHI site followed by plant consensus context for ATG of capsid protein gene and 15 further nucleotides of this gene—nts 366-383 of Ha SV RNA2).

HVP6C2 (Example 3)

The PCR product was made with VENT polymer (New England Biolabs). After gel purification, it was cut with BamHI and cloned into the vector. Orientation screened with EcoRI to identify insert in same direction as promoter giving plasmid pDHVCAPB. Expression was verified by Western blotting using anti- Ha SV antiserum. Both precursor P71 and processed P64 capsid protein were detected in protoplasts following transfection with pDHVCAPB, showing assembly of virus-like particles.

EXAMPLE 6

Identification of Midgut Binding Domains

Materials & Methods

A. Plasmid Construction

Was as described in Examples 3 and 4.

B. Western Blotting

Was as described in Examples 1 and 3.

C. Invitro Translation

In vitro transcripts of cloned CDNA of Ha SV RNA's was translated in vitro as in Examples 1 and 3.

D. Preparation of Brush Border Membrane Vesicles

Brush Border Membrane Vesicles were prepared from freshly isolated larvae midguts of H. Armigera by the method of M. Wolfersberger et al (1987) Comp. Biochem. Physiol. 86A: 301-308, as modified by S. F. Garczyuski et.al. (1991) Applied Environ. Micro-biol 57: 1816-2820. Brush Border Membrane Vesicles binding assays using invitro labelled protein or 125 I-labelled protein were as described in Garczynski et.al. (1991) or in H. M. Horton and Burand, J. P. (1993) J. Virol. 67: 1860-1868.

Results

i) Determination of Epitopes on the Capsid Surface

Comparison of the recently published sequence of the Nudaurelia ω virus (NwV) capsid protein with that of Ha SV shown that these proteins are closely related and fall into four distinct domains, which are alternatively variable and highly conserved. These domains are summarised as follows:

	Residues:
	HaSV	1-49	50-272	273-435	437-647
	NωV	1-46	47-269	270-430	431-645
	% identity:	37	81	34	81

Comparison of this observation with the alignment by Agrawal and Johnson (1992) between the NwV and the nodavirus BBV (whose crystal structure is known: Hosur et al (1987) Proteins: Structure, Function & Genetics 2: 167-176) showed that the variable region coincided with a region forming the most prominent surface protrusion on the BBV capsid. Both Ha SV and NwV carry large insertions at this point relative to BBV, and these insertions are largely different in sequence. Assuming that the alignment by Agrawal and Johnson (1992) is correct, then this means that Ha SV and NwV have a more prominent pyramid-like structures as a surface protrusion than do the nodaviruses, and the pyramid-like structures are different. As already noted, there is no immunological cross-reactivity between the two viruses, despite the high degree of identity. There is thus a strong implication of the variable domain as a surface protrusion which functions as the sole antigenic region.

To confirm this a 400 bp NarI fragment spanning the variable region was deleted from the capsid gene in the expression vector. With end-filling of these sites the deletion is in-frame, so that a truncated protein of ca. 57 KDa is produced in bacteria upon induction. This protein was recognized only poorly on Western blots by the antiserum against intact Ha SV particles made in rabbits. The central variable domain was recognized well by the antiserum when expressed in isolation from the rest of the capsid gene.

As shown in the table above the region of Ha SV capsid protein comprising residues 273-439 shows great divergence form the corresponding region of the NwV capsid protein, compared to its immediate flanking regions. Within this region an especially divergent domain is found from residue 351 to residue 411, which shows only 25% identity to the corresponding region of the NwV capsid protein. This region is flanked by the sequences corresponding to the b-sheet structural features b-E(residues 339-349) and b-F(residues 424-431) of the Ha SV capsid protein, based on the alignment the NwV and nodavirus capsid proteins by Agrawal and Johnson (1992), and is therefore likely to form the loop of the most prominent surface protrusion on the Ha SV capsid. This is based on comparison and correspondence to the nodavirus capsid protein structure and capsid structure as described by Wery J.-P. and Johnson, J. E. (1989) Analytical Chemistry 61, 1341A-1350A and Kaesberg, P., et al. (1990) J. Mol. Biol. 214, 423-435. This loop is thought to contain important epitopes. It is significant that this exterior loop on the nodavirus capsid protein is one of the most variable regions when capsid proteins sequences from a number of nodaviruses are compared (Kaesberg et al. 1990).

Finally, the present inventors have observed a significant level of immunological cross-reaction on Western blots, between antisera against the CryIA(c) Bt toxin and Ha SV capsid protein, whether obtained from virus or expressed in bacteria. Initial data from the NarI deletion mutant described above suggest that this binding is not to the central variable domain, but to other regions of the capsid protein. The only other region of the proteins which shows extensive sequence variability, the amino terminus, cannot be responsible for the binding, since both authentic capsid protein and the protein with an altered amino terminus expressed in bacteria are recognized by the anti Bt antisera.

ii) In-Vitro Binding Assays

The full-length clones for in vitro translation yielding highly 35 S or 3 H labelled proteins were constructed by replacing the bacterial translation interaction signal in the T7 plasmids above by the more active eucaryotic context sequence from the JHE gene. The labelled capsid protein made by in vitro translation of the in vitro transcripts may be tested for binding to brush border membrane vesicles (BBMV's). Conditions are optimised by testing different procedures. The deletion mutant lacking approximately 125 amino acids in the central region, and containing the variable domain, as well as others derived from it are also tested.

iii) Fusion Proteins Comprising Virus Capsid Midgut Binding Domains and Other Proteins

The idea behind these tests is to fuse the binding domain from the Ha SV capsid protein to either large proteins (preferably indigestible, causing protein to aggregate in or on the midgut cells) or toxin domains from other proteins with suitable properties but normally different binding specificities (e.g. Bt). In initial experiments, the gene for the complete capsid protein has been fused to the GUS gene, as has a deletion mutant containing essentially only the central portion of the capsid gene. The resulting fusion proteins are being expressed in bacteria and tested for GUS activity, and makes them sensitive probes for binding experiments on midgut tissue.

iv) Mapping Binding Sites Using Bt/ Ha SV Fusion Proteins

Analysis of deletion mutants of the CryIA(c) Bt toxin has identified domains which may be involved in determining the host-specificity of this Bt by acting as receptor-binding sites (Schnepf et al (1990) J. Biol. Chem. 265: 20923-20930; Li et al (1991), Nature 353: 815-21. The present inventors have obtained a clone of this toxin gene. Deletion mutants corresponding to those identified by Schnepf et al are constructed. Segments of the Ha SV capsid protein gene can then be inserted into these mutants, the protein expressed in bacteria and their insecticidal function assayed.

EXAMPLE 7

Viral Growth in Cell Culture

Materials & Methods

A. Cell Lines

The following cultured insect cell lines were tested for infection by Ha SV: Drosophila melanogaster, Helicoverpa armigera (ovarian derived), Heliothis zea (ovarian derived), Plutella xylostella, Spodoptera frigiperda ( Sf 9). All lines were grown under standard conditions. Upon reaching confluence, the culture medium was removed and all mono-layers covered with 1.5 ml of cell culture medium into which Ha SV had been diluted; the average multiplicity of infection (M.O.I.) was 10 4 . After adsorption at 26° C. for 2 h, the inoculum was removed, the cells carefully washed twice with phosphate buffered saline (pH 7.0) and incubation continued with 5 ml of 10%. Foetal calf serum in TC199 culture medium (Cyto Systems).

B. Northern Blotting Analysis

Virus replication in all the above cell lines was confirmed by northern blotting analysis. Total RNA was extracted from infected cells by the method of Chomczynski and Sacchi (1987). Anal. Biochem. 162: 156-159. The cells were lysed in 1 ml of lysis solution (4M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1M 2-mercaptoethanol). In order, 0.1 ml of 2M sodium acetate, pH 4, 1 ml of phenol (0.2M sodium acetate equilibrated), and 0.2 ml of chloroform-isoamyl alcohol mixture (49:1) were added with thorough mixing between reagents. This was then vortexed for 10 s and cooled on ice for 15 min. Tubes were centrifuged in an Eppendorf centrifuge at 14k for 15 min at 4° C. for at least 15 min to allow RNA precipitation. RNA was pelleted by centrifugation at 14k for 15 min, washed with 0.6 ml of ice-cold 70% ethanol, pelleted once again (10K, 10 min), air dried at room temperature and resuspended in DEPC (Sigma) treated millipore water. RNA was subject to denaturing agarose gel electrophoresis in the presence of formaldehyde according to Sambrook et.al. (1989). The gel was Northern transferred to a zeta-probe membrane (Biorad) as described by Sambrook et.al. (1989). The probe was prepared by random-priming the 3′ sequences of the Ha SV genome using DNA and cDNA clones pSHVR15GB and pT7T2p71 SR-1 as per manufacturer's instructions (Boehringer-Mannheim). Hybridization was carried out as described for the standard DNA probe protocol contained within the literature for the zeta-probe membrane (Biorad).

C. Vectors

Vectors as described below.

Results

It has been found that Ha SV will replicate in several continuous cell lines, of which the best is the Spodoptera frugiperda line Sf 9. Time course assays by Northern blotting in Sf 9 cells have shown that RNA 1 (SEQ ID No: 39) replication is clearly detectable within a few hours of infection. RNA 2 (SEQ ID No: 47) is present only in very small amounts early in infection and accumulates much more slowly than RNA 1 (SEQ ID No: 39) does. This observation is consistent with one made earlier in Ha SV-infected larvae, where RNA 2 (SEQ ID No: 47) replication was not observed until 3 days after infection.

Some apparent replication was also observed in Drosophila cells (DL2), but with the difference that more RNA 2 (SEQ ID No: 47) replication was observed at the early time points compared to the lepidopteran cell lines above.

Plasmids that express the Ha SV genome as RNA transcripts from full length cDNA clones have been constructed and tested. These clones, constructed by PCR and carefully checked, have restriction sited immediately adjacent to the ends of the sequence. Transcription is driven from a specially-re-engineered Drosophila HSP70 promoter.

i) Constructs for Expression in Insect Cells

The constructs are based on vectors carrying the Drosophila HSP 70 or actin promoters and suitable polyadenylation signals from Drosphila (Corces & Pellicer (1984) J. Biol. Chem. 259: 14812-14817) or SV40 (Angelichio et al (1991) Nucl. Acids. Res. 18: 5037-5043). Since transcription from such plasmids generates viral RNAs carrying long 3′ terminal extensions derived from sequences in the poladenylation signal fragment, it is necessary to achieve cleavage of the transcript immediately after the 3′sequence of the viral RNA. These plasmids gave no virus replication, presumably because of the 3′ terminal extension. The method of choice for obtaining authentic 3′ termini is based on introduction of DNA sequences encoding a cis-acting ribozyme into the constructs. With suitable engineering, such a ribozyme will cleave immediately 3′ to the viral sequences within the transcript. Suitable ribozymes, based on the hepatitis delta virus (Been M. D., Perrotta, A. T. & Rosenstein, S. P. Biochemistry 31, 11843-52 (1992) or the hairpin cassette ribozyme (Altschuler, M., Tritz R. & Hampel, A. Gene 122, 85-90 (1992) have been designed (see Example 4). This involves synthesis of overlapping oligonucleotides, which are then annealed and end-filled with the Klenow fragment of DNA polymerase, to create short DNA fragments encoding the desired ribozyme. These fragments carry restriction sites at their termini allowing them to be ligated into plasmids between the viral RNA cDNA (which has a 3′ restriction site added by PCR) and the restriction fragment carrying the poladenylation signal. Ribozyme function has been verified (Example 9).

The Drosophila HSP70 promoter was joined to the Ha SV RNA 1 sequence as follows. A BamHI restriction site was introduced into the promoter sequence as described on p.5 of this specification. Oligonucleotide HVR1B5P described in Example 8 was used to prime PCR of RNA 1 to yield a cDNA copy of the RNA carrying a BamHI restriction site 5′ to the RNA 1 sequence and separated from it by the nucleotides ACA which end the HSP70 promoter just before the start of transcription. This common BamHI site was used to link the HSP70 promoter and the Ha SV RNA 1 sequence. The resulting plasmid was completed by adding either the hairpin cassette ribozyme (giving plasmid pHSPR1HC) or the HDV ribozyme (giving plasmid pHSPR1HDV) plus the SV40 late polyadenylation sequence.

A similar approach was used to obtain plasmids for RNA 2 i.e. pHSPR2HC and pHSPR2HDV.

An alternative approach is to link the promoter and the Ha SV cDNAs using blunt end ligation of a DNA fragment and carrying the promoter and terminating at the last nucleotide before the start of transcription (the underlined residue in AC A ) and the cDNA fragments corresponding to either HASV RNA 1 or 2, as described for the plant expression plasmids in Example 9.

The latter approach was used to join the sarcoma virus (RSV) long terminal repeat (LTR) promoter to the Ha SV cDNAs for expression in insect cells. The RSV LTR promoter is active in many animal cells (Cullen, B. R. Raymond, K. & Ju, G. (1985) Mol. Cell. Biol. 5,438-447) and also in lepidopteran cell lines (D. Miller personal communication). It was obtained from plasmid pRSVCAT (Gorman, C., Padmanabhan, R. & Howard, B. H., (1983) Science 221, 551-553) as a 495 bp fragment carrying a 5′-XbaI site (added by PCR) and terminating at a blunt end with the sequence AA C , with the underlined residue corresponding to that immediately before the start of transcription. The resulting plasmids, pRSVR1HCLA and pRSVR2HCLA, carry the Ha SV RNA 1 and 2 cDNAs, respectively, and are otherwise like pHSPR1HC and pHSPR2HC, respectively. These plasmids carry the SV40 late polyadenylation signal. They allow efficient and precise expression of the Ha SV genomic RNAs in insect cells, for example if introduced using a baculovirus vector or by transfection.

EXAMPLE 8

Shedding of Infected Cells

Materials & Methods

A. Confocal Laser Scanning Microscopy (CLSM)

CLSM enables the visualisation and analysis of three-dimensional cell and tissue structures at the macro and molecular levels. The Leica CLSM used in this example is based on an MC 68020/68881 VME bus (20 MHz) with standard 2 Mbyte framestore and 4 Mbyte RAM and OS9 operating system with programmes written in C code. It incorporates a Leica Diaplan research microscope and using X10/0.45, X25/0.75, X40/1.30 and X63/1.30 Fluotar objectives has a claimed optical efficiency better than 90%. The confocal pinhole is software controlled over the range of 20 to 200 mm. Excitation at 488 and 514 nm is provided by a 2 to 50 mW argon-ion laser.

B. Immunocytochemistry (ICC)

For whole mount ICC, tissues were dissected under saline and fixed in fresh 4% formaldehyde in phosphate buffered saline (PBS) for at least 15 mins. After multiple washes in PBS they were permeablized either by 60 mins incubation in PBT (PLBS with 0.1% Triton X-100 plus 0.2% bovine serum albumin). After 30 mins blocking in PBT+N (5% normal goat serum) tissue was incubated in primary antibody diluted (1:40) in PBT+N for at least 2 hrs at room temperature then at 4° C. overnight. After extensive washing in PBT and 30 mins blocking in PBT+N the FITC conjugated secondary antibody diluted (1:60) in PBT+N was incubated for 2 hrs at room temperature plus overnight at 4° C. After multiple washes in PBT and PBS the tissue was cleared in 70% glycerol and mounted in 0.01% w/v p-phenylenediamine (Sigma#P1519) dissolved in 70% glycerol. All processing was at room temperature unless otherwise stated.

Results

The inventors' current model for the effect of Ha SV involves the detection by the insect midgut of infected cells, their identification as infected and their subsequent shedding in numbers sufficient to cause irreparable damage to the insect midgut. The evidence for this is based on the above and on the following direct observation of the fate of infected cells in midgut tissue over 1-3 days post infection. These results in repeat experiments were complicated by the discovery that another unrelated virus was present in the larval population being tested. Preliminary findings indicated that Ha SV infection activates or facilitates pathogenesis of the unrelated virus and together these cause severe disruption of the larval gut cells. Thus these two agents appear to act synergistically in causing gut cell disruption.

Midguts from larvae infected with Ha SV were treated with the antiserum to purified Ha SV particles (above) and examined under the Laser confocal microscope (described above). This established that some midgut cells were sufficiently infected with Ha SV to give strong fluorescence signals. Such cells were moreover clearly separating from the surrounding tissue, a sign that they were in the process of being shed.

Similar observation have been made with other insect viruses (Flipsen et al (1992) Society for Invertebrate Pathology Abstract #96) although in these cases the effect is too localised and weak to cause any anti-feeding effect apparently only the small RNA virus of the tetraviridae which are localised to the gut and cause more-or-less severe anti-feeding effects in their hosts (Moore, N. F. in Kurstak E. (Ed) (1991) Viruses of Invertebrates. Marcel Dekker, New York pp277-285) are capable of such an effect to an extent sufficient for pest control.

Following on from the immune-fluorescence work, in situ hybridization can be carried out to detect RNA replication in infected cells. Furthermore, larvae infected with a recombinant Ha SV expressing a foreign gene at early stages (by insertion of that gene into RNA 1 in place of the N-terminal portion of the replicase gene) can be studied. A correlation between virus replication and cell rejection can be confirmed by histochemical analysis of the midgut cells of the infected larvae. Thus the cell-shedding phenomenon offers a direct and rapid assay for early events in Ha SV-infected gut tissue. Extracts of baculo-vector infected insect cells carrying empty Ha SV particles can be fed to larvae directly and the midgut examined by toluidine blue staining and immune-fluorescence at intervals after infection. This will allow direct determination of whether the particles can bind the brush border membranes in intact gut, and whether such binding can induce the massive disruption evident in normally infected larvae. Control experiments using extracts from cells infected with the baculovector alone can be conducted to observe and distinguish effects due to the vector. The immune-fluorescence assay on midgut tissue allows analysis of binding to midgut brushborder membranes. Once determined for wild-type capsid protein expressed from a baculo-vector, deletion or replacement mutants can be inserted into the baculovectors. Suitable cell extracts from these can be used to infect larvae.

EXAMPLE 9

Engineered Virus and Uses

Materials & Methods

(as indicated in earlier Examples)

i) Engineered Virus as a Vector for Other Toxin Genes

This involves placing suitable genes under control of Ha SV replication and encapsidation signals. Genes which may be suitable include intracellular insect toxins such as ricin, neurotoxins, gelonin and diphtheria toxins. The toxin gene may be placed in the viral gene such that it is a silent (downstream) cistron on a polycistronic RNA, or in a minus strand orientation, requiring replication by the viral polymerase to be expressed. Standard techniques in molecular biology can be used to engineer these vectors.

A discussion of two recombinant Ha SV vectors which have been designed is given below:

for RNA 1 (SEQ ID No: 39):

The reporter gene (or one of the toxin genes mentioned above) is inserted in place of the amino-terminal portion of the putative replicase gene, such that the intiation codon used for the replicase (ie that at nucleotides 37-39 of the sequence) is now used to commence reporter gene translation. The fusion is achieved by the use of artificial NcoI restriction sites common to both sequences.

The short 36 nucleotide 5′-untranslated leader of RNA 1 (SEQ ID No: 39) (shown in upper case) is synthesised as the following sequence:

gg ggatcc acaGTTCTGCCTCCCCCGGACGGTAAATATAGGGGAACCATG G tctaga gg, (SEQ ID No: 53)

using two overlapping oligonucleotides comprising the first 31 (oligonucleotide HVR1B5P) nucleotides and the complement of the last 40 nucleotides (oligonucleotide HVR1NCO) respectively. These primers are annealed and end-filled by Klenow. The resulting fragment is then cut with BamHI and Xbal (sites underlined) and cloned with plasmid vector pBSIISK(−) to give pBSSKR1NCO.

The GUS gene carrying a NcoI site at the ATG codon was obtained as a NcoI-SacI fragment from plasmid pRAJ275 (Jefferson, R A J Plant Mol. Biol. Rep 5, 3387-405 (1987)). This SacI site is located just downstream from the coding sequence for the GUS gene.

The 5′ leader of Ha SV RNA 1 is excised as a BamHI-NcoI fragment from the plasmid pBSSKR1NCO, and is ligated together with the NcoI-SacI fragment carrying the GUS gene into plasmid pHSPR1HC or pHSPR1HDV or pDHStuR1HC carrying the full-length cDNA insert of RNA 1 (see above) which has been cut with BamHI and SacI. The resulting plasmid then carries a complete form of RNA 1 (SEQ ID No: 39) but with the amino-terminal portion of the replicase gene substituted by the GUS gene. It is desirable to produce a construct with approximately the same size as RNA 1 (SEQ ID No: 39) for encapsidation purposes.

Similar approaches are adopted for RNA 2 (SEQ ID No: 47), with the foreign, reporter or toxin gene fused to the initiation codon of either P17 or P71. In either case the context sequence of the introduced gene is modified to give the necessary expression level of that protein. The foreign gene is introduced into plasmids pHSPR2HC or pHSPR2HDV or pDHStuR2HC.

The above recombinants have been described specifically as insertions of a reporter gene (GUS). The toxin genes to be inserted are described on page 14 of the specification. These preferably further require a signal peptide sequence added at the amino-terminus of the protein.

ii) Capsid Technology

Identification of encapsidation (and replication) signals on virus RNA allows design of RNAs which can be encapsidated in Ha SV particles during assembly of virus in a suitable production system. The virus capsids then carry the RNA of choice into the insects midgut cells where the RNA can perform its intended function. Examples of RNAs which may be encapsidated in this manner include RNAs for specific toxins such as intracellular toxins, such as ricin, gelonin, diptheria toxins or neurotoxins. This strategy is based on the resistance of the virus particle to the harsh gut environment.

iii) Other Uses of the Capsid Particle

The capsid particles can be used as vectors for protein toxins. Knowledge of icosahedral particle structure elucidated by the inventors suggests that the amino and especially the C-termini are present within the capsid interior. It is possible to replace or modify the amino acid sequence corresponding to P7 such that it encodes a suitable protein toxin which is cleaved off the bulk of the capsid protein during capsid maturation. As with toxin-encoding mRNAs, the Ha SV capsid delivers it to the midgut cell of the feeding insect, where it exerts the desired toxic effect.

iv) Use of Ha SV in Plants

The use of Ha SV in the production of insect-resistant transgenic plants are shown in FIG. 12 . These inventions are based on the use of either the complete Ha SV genome, or of the replicase gene as a tool for the amplification of suitable amplifiable mRNAs (e.g. encoding toxin) or of the capsid protein as a means to deliver insecticidal agents. These strategies are now described in some detail.

a) Use of the Complete Ha SV Genome

Fragments of cDNA corresponding to the full-length Ha SV genome components RNAs 1 and 2 (SEQ ID Nos: 39 and 47) are placed in a suitable vector for plant transformation under the control of either a constitutive plant promoter (e.g. the CaMV 35S promoter mentioned above) or an inducible promoter or a tissue specific (e.g. leaf-specific) promoter. The cDNAs are followed by a cis-cleaving ribozyme and a suitable plant polyadenylation signal. Transcription and translation of these genes in transgenic plant tissues and cells leads to assembly of fully infectious virus particles to infect and kill feeding larvae.

The following experiments were conducted. The plasmids for expression used the CaMV 35S promoter to generate transcripts commencing at the first nucleotide of the Ha SV RNAs 1 and 2 (SEQ ID Nos: 39 and 47). The vector pDH5 1 (M. Pietrzak, R. Shilito, T. Hohn and I. Potrykus (1986). Nucleic Acids Research 14, 5857) which carries the CaMV 35S promoter followed by a multiple cloning site and the CaMV polyadenylation fragment was modified to make a suitable vector, pDH51Stu, carrying a StuI site at the immediate 3′ end of the CaMV 35S promoter. The promoter thereby terminates in the sequence GAGAGG C CT, with the underlined residue being that at which transcription would start. (Similar vectors have been described by Mori et al, J. General Virology 72, 243-246 (1991) and Dessens and Lomonossoff, ibid 74, 889-892 (1993).) The StuI site (AGG/CCT) is followed by a BamHI site (GGATCC). Cleavage of this vector with StuI and BamHI generates a vector DNA molecule with one blunt end (from StuI cleavage) and one sticky BamHI end. This allows ligation of cDNA molecules corresponding to the full-length Ha SV genomic RNAs, and carrying a blunt end at the 5′ end of the full-length cDNA and a BamHI site after the 3′-end of the full-length cDNA.

Suitable cDNA fragments carrying a blunt end corresponding to the 5′-terminal nucleotide of either RNA 1 or 2 (SEQ ID Nos: 39 and 47) were generated using PCR and an oligonucleotide primer corresponding to the 5′-terminal first 18 nucleotides of the sequence of either RNA 1 (SEQ ID No: 39) or RNA 2 (SEQ ID No: 47). The cDNA sequence corresponding to the 3′ terminal sequences of either RNA 1 (SEQ ID No. 39) or RNA 2 (SEQ ID No 47) were followed on these DNA fragments by sequences corresponding to one of the ribozymes whose sequences are shown in FIG. 8 and whose construction is described in Example 7. The 3′-terminal sequence corresponding to an XbaI site (TCTAGA) shown in these ribozyme sequences was followed on the suitable DNA fragments by a BamHI site, which upon cleavage with this enzyme yielded a sticky end capable of being ligated into the BamHI end of the vector cleaved as described above. There were therefore a total of four suitable DNA fragments for insertion into the vector:

RNA 1 (SEQ ID No: 39) followed by the hairpin cassette (HC) ribozyme

RNA 1 (SEQ ID No: 39) followed by the hepatitis delta virus (HDV) ribozyme

RNA 2 (SEQ ID No: 47) followed by the hairpin (HC) ribozyme

RNA 2 (SEQ ID No: 47) followed by the hepatitis delta virus (HDV) ribozyme.

These four fragments were individually ligated into the vector pDH51Stu cleaved with StuI and BamHI to generate four distinct plasmids as follows:

pDHStuR1HC

pDHStuR1HDV pDHStuR2HC pDHStuR2HDV

Transcription from the 35S promoter in these plasmids results in RNAs commencing at the first nucleotide of either the RNA 1 sequence (SEQ ID No: 39) or RNA 2 sequence (SEQ ID No: 47) and terminating in the CaMV polyadenylation fragment. Self-cleavage at the locations shown in FIG. 8 by the cis-acting ribozymes obtained within these transcripts generates RNA molecules with the 3′-termini corresponding to the natural virus termini.

After amplification and purification on CsCl gradients, thirty mg of each of these four plasmids was transfected by electroporation into aliquots of two million N. plumbaginifolia protoplasts (as described in Example 5) either individually or in the combinations listed below:

pDHStuR1HC+pDHStuR2HC

pDHStuR1HDV+pDHStuR2HDV

The production of infectious Ha SV particles within transfected protoplasts was then demonstrated by bioassay on heliothis larvae. After incubation at 25° C. for 3-5 days, the protoplasts were recovered by low speed centrifugation and applied directly to standard heliothis diet as surface contamination for bioassay as described in Example 1. Stunting was only observed when plasmids expressing Ha SV RNA 1 (SEQ ID No: 39) and RNA 2 (SEQ ID No: 47) were co-transfected, and then only in the case of those carrying the hairpin ribozyme to generate the viral 3′ ends (see Table 2). In contrast, constructs carrying the HDV ribozyme at the 3′ end were not infectious. The reasons for this have not been determined. As expected, expression of RNA 1 or 2 (SEQ ID Nos: 39 and 47) alone in protoplasts did not lead to the assembly of infectious particles. Western blot analysis of protoplasts transfected with the RNA 2 (SEQ ID No: 47) constructs did show production of limited amounts of the capsid protein.

Suitable control experiments confirmed that larval stunting was due to Ha SV particles generated de novo in the protoplasts. As shown in the Table 2, neither the protoplasts alone nor protoplasts mixed with plasmid DNA were capable of initiating stunting.

	TABLE 2
		No. of		No.
	Treatment	larvae	Escapes	stunted
1.	diet alone	24	0	0
2.	diet + HaSV	24	0	24
3.	diet + protoplasts	24	0	0
4.	diet + pDHStuR1HC	24	0	0
5.	diet + pDHStuR1HDV	24	0	0
6.	diet + pDHStuR2HC	24	0	0
7.	diet + pDHStuR2HDV	24	0	0
8.	diet + pDHStuR1HC +	24	0	22
	pDHStuR2HC*
9.	diet + pDHStuR1HDV +	24	0	0
	pDHStuR2HDV*
10.	pDHStuR1HC + pDHStuR2HC	24	0	0
	(but mixed with protoplasts)
*these plasmids were co-transfected with pDHVCAPB (see Example 5)

Ha SV infection of stunted larvae was confirmed by dot-blotting of RNA using Ha SV specific probes. After weighing, larva were sacrificed and total RNA extracted as follows. Each larva was homogenised in the presence of 260 ml deionised water, 24 ml 2M sodium acetate pH 4.0 and 200 ml phenol equilibrated with 2M sodium acetate pH 4.0. After centrifugation at 14 000 rpm for 15 min at 4° C., the supernatant (about 200 ml) was removed and extracted once with an equal volume of chloroform. After centrifugation, the supernatant (about 200 ml) was mixed with 20 ml of sodium acetate and 400 ml of absolute ethanol. The precipitate after centrifugation was vacuum dried and redissolved in 5-10 ml of sterile, DEPC-treated water. For dot-blotting, the RNA was mixed with 70 ml of DEPC-treated water and 30 ml of 10 mM EDTA, 30 mM NaOH. Ha SV RNA was determined and quantified by dot blotting (as described in Example 2) using a probe random primed DNA from clones corresponding to the terminal 1000 nucleotides of RNA 1 and 2. All larvae recorded as stunted in the bioassays were found to carry Ha SV and give signals comparable to those of the larvae fed purified Ha SV particles (Table 2). To confirm that the larvae were infected with Ha SV, ten aliquots of protoplasts were electroporated with plasmids pDHStuR1HC+pDHStuR2HC and the protoplasts fed (after incubation) to 150 heliothis larvae. The larvae were allowed to grow for one week, upon which significant stunting was observed in 50% of the larvae, and virus was then purified from these stunted larvae as described in Example 1. Analysis on CsCl gradients showed the production of distinct bands characteristic of Ha SV; analysis of the virus particles found in these bands by SDS-PAGE and Western blotting with Ha SV antiserum confirmed their identity as authentic Ha SV.

These results have therefore demonstrated that DNA plasmids capable of expressing the Ha SV genome in plant cells have been constructed. Once introduced into the cells, the plasmids are transcribed to yield Ha SV genomic RNA which can drive the assembly of particles able to infect heliothis larvae by the normal oral route. These constructs have therefore been shown to be suitable for use in engineering transgenic plants expressing Ha SV.

A variation on this strategy is to remove from the cDNA for RNA 2 (SEQ ID No: 47) the fragments encoding RNA encapsidation and/or replication signals. This results in either the assembly in the plant cells of Ha SV particles carrying only RNA 1 (SEQ ID No: 39), or of Ha SV particles carrying RNA 1 (SEQ ID No: 39) and a form of RNA 2 (SEQ ID No: 47) which cannot be replicated in the infected insect cell.

A further variation on this strategy is to modify the plant transgene encoding RNA 2 (SEQ ID No: 47) so that it is still replicatable and encapsidatable, but no longer express functional capsid protein. Ha SV capsids made in such plant cells will then be capable of making both the replicase and P17 (SEQ ID No: 48) in infected insect cells, but not of assembling progeny virus particles therein (such as shown in FIG. 12 ( d )). These measures confer inherent biological safety in the form of containment on the use of such transgenic plant material.

(b) Use of Portions of Ha SV Genome to Deliver Toxins to Insect Cells

This approach makes use of any of the systems described in (a) above. Plant cells contain an additional transgene encoding a suitable insect-specific, intracellular toxin (as described above). Such a toxin gene is expressed by plant RNA polymerase in either a positive or a negative sense (the latter is preferred) and in such a form that the RNA can be encapsidated by Ha SV capsid protein and/or replicated by the Ha SV replicase in infected insect cells (see FIGS. 12 a and 12 b ).

Transgenic plants would contain two different transgenes, making either unmodified capsid protein precursor or a modified form in which most of the carboxyterminal protein P7 is replaced by a suitable insect-specific toxin or one which is inactive as part of a fusion protein. (Gelonin or other ribosome-inactivating proteins, insect gut toxins or neurotoxins may be suitable here.) Expression from these two transgenes would be regulated so that only the required amounts of the modified and unmodified forms are made in the plant cell, and assembled in such proportions into the capsoids. One way to modulate the product ion of capsotixin fusion proteins is to make translation of the carboxyterminal toxin reading frame dependent on a translational frameshift or read-through of a termination codon. With an appropriate low frequency of frame-shifting (eg 0.1-2%), it could even be sufficient to use a single transgene, if it were possible to synthesise the P7 portion and the toxin portion as overlapping genes. Upon assembly (which we have demonstrated in insect cells using the baculovirus vectors) and maturation, the protein precursors are cleaved and release the mature P7 and the toxin, which remain within the capsoids. These proteins are not released until capsoid disassembly occurs in insect gut cells. The processed form of the toxin is then able to kill the pest.

(c) Ha SV Particles Devoid of Nucleic Acid Carrying One or More Suitable Protein Toxins and/or Their mRNA

A protein toxin (or toxins) is expressed as a fusion with the capsid protein. The fusion protein then assembles into capsid carrying the toxin(s). These capsids present in the plant tissue exert an antifeeding effect on insects attaching the plant.

EXAMPLE 10

Expression of Ha SV in Other Delivery Vectors

Materials & Methods

(as indicated in earlier Examples)

Constructs similar to those for plant expression are introduced into yeast or bacteria by standard techniques. Virus particles are assembled for either fully infectious virus or any of the modified or biologically contained forms described in Example 9. Microbes produced in suitable fermentation or culture facilities and carrying such forms of the virus are then delivered to the crop by spraying. The microbial cell wall provides extra protection for the virus particles produced within the microbe.

Well established techniques exist for culture and transformation of yeast (Ausubel, F. M. et al. (eds) Current Protocols in Molecular Biology. J. Wiley & Sons, N.Y., 1989). An example of a yeast expression vector is pBM272, which contains the URA3 selectable marker (Johnston, M. & Davies, R. W. Mol. Cell. BIol. 4, 1440-8, (1984); Stone, D. & Craig, E. Mol. Cell. Biol. 10, 1622-32 (1990). Another example of an expression vector is pRJ28, carrying the Trp1 and Leu2 selectable markers.

Yeast has recently been shown to support replication of RNA replicons derived from a plant RNA virus, brome mosaic virus (Janda, M. & Ahlquist, P. Cell 72, 961-70 (1993). Since the BMV replicase is distantly related to that of Ha SV, and the two viruses are likely to replicate by similar strategies within cells, yeast cells probably contain all the cellular factors required for Ha SV to generate infectious virus.

For bacteria, suitable expression vectors have been described above.

EXAMPLE 11

The Transvirus Approach for Insect Pest Control: Making Transgenic Plants Expressing Ha SV

1. Vector Construction

A special binary vector was constructed for transforming plants with the Ha SV genome. This vector is based on pART27 (A. Gleave (1992) Plant Mol.Biol.20, 1203-1207), which was modified to (1) carry an alternative origin of replication for the host Agrobacterium tumefaciens and (2) incorporate restriction sites in the multiple cloning site for restriction enzymes Asc I and Pac I which recognise rare (8 bp) sequences.

For engineering the multiple cloning site, pART27 was cut with SpeI and NotI. Ten picomoles of each of the two oligos whose sequence follows (TOP and BOTTOM) were annealed in 10 microliters of water (heated to 80° C. for 2 min and allowed to cool slowly to room temperature). The sticky ends on these annealed oligonucleotides allowed the insert to be cloned into pART27 (giving pART27mod) as described in Example No. 3 and 9.

Sequence of oligonucleotide:

TOP: 5′-GGCCGCTTAATTAAGGATCCGGCGCGCCA-3′ (SEQ ID NO:54)

BOTTOM: 3′-CGAATTAATTCCTAGGCCGCGCGGTGATC-5 (SEQ ID NO:55)

(The PacI recognition sequence is TTAATTAA (SEQ ID No:56) and that for AscI is GGCGCGCC SEQ ID No: 57). A 4 kbp SalI fragment from plasmid pART27mod (containing the right border, IacZ marker (+multiple cloning site)nptII gene for kanamycin resistance under control of the nos promoter and polyadenylation signal and the left border) was cloned into the 13 kbp vector pKT231 linearised with XhoI. Plasmid pKT231 carries the IncQ origin of replication for the host Agrobacterium tumefaciens and a resistance (marker) gene for streptomycin/spectinomycin. (Bagdasarian, M. & Timmis, K. N. (1982) Curr. Topics Microbiol. Immunol. 96, 46-67). These two fragments were ligated using standard protocols (eg in Example No 3) and transformed into E. coli strain DH5α using standard protocols (eg in Example No 3). The resultant plasmid was named pJDML1.

2. Cloning Ha SV Genes into Transfer Plasmid

Construction of Transfer Vectors With Ha SV Genes

Before the Ha SV gene cassettes could be cloned into binary transfer vectors pART27 mod or pJDML1, they were re-cloned into the vector plasmid pBJ33 to provide flanking AscI and PacI sites. Plasmid pBJ33 (provided by Bart Janssen) is based on pBC SK(+) supplied by Stratagene), but with a multiple cloning site modified to contain the following sites:

SacI/PacI/AscI/SacII/XbaI/SpeI/BamHI/PstI/EcoRI/EcoRV/HindIII/ClaI/SaII/XhoI/Ap aI/PacI/AscI/KpnI.

The cDNA fragment corresponding to complete Ha SV RNA 1 behind the 35S promoter and terminating in the hairpin cassette ribozyme and the CaMV polyadenylation signal fragment (approx 6 kpb in total) was excised from plasmid pDHStuR1HC (Example 9) with EcoRI and cloned into EcoRI-cut vector pBJ33 to give plasmid pBJ33R1HC. Similarly, the cDNA fragment corresponding to complete Ha SV RNA 2 behind the 35S promoter and terminating in the hairpin cassette ribozyme and the CaMV polyadenylation signal fragment (approx 3.3 kbp in total) was excised from plasmid pDHStuR2HC (Example 9) as two fragments, one (covering the 35S promoter and the first 500 bp of the RNA 2 sequence) of about 1 kbp with EcoRI and R5rII and the second (covering the remainder of the RNA 2 sequence, the ribozyme and the polyadenylation signal) of about 2.3 kbp with RerII and HindIII. These two fragments were simultaneously ligated into EcoRI and HindIII-cut vector pBJ33 to give plasmid pBJ33R2HC.

A 1.9 kbp fragment comprising the 5′ 1.7 kbp of the Ha SV capsid gene, together with the polyadenylation fragment, were excised from expression plasmid pDHVCAPB (described in Example 5) as a Eco RI-KpnI fragment and cloned into pTZ19U (pharmacia) cut with EcoRI and KpnI, giving pTZ19UEVCAPB. This portion of the Ha SV capsid gene expression cassette was then re-excised as a HindIII-EcoRI fragment and cloned into PBJ33 cut with these enzymes. This plasmid (pBJ33EVCAPB) was then linearized with EcoRI and the ca. 800 bp EcoRI fragment from pDHVCAPB carrying the 35S promoter and the 5′ 250 bp of the capsid gene inserted, followed by screening for orientation. The resulting plasmid carrying the reassembled complete capsid gene expression cassette was named pBJ33VCAPB.

Assembling Binary Plasmids

The RNA 1 expression cassette was excised from plasmid pBJ33R1HC with AscI and PacI and cloned into pART27 mod cut with AscI and PacI to give pMLR1. The RNA 2 expression cassette was also cloned as an AscI-PacI fragment into pJDML1 cut with AscI and PacI to give pJDMLR2.

The capsid protein gene cassette was excised from pBJ33 VCAPB with PacI and cloned into plasmid pMLR1 cut with PacI. Resulting plasmids were screened for orientation and the plasmid with the capsid gene and RNA1 in the same orientation was named pMLR1V. The complete fragment carrying the Ha SV capsid gene and RNA 1 expression cassettes in pMLR1V was excised with AscI and cloned into pJDMLR2 linearised with AscI to give p Ha SV1 (29 kpb). This plasmid carries the Ha SV capsid gene expression cassette and the Ha SV RNA 1 and RNA 2 expression cassettes in this order and all in the same orientation. The kanamycin resistance gene is located upstream of the capsid gene and in the opposite orientation.

Table of constructs generated:

			#Plants
			(independent
Vector	Insert(s)	Name	transformants)	Comments
pART27mod	RNA 1	pMLR1	15	control
pJDML1	R1 + R2 +	pHaSV1	30	complete virus
	CAP
pART27mod	R1 + CAP	pMLR1V	15	subvirus
pJDML1	R1 + CAP	pJDMLR1V	30	subvirus
pART27mod	RNA2	pMLR2	15	control
pJDML1	RNA2	pJDMLR2	15	control
pART27mod	CAP	pMLVF	15	control
(CAP = HaSV capsid gene)

3. Plant Transformation and Regeneration

Binary transfer vectors (above) were transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation (Lin, J. J. (1994) FOCUS 16, 18-19; Lin, J. J. (1994) Plant Science 101, 11-15). Leaf discs from Nicotiana tabacum grown under sterile conditions were transformed using cocultivation with transformed A. tumefaciens (Horsch, R. B. et al. (1984) Science 23, 496-498; Horsch, R. B. et al. (1988) Plant Molecular Biology Manual A5:1-9; as modified by Lisa Molvig (pers. comm.)) and grown on kanamycin. After transfer of regenerating shoots for further selection on kanamycin medium, kanamycin-resistant roots were selected and then tissue from these plants used to verify Ha SV gene expression. The numbers of plants selected are shown in the table above for each of the constructs.

4. Western, Northern and Southern Blotting on Transgenic Plants

For western blots: A small amount (0.1 g) of fresh leaf material from each plant was extracted by grinding in 0.2 ml of plant extraction buffer (0.2M NaCl, 0.1M Tes, pH 7.65, 1 mM PMSF, 2% b-mercaptoethanol, 1 mM EDTA). After centrifugation to pellet plant debris the supernatant was collected and 10 μl aliquots run on a SDS-gel for blotting and immuno-analysis with antibody against Ha SV as described in Example 1. The results for the first plants assayed are given in Table 3.

For Northern blots: Total leaf RNA was extracted from 0.15 g of fresh leaf material. The leaf material was ground under liquid nitrogen to a powder and then extracted by further grinding in 0.45 ml NTES buffer (0.1 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS) plus 0.45 ml Tris pH8.0-saturated phenol/chloroform. The slurry was vortexed, centrifuged for 3 min and the aqueous phase mixed with 1 volume of isopropanol to precipitate RNA and DNA. After resuspending the pellet in 0.1 ml water, 1 volume of 4M LiCl was added and the mix stood on ice overnight before centrifugation to pellet RNA. The RNA was then analysed by gel electrophoresis according to the methods in Examples 1 and 2. Ha SV specific RNAs were detected by Northern blottings as described in Example 2 and by using riboprobes made to detect the 3′-terminal 1000 nucleotides of each of RNA 1 and 2, made using the Promega Riboprobe kit and used as specified by the supplier.

For Southern Blots: to Detect Ha SV Genes in Plant Genomic DNA

To recover plant genomic DNA, the supernatant from LiCl precipitation (above) was mixed with 2 volumes of ethanol. The pellet was redissolved and the DNA cut with BamHI before analysis on agarose gels and transfer to nylon membrane as described by Sambrook et al (1989) and by the manufacturer (Zetaprobe/BioRad). Ha SV-specific bands were detected described above.

5. Bioassays on Leaf Material

Two small leaves (2-3 cm in length) were selected from each transformed plant selected, and placed in petri dishes containing 1.5% agarose in water. Three to 8 neonate larvae were placed in each petri dish and observed for 3 days. At the end of this time, larvae were weighed and then total RNA extracted as described in Example 1. The extent of leaf damage was quantified by measuring the area of leaf consumed by each group of larvae over the three days of the assay (see Table 3).

TABLE 3

Preliminary Bioassay of Ha SV Transgenic Plants

Three to 8 larvae were placed on a small leaf (from a newly regenerated plant) in a petri dish with no provision of fresh food, after 3 days, larvae were sacrificed and northern blotted; also, protein extracted from leaves of the plants were western blotted using anti- Ha SV antisera.

TABLE 3
Preliminary bioassay of HaSV transgenic plants
Three to 8 larvae were placed on a small leaf (from a newly regenerated plant) in a petri dish
with no provision of fresh food, after 3 days, larvae were sacrificed and northern blotted; also,
protein extracted from leaves of the plants were western blotted using anti-HaSV antisera.
	Transform-	Western Blot for	Northern blot for	Larval	Leaf Damage
	ation	HaSV capsid pro-	HaSV RNA	Weight	(mm 2
Plant	Plasmid	tein in plant (+/−)	in plant	(mg)	consumed/larvae)
Negative		−	−	2.1 −	61
Controls				2.7 ± 0.8*
1.1	pJDMLR1V	+		1.1 ± 0.2	29
(subvirus)
(RNA1 = p
71)
3.2 (whole	pHaSV1	+		1.0 ± 0.4	38
virus)
3.4 (whole	pHaSV1	+		1.2 ± 0.4	32
virus)
*Diet was limiting (ran out of food) in some cases

TABLE 4

Further Bioassay of Ha SV Transgenic Plants

Four-6 individual larvae were fed leaf disc (50 mm 2 ) from control or transgenic plants at one disc each per day for 4 days, before transferred to artificial diet for a further 3 days. RNA was then extracted from the larvae and Northern blotting with Ha SV-specific probes used to verify the presence of Ha SV in the larvae.

TABLE 4
Further bioassay of HaSV transgenic plants
Four-6 individual larvae were fed leaf disc (50 mm 2 ) from control or
transgenic plants at one disc each per day for 4 days, before transferred
to artificial diet for a further 3 days. RNA was then extracted from the
larvae and Northern blotting with HaSV- specific probes used to verify
the presence of HaSV in the larvae.
		Western blot for
		HaSV capsid protein	Mean larval
Plant	Transformed with	in plants (+/−)	weight (mg)
negative control		−	12.4
positive control		−	0.1
(leaf + HaSV)
3.2	pHaSV1	+	0.9
3.10	pHaSV1	+	4.8
3.11	pHaSV1	+	8.2

Efficacy of Ha SV as a transvirus in Plants

Factors affecting the efficacy of Ha SV are the viral dose required, the expression levels achieved in plants and the leaf damage observed. These need to be considered separately at this stage due to uncertainty about the efficiency of Ha SV assembly in plants and because larvae will continue feeding for about one day after receiving a toxic dose of Ha SV.

I. Dose of Virus

Infection with Ha SV requires neonate larvae to eat up to 10 000 particles. Assuming that transgenic plants make only 1 particle per cell, this means the larvae must consume up to 10 000 leaf cells.

Since a small tobacco leaf contains about one million cells, larvae could acquire a toxic dose by consuming just 1% of the leaf

This dose would correspond to as little as 0.000 000 5% of the soluble protein in these cells (330×10 −13 g of Ha SV per leaf in 7×10 −3 g soluble plant protein per leaf).

II. Expression Levels

Assuming standard levels of 1% expression and complete incorporation into virus particles, there should be about 10 8 particles per cell (7×10 −9 g of protein per cell over 330×10 −19 g per Ha SV particle).

However, at present only part of this protein is likely to form infectious virus. If 1% does, then there would be 10 6 particles per cell, well above the toxic dose.

Initial results from Western blots suggest current expression at least exceed 01.1% of soluble cell protein. Processing of the precursor protein appears to occur to a variable extent, suggesting that particle assembly has also occurred.

The dose of infectious virus delivered by transgenic plants must be quantified by appropriately standardised bioassays.

Optimisation of the infectious virus level will be achieved by improving virus assembly rather than just boosting expression of components—this represents a fundamental difference to the situation with toxins like Bt.

III. Leaf Damage

While as little as 1% of the leaf (and more likely far less) may be sufficient to deliver a toxic dose of Ha SV, larvae will keep feeding for a limited period after becoming infected. This makes it necessary to determine the extent of leaf damage empirically.

Our initial observations were that plants making detectable levels of Ha SV capsids showed reduced susceptibility to larval feeding; this has not been quantified yet, and the assay was a severe one.

Consumption of leaf material by infected larvae may be estimated indirectly using our data on larval growth and frass production, which are approximately equal. Since neonate frass production is too low to quantify, the data were obtained from 4-day old larvae. These produce 30 mg of frass over 7 days, compared to 400 mg for uninfected controls. Neonate growth and frass production may be estimated at 10% of this figure.

Assuming that 1 mg growth or frass—3 mg leaf material, an infected neonate will consume about 5% of a small tobacco leaf (20 mg of a total fresh weight of 350 mg) over seven days compared to over 60% for an uninfected control (240 mg of 350 mg).

Biosafety Considerations

It is believed that the approach of controlling pests by making an insect virus in transgenic plants is not dangerous to the environment. This is despite our very tentative observation that some Ha SV replication is observed in protoplasts. There has been widespread debate recently concerning the safety of protecting crops against plant viruses by inserting transgenes expressing viral proteins into the plants. Falk, B. W. and Bruening, G., 1994 (Science 263, 1395-1396) identified 3 possible mechanisms which might result in the appearance of novel viruses. These mechanisms are transencapsidation, phenotypic mixing and heterologous recombination.

Transencapsidation or phenotypic mixing involving Ha SV plants are not likely to cause problem because:

the Ha SV capsid gene is not acquired by the transencapsidated plant virus genome.

such an event would yield a virus only capable of “infecting” heliothis larvae, which are not efficient vectors to enlarge the host range of a plant virus.

Heterologous recombination is not perceived as a problem because

It requires substantial sequence similarity and has only been observed within plant virus families. The tetraviruses are an insect specific virus family showing minimal sequence homology to any plant RNA virus.

Even if interfamily recombination occurred, this would generate a combination of genes for which there is no precedent in either viruses infecting both plants and insects or plants alone ( Ha SV would not encounter any other insect-specific virus in transgenic plants); these viruses require functions and genes which it is physiologically impossible to generate from such recombinations.

This is because:

1) the four families of insect-vectored plant viruses which replicate in their vectors are much more complicated than Ha SV, both in particle structure and in genome organisation. All these viruses have negative-stranded or double-stranded RNA genomes and at least 4-5 genes.

2) even the four families of insect-vectored plant viruses which circulate in their vectors without replicating are more complicated than Ha SV in genome organisation, number of genes and expression strategies; although they have (+)-stranded RNA genomes, they are found in a different virus superfamily, with replicases essentially unrelated to that of Ha SV. Their capsids are unrelated to that of Ha SV and are not uncoated in the insect vectors.

All the simple, (+) stranded plant viruses which more closely resemble Ha SV (which include some passively transmitted by sucking insects, ie without entering the vector).

must have a plant cell—cell movement protein for which there is no direct functional equivalent in Ha SV.

have replicases specifically adapted to plant cells, and with minimal overall amino acid sequence homology (under 25%) to that of Ha SV.

have capsids specifically adapted for long range movement in plants and vectoring by insects without entering these or being uncoated in them; these capsid genes have no detectable sequence homology to Ha SV or other insect capsids.

Sub-Virus

Containment Strategies

Although the expression of Ha SV in transgenic plants is not considered to present any environmental hazard, some Ha SV constructs we have used to engineer plants contain essentially suicidal versions of both RNA 1 and 2. This has been achieved for RNA 2 by deleting all sequences apart from those directly encoding the capsid protein and demonstrating that effective virus can still be assembled in plant cells. This alone will prevent transmission of progeny virus, since infected larvae respond as though they had ingested normal virus, but are unable to produce infectious progeny virus. The virus produced in plants therefore only infects targets feeding on the crop plant and can neither infect other species nor persist in the environment.

It is also possible to engineer subviral forms of RNA 1 which retain efficacy but do not allow production of viable progeny virus. (For example, remove replication signals from RNA 1).

1) Results

The subvirus approached was tested using the following combination of plasmids transiently expressed in protoplast and followed by bioassay as described above.

		Bioassays weights	HaSV RNAs 1 & 2
		(mg) of larvae	detected by Northern
		fed on protoplast	blotting of RNA
	Plasmids	extracts	extracted from larvae
1.	pDHStuR1HC +	29 ± 15	+
	pDHVCAPB
2.	pDHStuR1 HDV +	57 ± 25	(−)
	pHV CAPB
3.	Control: (diet only/diet +	85 ± 15	−
	protoplasts
4.	pDHStuR1 HC +	33 ± 28	+
	pDHStuR2 HC +
	pDHVCAPB
5.	pDHStuR1 HDV +	64 ± 22	−
	pDHStuR2 HDV +
	pDHVCAPB

ii) RNA extraction from larvae showed

(a) that larvae fed protoplasts transfected with pDHStuR1HC+pDHStuR2HC+DHVCAPB contained both RNA1 and 2 of Ha SV in intact form.

(b) that larvae fed protoplasts transfected with pDHStuR1HC+DHV CAPB (subvirus) contained a very small amount of intact Ha SV RNA1 and a considerably greater amount of degraded RNA1.

(c) that larvae fed protoplasts transfected with pDHStuR1HDV+pDHStuR2HDV+pDHVCAPB contained no Ha SVRNA with one exception.

(d) that larvae fed protoplasts transfected with pDHStuR1HDV+pDHVCAPB contained no Ha SV RNA.

Conclusions

The HC ( Ha SV expression) constructs with the hairpin cassette ribozyme give infectious particles with both RNAs; the HDV expression constructs do not under these conditions.

That the subvirus approach results in RNA1 replicating in larvae but this RNA is degraded because it cannot be encapsidated due to missing replicatable RNA2.

That subvirus approach gives stunting as effectively as does the complete virus approach under these conditions.

EXAMPLE 12

Capsovector

The aim of this section is to describe the invention of capsovectors and present supporting data. In addition ideas for their further improvement will be presented.

The capsovector is a virus-like particle (VLP) from a small RNA insect virus with a pest insect host whose properties will facilitate the entry (or vector) into the cells of the pest insect either, or both, RNA or a protein that will induce toxicity in the insect. Capsovectors can be produced in transgenic crop plants targeted by the host insect pests or produced for spray applications by transgenic plants or recombinant microorganisms.

There are two types of capsovectors, ones that vector RNA moieties (RNA capsovectors) and ones that vector proteinaceous moieties (protein capsovectors). Because of their distinct properties, RNA and protein capsovectors will be dealt with separately. However, the description of the protein capsovector will rely heavily on the preceding description of the RNA capsovector.

In this invention, the Helicoverpa armigera stunt virus ( Ha SV) of the Tetraviridae will be used as the model insect small RNA virus. However, this does not exclude other types of insect small RNA viruses being used in a similar manner.

Characteristics of Ha SV Pertinent to Capsovectors

Ha SV is a member of the Tetraviridae family of viruses and infects only midgut cells of young larvae of heliothine insects (Hanzlik et al., 1993) after ingestion with food. Numerous attempts to grow the virus in non-gut tissue, other insects and cultured cells have failed. This believed to be due to the ability of the Ha SV capsid protein to bind and enter only midgut cells of the host insect (Hanzlik et al., 1995).

Ha SV has been characterised in great detail at the molecular level. Its physical characteristics have been determined (Hanzlik et al., 1993) and its complete genome sequenced (Gordon, et al., 1995; Hanzlik, et al., 1995).

Ha SV virions are extremely simple in composition: two capped, single-stranded RNAs (5.5 kb and 2.5 kb) encased in a 36 nm icosahedral capsid. The capsid is composed of 240 copies of a 71 kDa precursor protein (p71) that is cleaved upon capsid formation into two 64 kDa and 7 kDa proteins (p64 and p7, respectively). The smaller protein, p7, is located within the particle along with the genomic RNA. The positive sense RNA genome has only three known genes with the larger RNA strand encoding the 187 kDa viral replicase and the smaller strand encoding the precursor capsid protein and another 17 kDa protein of unknown function.

Much is known about the physical structure of the Ha SV particle because substantial work on the physical characteristics of Ha SV (Hanzlik et al., 1993) as well as the crystal structure of close relative of Ha SV, the Nudaurelia ω Virus (NωV), being solved to 3.6 angstrom resolution. The overall identity between the known sequences of the two viruses is 67% (Hanzlik et al., 1995) with the main differences in the amino acid sequences of the two capsid proteins residing in a 165 residue region in the middle. The solved structure shows this dissimilar region to be IgG-like and located on the outside of the capsid. It is believed to be at least partly responsible for host specificity of the virus by being able to bind to different receptors on different hosts. Also giving insights into the structure s of Ha SV and NωV is the similarity of their capsids to those of the highly characterised nodaviruses (Agrawal and Johnson, 1992).

Virus-like particles VLPs have been made from the capsid gene of HASV and NwV (Agrawal and Johnson, 1995). The work has shown that by expressing only the coat protein (p71) sequences of the virus, mature VLPs can be made with p71 processed into p64 and p 7 , and which encapsidates their mRNAs. Work (described below) has shown that the Ha SV VLPs possess many of the same properties of the HASV virion. The invention of capsovectors exploits this similarity.

RNA Capsovectors

An RNA capsovector particle resembles a normal virus particle in that it is an assemblage of coat proteins and an RNA strand(s) constructed in such a manner that the proteins encase, protect, and vector the RNA strand to inside a host insect cell. Upon entry into the host cell, the RNA strand is uncoated and translated into protein. The main difference between the capsovector particle and the virus particle resides in what types of protein the RNA strand encodes, if any. That contained within virus particles is aimed at replication of new virions replication with any toxicity is a by-product of this process; that contained within capsovectors is aimed at rapid toxicity to the cell and not at replicating new capsovectors.

Important to the toxic activity of a RNA capsovector are the properties of viral genes that lead to the activities of encapsidating, protecting, entry, uncoating and translating of the viral RNA in the host cell. These activities are necessary for successful virus infection yet do not involve replication. Because of the simplicity of Ha SV, all of these properties are contained in one gene and its product, the coat protein, p71. Generally, the coat protein gene of other small RNA viruses have the same capacity and therefore are also suitable for capsovectors. The p71 gene of Ha SV is employed in illustrating the capsovector invention and its properties are explained and demonstrated under their respective headings below.

The capsovector adds toxicity to the virus functionalities by using sequences exogenous to Ha SV or viruses on the RNA contained within the capsovectors. These toxic sequences are aimed at inducing rapid and direct toxicity to the cell the capsovector has entered. The sequences are either translated into protein or fold the RNA into appropriate secondary structures which then causes a toxic lesion in the cell. These sequences are explained below as well.

It must be pointed out here that viruses avoid inducing rapid and direct toxicity to cells they have entered as this is deleterious to viral replication. Because the cell must be viable for the production of new viruses, many viruses do not induce cell death until late in their infections if at all. Indeed, infection of hosts by many insect small RNA viruses different from Ha SV does not result in any discernible response in the host insect upon its being infected. These innocuous viruses can still be exploited, however, because the viruses are able to perform the previously mentioned functions of encapsidation and protection of the labile RNA genome, ability of the particles to enter host cells, uncoating and subsequent translation of the RNA genome. Appropriate placement of toxin sequences with those of the coat proteins of these viruses as described in this invention can result in toxicity and subsequent control of the insect pest host.

Encapsidation. Encapsidation is defined here as the process of forming a virus particle or a virus-like particle that incorporates RNA into its interior. For this process to occur, interactions among the capsid proteins (encoded by the RNA) and between the proteins and specific regions of the RNA must occur that result in the ordered aggregation of 240 copies of the coat protein and the RNA strand(s). The specific regions on the RNA are either or both a defined primary sequence or a specific secondary structure arrived at by a number of primary sequences.

All three types of interactions, protein/protein, RNA/protein and RNA folding into secondary structures, reside in the capsid gene ORF of Ha SV. This is shown by the following data:

1. When the ORF the Ha SV capsid protein is expressed in insect cells with a recombinant baculovirus, VLPs can be observed with transmission electron microscopy (TEM) in sections of fixed, positively-stained, infected cells. They are highly similar in morphology and dimensions to native virions observed in gut tissue of diseased insects. The VLP morphology is that of a smooth surfaced sphere with a diameter of 35-40 nm identical to virus particles observed in diseased tissue. Also noted is that a fraction of the particles have dark, electron dense cores. The fraction of VLPs having electron dense cores is smaller than that observed for virus particles observed in diseased tissue. The electron dense cores indicate that the particles contain electron dense, non proteinaceous RNA.

Methods and Materials:A recombinant baculovirus able to express p71 was made by the following procedure. An amplicon containing the p71 ORF was obtained from a PCR reaction made with HP64NEUK (GGCCGGATCCAGACATGCTGGAGTGGCGTCAC) and HVP6C2 (GGGATCCCTAATTGGCACGAGCGGCGC) off a DNA template consisting of the pT7T2p7l plasmid used for the bacterial expression of the capsid gene. This fragment contains the p71 ORF flanked with BamHI sites and the initiating AUG placed behind a more favourable context for expression in eukaryotes. This fragment was restricted with BamHI and cloned into the baculovirus expression vector, pVL94 1, which was transfected into Sf9 cells with linearized wildtype baculovirus DNA. A recombinant baculovirus, Bacp71, was obtained by standard means (King and Possee, 1992).

For TEM, Sf9 cells infected for three days with Bacp71, were harvested, fixed with gluteraldhyde, embedded in LR White resin and sectioned for examination with TEM using standard procedures. The sections were examined with a JEOL 100CX transmission electron microscope.

2. When the VLPs are purified and characterised, they also show highly similar characteristics to native Ha SV virions. Buoyant densities of the particles are similar 1.296 g/ml for virions and 1.29 g/ml for VLPs. Particle morphologies as seen by negative stained TEM are highly similar with the micrographs for both virions and VLPs showing spheres of 35-40 nm spheres possessing electron dense interiors. The electron dense interiors also indicate the presence of RNA.

Methods and Materials: After a three day infection with Bacp71 in a 220 cm 2 flask, Sf9 cells were harvested, pelleted, washed, and lysed with a freeze-thaw cycle in 10 ml of buffer. This was centrifuged at 10,000×g for 10 min and the supernatant was recovered and recentrifuged on top of a 30% sucrose cushion. The pellet was then redissolved in 0.5 ml of buffer (50 mM Tris pH7.5, 5 mM CaCl) overnight and placed on top of a solution in a SW41 tube (Beckman) consisting of 5 mls each of 60% and 30% of CsCl in buffer. This was centrifuged overnight at 40,000 rpm and the sole band located in the middle of the tube was removed, placed into 10 ml of buffer and pelleted by centrifugation at 35,000 rpm in a SW41 rotor. The pellet was dissolved in 50 ml of buffer.

VLPs in the solution were examined on a JEOL 100CX microscope with standard procedures after negative staining with uranyl acetate..

3. The VLPs contained RNA. The above observations indicated that the VLPs in the were electron dense and that RNA was within the VLPs. These observations were confirmed when the VLPs were extracted for RNA and the RNA analysed by agarose gel electrophoresis. The RNA was 2900 bases in length, the expected size of the mRNA transcribed from the baculoviral genome. When the RNA was probed with a radioactively labelled probe specific for p71 sequences, strong hybridisation occurred, showing that the RNA was the p71 mRNA.

Methods and Materials: RNA was removed from the purified VLPs with extraction with phenol, phenol/CH3Cl and CH3Cl and ethanol precipitation. This was then run on an 1% formaldehyde agarose gel. A northern blot of the gel was done with standard procedures. The 32 P-labelled probe was prepared from clone HR326 which contains the p71 sequence.

4. The p71 mRNA extracted from the VLPs are shown to have heterologous, non- Ha SV sequences. This is shown by the recovered RNA strand having a length of 2900 bases which is equal to the predicted size of the mRNA transcribed from the baculoviral genome and expressing the p71 ORF. Only 1900 bases of the mRNA are sequences from the p71 ORF, the rest being sequences from the baculovirus expressing the gene. When a radioactively labelled probe specific for the polyhedrin gene located between the p71 gene the polyadenylation signal for the polyhedrin gene was hybridized to the RNA extracted from the VLPs, a strong hybridation signal was seen on the autorad. This shows that the signals required for encapsidating RNA are present in the p71 ORF and that non- Ha SV sequences can be placed inside the VLP.

Methods and Materials: RNA extracted from VLPs were northern blotted by standard procedures and probed with a labelled 950 bp Hind III fragment from the baculoviral transfer vector pVL941 having sequences 3′ to the inserted p71 gene and 5′ to the polyadenylation site of the polyhedrin gene.

5. The RNA having the capsid protein ORF is specifically encapsidated. No other RNA except for the p71 mRNA is present in the VLPs. This is shown by the failure of another highly transcribed region of the baculoviral genome (therefore also present in great abundance inside the baculoviral infected cell) failing to hybridise to the VLP RNA. When a probe specific for the p10 mRNA, a late gene product from the baculovirus, is hybridised to the RNA extracted from the purified VLPs, no signal occurs on the northern blot.

Methods and Materials: RNA extracted from VLPs were northern blotted by standard procedures and probed with a 32 P-labelled 245 bp Xba I/Eco RI fragment from the baculoviral transfer vector pAcUW31 having sequences 3′ to the start of transcription of the p10 promoter for AcMNPV baculovirus.

Thus RNA sequences contained within the ORF of Ha SV capsid protein (p71) can produce VLPs that encapsidate only RNAs having the p71 sequence. If there are exogenous sequences not from Ha SV also on the p71 mRNA these then can be encapsidated. This shows that sequences for toxins can be placed within the VLP if it possesses a p71 sequence.

Protection: Normally labile RNA contained within the capsovector must be protected from degradation during the period between its encapsidation inside the cell where it was produced and its entry into the cell where it will effect toxicity. This is particularly important because part of this period is spent in the insect gut that is a highly degradative milieu for both RNA and protein. This function is performed by the 240 copies of the capsid protein which form a protective shell around the virion RNA.

The protective properties of the Ha SV coat protein for both the virion and the VLP was shown by western blots of the capsid protein from Ha SV particles, Ha SV VLPs, and lipophorin before and after timed exposure to the gut contents of heliothis larvae: The data shows that:

1. When a non-viral globular protein like lipophorin is exposed to the contents of the heliothis midgut, rapid degradation of the protein occurs within 10 minutes.

2. When either an Ha SV virion or VLP is exposed in the same manner, minimal degradation of the protein occurs despite extended exposure times (>2 hr). Thus, when translated, the RNA sequences contained within the p71 ORF lead to protection of the protein and RNA of the VLP.

Methods and Materials: The midguts of fifth instar Heliothis armigera were excised, their contents removed and centrifuged at 14,500×g for 15 min. Into 10 ml of the contents were placed 1 ml of a solution having 1 mg protein of either lipophorin, Ha SV virions or Ha SV VLPs. At timed intervals 1 ml of this solution was removed and immediately boiled in SDS-PAGE sample buffer. SDS-PAGE and immunoblots with the respective antisera were then carried out with standard procedures.

Cell Entry: Studies of animal host/virus systems have shown that entry of virions into cells is mediated by cellular receptors located on their exteriors and viral acceptor proteins or VAPs (Lentz, 1990). The Ha SV VAP is by exclusion of any other possibility, p71, it being the sole protein component of the Ha SV capsid (Hanzlik et al., 1993, 1995). In particular, it is believed that a specific region of p71, between residue 274 and 439 of the protein sequence, is responsible for the binding of Ha SV to the presently unknown host cell receptor.

Experimental verification of the ability of Ha SV particles to bind to midgut cell extracts is obtained when the Ha SV particles are radioactively labelled with 125 I and incubated with midgut brush border membranes. If membranes are incubated with 103,000 cpm of labelled particles, 8,500 cpm remain with the membranes after washing; this compares to 1,700 cpm if the membranes are boiled before the incubation or to 2,100 cpm if the membranes are preincubated for one hour with unlabelled Ha SV.

Methods and Materials: Ha SV particles were labelled with 125 I and Chloromine T (Pierce) according to the manufacturer's instructions. Labelling of the capsid was verified by autoradiography of dried SDS-PAGE gels after digesting the protein with gut juice from larvae of H. armigera . The lack of degradation of p64 as seen as a band at 64 kDa on the autorad verified labelled, intact virions of Ha SV. Brush border membranes were prepared according to Wolfersberger et al. (1987). Binding assays were conducted by placing 50 mg of membrane protein into 100 ml of binding buffer (17 mM Tris, pH 7.5, 5 mM EGTA, 300 mM mannitol) and incubating them for one hr with 100,000 cpm of labelled Ha SV particles. This mixture was centrifuged at 14,000×g for 5 min and the pellet washed three times with 1 ml of buffer. The pellet was dissolved overnight in 100 ml of Solvable (DuPont) tissue solubilizer and then counted in a gamma counter (Packard Instruments).

Uncoating: Non-enveloped viruses like Ha SV face a quandary when they are assembled: The virions' assembly must occur in the very place where they must disassemble and expose the RNA for their translation, ie. inside the host cells. The mechanism used by Ha SV is the maturation cleavage, a process which has been shown to occur for picornaviruses (Kirkegaard and Compton, 1990), nodaviruses (Schneemann et al., 1992) and tetraviruses which have a capsid structure similar to nodaviruses (Agrawal and Johnson, 1992).

The maturation cleavage occurs after capsid assembly (Gallagher and Rueckert, 1988). The cleavage event has been shown to be necessary for uncoating and subsequent infection of the host cell and is believed to cause conformational changes in the capsid's structure (Schneemann et al., 1992). The changes lead the capsid to disassemble and uncoat the RNA when re-exposed to intracellular conditions upon its entry into a new host cell. The maturation cleavage is mediated by the encapsidated RNA in the interior of the capsid (Wery et al., 1994). The maturation cleavage displays itself in Ha SV by the appearance of a 64 kDa protein and the disappearance the 71 kDa precursor similar to the nodaviruses (Gallagher and Rueckert, 1988)).

That this process occurs for Ha SV VLPs is shown by observing an immunoblot of extracts of cells infected with Bacp71 and expressing p71 and proteins extracted from purified Ha SV virions and purified Ha SV VLPs. The blot shows that for the cell extract, p71 is expressed in the majority with minor expression of lower molecular weight products that are presumed degradation products. For proteins extracted from Ha SV virions, the 64 kDa cleavage produce is present in the great majority with only a minor presence of the 71 kDa precursor. For proteins from VLPs, the 64 kDa and 71 kDa proteins are present approximately equal amounts showing that the maturation cleavage does occur although not as efficiently as with viral RNA present inside the particles.

Thus Ha SV VLPs are in a condition to be uncoated when they enter a host cell. This process is mediated by RNA sequences in the coat protein ORF.

Methods and Materials: An extract of cells infected with Bacp71 for three days was made by pelleting cells from a 25 cm 2 flask and lysing them in phosphate-buffered saline with freeze-thaw. SDS-PAGE and immunoblotting with anti-sera against Ha SV was performed on the cell extract and proteins from VLPs and virions according to standard procedures.

Translation: As a general rule, ribosomes responsible for translating proteins initiate translation by binding to the cap structure of an mRNA then scanning to the first MET placed in an appropriate context. This presents an initial difficulty in translating a toxin sequence on an RNA from a protein product (the VLP) translated from the region of the mRNA where the encapsidation signal resides (see above).

This difficulty can be dealt with in either of two ways: trans-encapsidation or through the use of internal ribosome entry sites (IRESs).

1. Trans-encapsidation. Trans-encapsidation is the process whereby an RNA strand is produced with the toxin sequence placed in good translatable context before the p71 encapsidation sequence. Translation of an RNA sequence is not required for its encapsidation by proteins produced from another mRNA. In this way a VLP produced from an RNA having a translatable p71 encapsidates an RNA with a nontranslatable p71 sequence but possessing the encapsidation signal that ensures the encapsidation of the strand with a translatable toxin sequence.

2. IRESs. IRESs are one of the exceptions to the general rule noted above for translation They are sequences on an RNA strand that allow ribosomes to bind an RNA internally instead of at the 5′ cap to initiate protein translation. They are located in the 5′ regions of picornavirus vRNAs and specialised genes from certain organisms (Jackson et al., 1994)). They have been employed to allow the translation of two proteins from the same mRNA (Lipsick and Smarda, 1993) and thus can be employed to express a toxin from a sequence located after a translatable p71. An advantage to this approach is that many IRESs are host specific. If the IRES used in the RNA capsovector is able to function only in the target organism, the toxin is produced only in the target organism and not in the organism producing the RNA capsovector. A picornavirus with a targeted pest insect host will possess a suitable IRES for a capsovector. For example, an IRES from a picornavirus with a Heliothis host will be used in a capsovector constructed with p71 from Ha SV and a cytotoxin such as the ricin A fragement. This particular capsovector will affect only heliothine insects and no others as well as not producing a ricin A fragment in the plant producing the capsovector.

Translation Into Toxicity

Ultimately, all of the above abilities of encapsidation, protection, entry, and uncoating must be induced or accomplished by sequences in the RNA strand that produce the capsovector. Also important are sequences on the RNA, derived from other viral and non-viral sources as well as from Ha SV, that are responsible for the toxic activity and if required, translation into protein that confer the toxicity. These types of sequences will be dealt with in separate sections.

RNA sequences leading to toxicity of the organism or cell can either be translated into protein or the sequences cause secondary structures to made on the RNA strand that lead to toxicity.

1. Toxicity from protein sequences. Both types of toxins, nerve toxins, specific for insects and work at the level of the organism (Binnington and Baule, 1993) and cytotoxins (Stripe and Barbeiri, 1986), toxic only to the cell the capsovector has entered, can be used with RNA capsovectors. However, the former type of toxin will have to have a secretion signal appropriate to midgut cells. Examples of these proteins are described in Maeda et al., 1991.

2. Toxicity from RNA secondary structure sequences. RNA secondary structures will have the ability to cause toxicity to the cell to which they have been vectored by RNA capsovectors. These structures are caused by primary sequences; however, any number of primary sequences on the RNA strand can lead to the same toxic secondary structure. There are three types of these sequence structures that will be appropriate for RNA capsovectors: antisense sequences, ribozymes and mimicking structures. The first two types are reviewed by Eguchi et al., 1991 and elsewhere herein, respectively and aimed at preventing the expression of key cellular enzymes. The latter is novel and will be detailed.

It is the activity of the Ha SV replicase and not virosis or accumulation of viruses that causes the midgut cell to cease functioning. This is shown by data generated from the following experiment. When protoplasts are transfected with genes that make a replicatable RNA1 and only the capsid protein and not a replicatable RNA2 (R1-HC and VCAPB according to procedures listed above), stunting occurs. When the stunted larvae are extracted for RNA which is then northern blotted with probe for Ha SV nucleic acid, only RNA1 of Ha SV is seen to be present. Stunting does not occur when the protoplasts are transfected with genes that do not make a replicatable RNA1 (lacking an effective ribozyme to cleave after the last viral base in the gene) and only the capsid protein and not a replicatable RNA2 (R1-HDV and VCAPB according to procedures listed elsewhere in patent). When the stunted larvae are extracted for RNA which is then northern blotted with probe for Ha SV nucleic acid, no Ha SV RNA is seen to be present.

These data are consistent with RNA1 being encapsidated and able to enter midgut cells of the larvae. RNA1 is able to self-replicate but not produce virions as there is no replicatable RNA2 which has the p71 ORF. The self-replication leads to antibiosis due to apoptosis of the midgut cells having the replicating RNA1. The particles made in the same manner but having RNA1 not able to self-replicate (due to the 3′ sequences left by the defective ribozyme) were unable to stunt the larvae.

As shown in other systems, the activity of the replicase may be mimicked by placing a by-product of replicase activity into the cell. This action “tricks” the cell into initiating anti-viral measures such as shut-down of protein synthesis or cell death by apoptosis (the activity believed to be responsible for the antibiosis caused by Ha SV).

One such by-product of the replicase's activity is double stranded RNA, an intermediate of RNA replication of tetraviruses (du Plessis et al., 1991). Other systems have shown that transfection of double stranded RNA into cells causes anti-viral measures to be initiated in them.

Double-stranded RNA can be delivered to midgut cells by RNA capsovectors by a synthetic gene construct which produces a large stem-loop structure when transcribed. This structure is made by making the 3′ half the reverse complement of the 5′ half which then self-hybridises into the stem-loop. The RNA can be of viral, from either Ha SV strand or of non-viral origin.

Protein Capsovectors

Protein capsovectors are VLPs composed of a modified capsid protein or of a mixture of the modified capsid protein and the unmodified capsid protein. The modified coat protein is the coat protein, p71, fused to a fragment of a cytotoxin of either a plant or bacterial origin. When expressed, the coat proteins and coat protein-toxin fusions will self-assemble into the protein capsovector. Similar to the RNA capsovectors, protein capsovectors will not be self-replicating entities like viruses. Upon being eaten by an insect pest, the structure of the capsovector will vector the toxin moiety to inside the midgut cell by preventing proteolysis of the toxin moiety in the midgut and entering the midgut cells in a manner similar to what occurs for a virus particle. Upon entry, the capsovector will expose the cytotoxic moiety which will then kill the cell. Large numbers of midgut cells killed by capsovectors will cause antibiosis to the feeding insects. It is believed that a single gene will be able to express the capsovector.

In concept, protein capsovectors are similar to the immunotoxins successfully used in human cancer therapy where cytotoxic moieties are vectored to specific cells by fusion to a specific binding moiety such as antibodies or cytokines. By themselves, the cytotoxin fragments do not display toxicity even when injected intravenously. Only when they are attached or fused to a binding element does cytotoxicity occur to those cells to which the element binds. The binding element of the protein capsovector is the VAP part (see above) of the Ha SV VLP which is able to bind and enter midgut cells of heliothis larvae.

However, the protein capsovector is distinct from immunotoxins in that its structure also protects the toxin moiety from degradation in addition to binding to the midgut cell. The toxin fragment will be contained within the capsid shell until the capsovector enters the midgut cell. Also, it is the capacity of capsovectors to protect the toxin moiety from degradation in the midgut lumen that makes it distinct from other insect control factors that are fused to elements that only “interact” with midgut cells.

In the following sections, each of the two elements of a capsovector, the cytotoxic fragment and a viral coat protein gene, will be described followed by descriptions of how the capsovector are constructed with data supporting their feasibility.

The Toxin: Several cytotoxin fragments suitable for protein capsovectors are available in readily accessible form. They have extensive literature describing their activity in various fusions for use in immunotoxins (Thorpe et al., 1982). For expression in plants, plant-derived fragments of proteinaceous toxins, such as ricin A fragment, which are not toxic to plant cells but toxic to animal cells will be the most suitable. For expression in microorganisms, toxins of bacterial origin may be the most suitable, it being that they are not toxic to the microorganism producing the protein capsovectors.

As described in the section on RNA caspsovectors, p71 has the ability to self-assemble into VLPs when expressed in various non-host expression systems. Also shown was the ability of the VLPs and virions to resist degradation and bind to, then enter, midgut cells. The critical question concerning the feasibility of the protein capsovector using p71, the Ha SV coat protein, is the ability of VLPs to form with the modified coat proteins fused to toxins.

Construction of model protein capsovectors with capsid protein, p71 and a reporter peptide: The cytotoxin fragment can be fused to p71 in a number of ways. In order to test the possibilities, a reporter peptide fragment was used in place of the toxins. This allows a more rapid characterisation of the products as immunodetection of the exogenous fragment is facilitated by a commercially available monoclonal antibody specific for the fragment (IBI FLAG Biosystem). Two constructs were made using standard techniques. The FlagT construct placed the reporter fragment, sized 2501 Da, at the C-terminus of p71 (FIG. 8 ). The FlagM construct placed a reporter fragment, sized 1243 Da, in the middle of the p71 sequence near the site where p71 is cleaved into p64 and p7 (FIG. 8 ). Both constructs should have resulted in the reporter fragment being placed into the interior of the VLP where p7 is located.

Formation of VLPs made with modified p71: Recombinant baculoviruses were made with the modified p71 genes by standard techniques (King and Possee, 1992) and used to infect Sf9 cells. When these cells were processed and examined as before with TEM, particles highly similar to VLPs made with unmodified p71 and Ha SV virions were observed. The particles were purified on CsCl gradients and were examined with SDS-PAGE, immunoblots, negatively stained TEM. In addition, the particles were tested for their ability to protect the FLAG epitope on the fused peptide fragment. These experiments showed:

1. The purified particles were highly similar to Ha SV virions and VLPs made with unmodified p71 and showed the characteristic 35-40 nm diameter spheres with a fraction having the electron dense cores. The FlagT particle possessed a buoyant density of 1.31 g/ml compared to 1.29 g/ml for unmodified VLPs and virions.

2. Proteins extracted from the particles and observed with SDS-PAGE had the molecular weights predicted from the constructs (1243 Da and 2501 Da for FlagM and FlagT respectively. In addition, post-assembly cleavage into p64 was observed for the FlagM particle. No processing was seen in the FlagT particle.

3. Proteins extracted from the particles reacted with both the anti-p71 antisera and with the FLAG monoclonal antibody on immunoblots.

4. The FLAG epitope was protected during exposure to heliothis midgut contents. This is shown by the FLAG epitope remaining at the original molecular weight and therefore undegraded.

Hybrid VLPs: Although VLPs can be made with only modified p71 fused to the reporter peptide, and protection of the exogenous reporter peptide occurs, a protein capsovector made with both native p71 and modified p71 fused to a cytotoxin may function better. At present it is not clear what properties of the “native” VLP, if any, are altered with the addition of the exogenous, fused peptides to p71. If any deleterious properties arise such as poor stability of the particle, a resolution to the problem will be to produce a hybrid particle. This will minimise any disruption of desirable properties of the native VLP by the modified p71.

Hybrid VLP Expression in Plants: Three ways can be envisioned to express, either in a transgenic plant, the hybrid capsovector which requires two distinct, but closely related proteins. The most obvious is two insert two genes into the plant, one for each protein. However, the use of elements from plant viruses can make it possible to express a capsovector from a single gene. These elements are suppressible stop contexts and frame-shift sequences that are detailed by Sleat and Wilson (1992). The use of these elements make it feasible to precisely regulate the ratio of coat protein to coat protein-toxin fusion.

REFERENCES

Agrawal, D. K. & Johnson, J. E. (1992) Sequence and analysis of the capsid protein of Nudaurelia capensis ω virus, an insect virus with T=4 icosahedral symmetry. Virology 190, 806-804.

Agrawal, D. K. & Johnson, J. E. (1995) Assembly of the T=4 Nudaurelia capensis ω virus capsid, post-translational cleavage, and specific encapsidation of its mRNA in a baculovirus expression system. Virology (in press)

Binnington, K. C. and Baule, V. J. (1993) Naturally occurring insecticidal molecules as candidates for genetic engineering in Molecular approaches to fundamental and applied entomology (Oakeshott, J. and Whitten M., eds) Springer-Verlag, New York.

Eguchi, Y., Itoh, T., and Tomizawa, J. (1991) Antisense RNA. Annual Reviews of Biochemistry 60, 631-652

Gallagher, T. M. and Rueckert, R. R. (1988) Assembly-dependent maturation cleavage in provirions of a small icosahedral insect ribovirus. Journal of Virology, 62, 3399-3406.

Gordon, K. H. J., Johnson, K. N. & Hanzlik, T. N. The larger genomic RNA of Helicoverpa armigera stunt virus encodes the viral RNA polymerase and has a novel 3′-terminal tRNA-like structure. Virology (in press)

Hanzlik, T. N., Dorrian, S. J., Gordon, K. H. J., & Christian, P. D. (1993) A novel small RNA virus isolated from the cotton bollworm, Helicoverpa armigera. Journal of General Virology 74, 1805-1810

Hanzlik, T. N., Dorrian, S. J., Gordon, K. H. J., & Christian, P. D. Sequence of RNA2 of the Helicoverpa armigera stunt virus (Tetraviridae) and bacterial expression of its genes Journal of General Virology (in press).

Jackson, R. J., Hunt, S. L., Gibbs, C. L. and Kaminski, A. (1994) Internal initiation of translation of picornaviral RNAs. Molecular Biology Reports 19, 147-159

King, L. A. and Possee, R. D. (1992) The Baculovirus Expression System . Chapman and Hall, London

Kirkegaard, K. and Compton, S. R. (1990) Defined mutations in the poliovirus capsid proteins cause specific defects in RNA encapsidation, RNA uncoating and VP0 cleavage in New Aspects of positive strand RNA viruses . (Brinton, M. A. and Heinz, F. X., eds.) pp. 245-249 American Society for Microbiology, Washington, D.C.

Lentz, T. L. (1990) The recognition event between virus and host cell receptor: a target for antiviral agents. Journal of General Virology 71, 751-766

Maeda, S., Volrath, S. L., Hanzlik, T. N., Harper, S. A., Maddox, D. W., Hammock, B. D., and Fowler, E. Insecticidal effects of an insect-selective neurotoxin expressed by a recombinant baculovirus. (1991) Virology 184: 777-780.

du Plessis, D. H., Mokhosi, G. and Hendry, D. A. (1991) Cell-free translation and identification of the replicative form of Nudaurelia b virus RNA. Journal of General Virology 72, 267-273.

Schneeman, A. Zhong, W. Gallagher, T. M. and Rueckert, R. R. (1992) Maturation cleavage required for infectivity of a nodavirus. Journal of Virology 66, 6728-6734

Sleat, D. E. and Wilson, T. M. A. (1992) Plant virus genomes as sources of novel functions for genetic manipulations, in Genetic Engineering with Plant Viruses (T. M. Wilson and J. W. Davies, eds.) CRC Press Boca Raton.

Smarda, J. and Lipsick, J. S. (1993) Dicistronic selection for nuclear proteins in living animal cells. Gene 137, 145-149.

Stripe, F. and Barbieri, L. (1986) Ribosome-inactivating proteins up to date. FEBS Letters, 195, 1-8.

Thorpe, P. E. Edwards, D. C. Davies, A. J. S. and Ross, W. C. J. (1982) in Monoclonal Antibodies in Clinical Medicine (McMichael, A. J. and Fabre, J. W., eds.) pp.167-201, Academic Press, London.

Wery, J-P., Reddy, V. S., Hosur, M. V., and Johnson, J. E. (1994) The refined three-dimensional structure of an insect virus at 2.8 A resolution. J. Mol. Biol. 235, 565-586.

Wolfersberger, M., Luthy, P., Maurer, A. Parenti, P. Sacchi, F. V. Giordan, B. and Hanozet, G. M. (1987) Comp. Biochem. Physiol. 86A, 301-308.

57 13 base pairs nucleic acid unknown unknown DNA unknown 1GGATCCACAG NNN 13 28 base pairs nucleic acid unknown unknown DNA unknown 2ATGGGCGATG CCGGCGTCGC GTTCACAG 28 27 base pairs nucleic acid unknown unknown DNA unknown 3ATGGAGGATG CTGGAGTGGC GTCACAG 27 27 base pairs nucleic acid unknown unknown DNA unknown 4ATGAGCGAGG CCGGCGTCGC GTCACAG 27 30 base pairs nucleic acid unknown unknown DNA unknown 5CCATCGATGC CGGACTGGTA TCCCAGGGGG 30 31 base pairs nucleic acid unknown unknown DNA unknown 6CCATCGATGC CGGACTGGTA TCCCGAGGGA C 31 39 base pairs nucleic acid unknown unknown DNA unknown 7CCATCGATGA TCCAGCCTCC TCGCGGCGCC GGATGGGCA 39 39 base pairs nucleic acid unknown unknown DNA unknown 8GCTCTAGATC CATTCGCCAT CCGAAGATGC CCATCCGGC 39 39 base pairs nucleic acid unknown unknown DNA unknown 9CCATCGATTT ATGCCGAGAA GGTAACCAGA GAAACACAC 39 39 base pairs nucleic acid unknown unknown DNA unknown 10GCTCTAGACC AGGTAATATA CCACAACGTG TGTTTCTCT 39 45 base pairs nucleic acid unknown unknown DNA unknown 11GGGGGGAATT CATTTAGGTG ACACTATAGT TCTGCCTCCC CGGAC 45 27 base pairs nucleic acid unknown unknown DNA unknown 12GGGGGGATCC TGGTATCCCA GGGGGGC 27 28 base pairs nucleic acid unknown unknown DNA unknown 13CCGGAAGCTT GTTTTTCTTT CTTTACCA 28 46 base pairs nucleic acid unknown unknown DNA unknown 14GGGGGATCCG ATGGTATCCC GAGGGACGCT CAGCAGGTGG CATAGG 46 52 base pairs nucleic acid unknown unknown DNA unknown 15AAATAATTTT GTTACTTTAG AAGGAGATAT ACATATGAGC GAGCGAGCAC AC 52 55 base pairs nucleic acid unknown unknown DNA unknown 16AAATAATTTT GTTTAACCTT AAGAAGGAGA TCTACATATG CTGGAGTGGC GTCAC 55 30 base pairs nucleic acid unknown unknown DNA unknown 17GGAGATCTAC ATATGGGAGA TGCTGGAGTG 30 17 base pairs nucleic acid unknown unknown DNA unknown 18GTAGCGAACG TCGAGAA 17 31 base pairs nucleic acid unknown unknown DNA unknown 19GGGGGATCCT CAGTTGTCAG TGGCGGGGTA G 31 28 base pairs nucleic acid unknown unknown DNA unknown 20GGGGATCCCT AATTGGCACG AGCGGCGC 28 29 base pairs nucleic acid unknown unknown DNA unknown 21AATTACATAT GGCGGCCGCC GTTTCTGCC 29 29 base pairs nucleic acid unknown unknown DNA unknown 22AATTACATAT GTTCGCGGCC GCCGTTTCT 29 19 amino acids amino acid unknown unknown protein unknown 23Phe Ala Ala Ala Val Ser Ala Phe Ala Ala Asn Met Leu Ser Ser Va1 5 10 15Leu Lys Ser 20 amino acids amino acid unknown unknown protein unknown 24Pro Thr Leu Val Asp Gln Gly Phe Trp Ile Gly Gly Gln Tyr Ala Le1 5 10 15Thr Pro Thr Ser 20 6 amino acids amino acid unknown unknown protein unknown 25Phe Ala Ala Ala Val Ser1 5 23 base pairs nucleic acid unknown unknown DNA unknown 26GCGCCCCCUG GGAUACCAGG AUC 23 17 base pairs nucleic acid unknown unknown DNA unknown 27TCAGCAGGTG GCATAGG 17 32 base pairs nucleic acid unknown unknown DNA unknown CDS 6..32 28CCCAT ATG GGC GAT GCC GGC GTC GCG TCA CAG 32 Met Gly Asp Ala Gly Val Ala Ser Gln 1 5 9 amino acids amino acid linear protein unknown 29Met Gly Asp Ala Gly Val Ala Ser Gln 1 5 32 base pairs nucleic acid unknown unknown DNA unknown CDS 6..32 30CCCAT ATG AGC GAG GCC GGC GTC GCG TCA CAG 32 Met Ser Glu Ala Gly Val Ala Ser Gln 1 5 9 amino acids amino acid linear protein unknown 31Met Ser Glu Ala Gly Val Ala Ser Gln 1 5 27 base pairs nucleic acid unknown unknown DNA unknown CDS 1..27 32ATG GGA GAT GCT GGA GTG GCG TCA CAG 27Met Gly Asp Ala Gly Val Ala Ser Gln 1 5 9 amino acids amino acid linear protein unknown 33Met Gly Asp Ala Gly Val Ala Ser Gln 1 5 27 base pairs nucleic acid unknown unknown DNA unknown 34GGGGGATCCC GCGGATTTAT GAGCGAG 27 32 base pairs nucleic acid unknown unknown DNA unknown 35GGGGGATCCC GCGGAGACAT GAGCGAGCAC AC 32 34 base pairs nucleic acid unknown unknown DNA unknown 36GGGGGATCCA GCGACATGAG AGATGCTGGA GTGG 34 34 base pairs nucleic acid unknown unknown DNA unknown 37GGGGGATCCA GCGACATGAG AGATGCTGGA GTGG 34 26 base pairs nucleic acid unknown unknown DNA unknown 38GGGGGATCCG TTCTGCCTCC CCGGAC 26 5312 base pairs nucleic acid unknown unknown DNA unknown CDS 37..5148 39GTTCTGCCTC CCCCGGACGG TAAATATAGG GGAACA ATG TAC GCG AAA GCG ACA 54 Met Tyr Ala Lys Ala Thr 1 5GAC GTG GCG CGT GTC TAC GCC GCG GCA GAT GTC GCC TAC GCG AAC GTA 102Asp Val Ala Arg Val Tyr Ala Ala Ala Asp Val Ala Tyr Ala Asn Val 10 15 20CTG CAG CAG AGA GCA GTC AAG TTG GAC TTC GCC CCG CCA CTG AAG GCA 150Leu Gln Gln Arg Ala Val Lys Leu Asp Phe Ala Pro Pro Leu Lys Ala 25 30 35CTA GAA ACC CTC CAC AGA CTG TAC TAT CCG CTG CGC TTC AAA GGG GGC 198Leu Glu Thr Leu His Arg Leu Tyr Tyr Pro Leu Arg Phe Lys Gly Gly 40 45 50ACT TTA CCC CCG ACA CAA CAC CCG ATC CTG GCC GGG CAC CAA CGT GTC 246Thr Leu Pro Pro Thr Gln His Pro Ile Leu Ala Gly His Gln Arg Val 55 60 65 70GCA GAA GAG GTT CTG CAC AAT TTC GCC AGG GGA CGT AGC ACA GTG CTC 294Ala Glu Glu Val Leu His Asn Phe Ala Arg Gly Arg Ser Thr Val Leu 75 80 85GAG ATA GGG CCG TCT CTG CAC AGC GCA CTT AAG CTA CAT GGG GCA CCG 342Glu Ile Gly Pro Ser Leu His Ser Ala Leu Lys Leu His Gly Ala Pro 90 95 100AAC GCC CCC GTC GCA GAC TAT CAC GGG TGC ACC AAG TAC GGC ACC CGC 390Asn Ala Pro Val Ala Asp Tyr His Gly Cys Thr Lys Tyr Gly Thr Arg 105 110 115GAC GGC TCG CGA CAC ATT ACG GCC TTA GAG TCT AGA TCC GTC GCC ACA 438Asp Gly Ser Arg His Ile Thr Ala Leu Glu Ser Arg Ser Val Ala Thr 120 125 130GGC CGG CCC GAG TTC AAG GCC GAC GCC TCA CTG CTC GCC AAC GGC ATT 486Gly Arg Pro Glu Phe Lys Ala Asp Ala Ser Leu Leu Ala Asn Gly Ile135 140 145 150GCC TCC CGC ACC TTC TGC GTC GAC GGA GTC GGC TCT TGC GCG TTC AAA 534Ala Ser Arg Thr Phe Cys Val Asp Gly Val Gly Ser Cys Ala Phe Lys 155 160 165TCG CGC GTT GGA ATT GCC AAT CAC TCC CTC TAT GAC GTG ACC CTA GAG 582Ser Arg Val Gly Ile Ala Asn His Ser Leu Tyr Asp Val Thr Leu Glu 170 175 180GAG CTG GCC AAT GCG TTT GAG AAC CAC GGA CTT CAC ATG GTC CGC GCG 630Glu Leu Ala Asn Ala Phe Glu Asn His Gly Leu His Met Val Arg Ala 185 190 195TTC ATG CAC ATG CCA GAA GAG CTG CTC TAC ATG GAC AAC GTG GTT AAT 678Phe Met His Met Pro Glu Glu Leu Leu Tyr Met Asp Asn Val Val Asn 200 205 210GCC GAG CTC GGC TAC CGC TTC CAC GTT ATT GAA GAG CCT ATG GCT GTG 726Ala Glu Leu Gly Tyr Arg Phe His Val Ile Glu Glu Pro Met Ala Val215 220 225 230AAG GAC TGC GCA TTC CAG GGG GGG GAC CTC CGT CTC CAC TTC CCT GAG 774Lys Asp Cys Ala Phe Gln Gly Gly Asp Leu Arg Leu His Phe Pro Glu 235 240 245TTG GAC TTC ATC AAC GAG AGC CAA GAG CGG CGC ATC GAG AGG CTG GCC 822Leu Asp Phe Ile Asn Glu Ser Gln Glu Arg Arg Ile Glu Arg Leu Ala 250 255 260GCC CGC GGC TCC TAC TCC AGA CGC GCC GTC ATT TTC TCC GGC GAC GAC 870Ala Arg Gly Ser Tyr Ser Arg Arg Ala Val Ile Phe Ser Gly Asp Asp 265 270 275GAC TGG GGT GAT GCG TAC TTA CAC GAC TTC CAC ACA TGG CTC GCC TAC 918Asp Trp Gly Asp Ala Tyr Leu His Asp Phe His Thr Trp Leu Ala Tyr 280 285 290CTA CTG GTG AGG AAC TAC CCC ACT CCG TTT GGT TTC TCA CTC CAT ATA 966Leu Leu Val Arg Asn Tyr Pro Thr Pro Phe Gly Phe Ser Leu His Ile295 300 305 310GAA GTC CAG AGG CGC CAC GGC TCC AGC ATT GAG CTG CGC ATC ACT CGC 1014Glu Val Gln Arg Arg His Gly Ser Ser Ile Glu Leu Arg Ile Thr Arg 315 320 325GCG CCA CCT GGA GAC CGC ATG CTG GCC GTC GTC CCA AGG ACG TCC CAA 1062Ala Pro Pro Gly Asp Arg Met Leu Ala Val Val Pro Arg Thr Ser Gln 330 335 340GGC CTC TGC AGA ATC CCA AAC ATC TTT TAT TAC GCC GAC GCG TCG GGC 1110Gly Leu Cys Arg Ile Pro Asn Ile Phe Tyr Tyr Ala Asp Ala Ser Gly 345 350 355ACT GAG CAT AAG ACC ATC CTT ACG TCA CAG CAC AAA GTC AAC ATG CTG 1158Thr Glu His Lys Thr Ile Leu Thr Ser Gln His Lys Val Asn Met Leu 360 365 370CTC AAT TTT ATG CAA ACG CGT CCT GAG AAG GAA CTA GTC GAC ATG ACC 1206Leu Asn Phe Met Gln Thr Arg Pro Glu Lys Glu Leu Val Asp Met Thr375 380 385 390GTC TTG ATG TCG TTC GCG CGC GCT AGG CTG CGC GCG ATC GTG GTC GCC 1254Val Leu Met Ser Phe Ala Arg Ala Arg Leu Arg Ala Ile Val Val Ala 395 400 405TCA GAA GTC ACC GAG AGC TCC TGG AAC ATC TCA CCG GCT GAC CTG GTC 1302Ser Glu Val Thr Glu Ser Ser Trp Asn Ile Ser Pro Ala Asp Leu Val 410 415 420CGC ACT GTC GTG TCT CTT TAC GTC CTC CAC ATC ATC GAG CGC CGA AGG 1350Arg Thr Val Val Ser Leu Tyr Val Leu His Ile Ile Glu Arg Arg Arg 425 430 435GCT GCG GTC GCT GTC AAG ACC GCC AAG GAC GAC GTC TTT GGA GAG ACT 1398Ala Ala Val Ala Val Lys Thr Ala Lys Asp Asp Val Phe Gly Glu Thr 440 445 450TCG TTC TGG GAG AGT CTC AAG CAC GTC TTG GGC TCC TGT TGC GGT CTG 1446Ser Phe Trp Glu Ser Leu Lys His Val Leu Gly Ser Cys Cys Gly Leu455 460 465 470CGC AAC CTC AAA GGC ACC GAC GTC GTC TTT ACT AAG CGC GTC GTC GAT 1494Arg Asn Leu Lys Gly Thr Asp Val Val Phe Thr Lys Arg Val Val Asp 475 480 485AAG TAC CGA GTC CAC TCG CTC GGA GAC ATA ATC TGC GAC GTC CGC CTG 1542Lys Tyr Arg Val His Ser Leu Gly Asp Ile Ile Cys Asp Val Arg Leu 490 495 500TCC CCT GAA CAG GTC GGC TTC CTG CCG TCC CGC GTA CCA CCT GCC CGC 1590Ser Pro Glu Gln Val Gly Phe Leu Pro Ser Arg Val Pro Pro Ala Arg 505 510 515GTC TTT CAC GAC AGG GAA GAG CTT GAG GTC CTT CGC GAA GCT GGC TGC 1638Val Phe His Asp Arg Glu Glu Leu Glu Val Leu Arg Glu Ala Gly Cys 520 525 530TAC AAC GAA CGT CCG GTA CCT TCC ACT CCT CCT GTG GAG GAG CCC CAA 1686Tyr Asn Glu Arg Pro Val Pro Ser Thr Pro Pro Val Glu Glu Pro Gln535 540 545 550GGT TTC GAC GCC GAC TTG TGG CAC GCG ACC GCG GCC TCA CTC CCC GAG 1734Gly Phe Asp Ala Asp Leu Trp His Ala Thr Ala Ala Ser Leu Pro Glu 555 560 565TAC CGC GCC ACC TTG CAG GCA GGT CTC AAC ACC GAC GTC AAG CAG CTC 1782Tyr Arg Ala Thr Leu Gln Ala Gly Leu Asn Thr Asp Val Lys Gln Leu 570 575 580AAG ATC ACC CTC GAG AAC GCC CTC AAG ACC ATC GAC GGG CTC ACC CTC 1830Lys Ile Thr Leu Glu Asn Ala Leu Lys Thr Ile Asp Gly Leu Thr Leu 585 590 595TCC CCA GTC AGA GGC CTC GAG ATG TAC GAG GGC CCG CCA GGC AGC GGC 1878Ser Pro Val Arg Gly Leu Glu Met Tyr Glu Gly Pro Pro Gly Ser Gly 600 605 610AAG ACG GGC ACC CTC ATC GCC GCC CTT GAG GCC GCG GGC GGT AAA GCA 1926Lys Thr Gly Thr Leu Ile Ala Ala Leu Glu Ala Ala Gly Gly Lys Ala615 620 625 630CTT TAC GTG GCA CCC ACC AGA GAA CTG AGA GAG GCT ATG GAC CGG CGG 1974Leu Tyr Val Ala Pro Thr Arg Glu Leu Arg Glu Ala Met Asp Arg Arg 635 640 645ATC AAA CCG CCG TCC GCC TCG GCT ACG CAA CAT GTC GCC CTT GCG ATT 2022Ile Lys Pro Pro Ser Ala Ser Ala Thr Gln His Val Ala Leu Ala Ile 650 655 660CTC CGT CGT GCC ACC GCC GAG GGC GCC CCT TTC GCT ACC GTG GTT ATC 2070Leu Arg Arg Ala Thr Ala Glu Gly Ala Pro Phe Ala Thr Val Val Ile 665 670 675GAC GAG TGC TTC ATG TTC CCG CTC GTG TAC GTC GCG ATC GTG CAC GCC 2118Asp Glu Cys Phe Met Phe Pro Leu Val Tyr Val Ala Ile Val His Ala 680 685 690TTG TCC CCG AGC TCA CGA ATA GTC CTT GTA GGG GAC GTC CAC CAA ATC 2166Leu Ser Pro Ser Ser Arg Ile Val Leu Val Gly Asp Val His Gln Ile695 700 705 710GGG TTT ATA GAC TTC CAA GGC ACA AGC GCG AAC ATG CCG CTC GTT CGC 2214Gly Phe Ile Asp Phe Gln Gly Thr Ser Ala Asn Met Pro Leu Val Arg 715 720 725GAC GTC GTT AAG CAG TGC CGT CGG CGC ACT TTC AAC CAA ACC AAG CGC 2262Asp Val Val Lys Gln Cys Arg Arg Arg Thr Phe Asn Gln Thr Lys Arg 730 735 740TGT CCG GCC GAC GTC GTT GCC ACC ACG TTT TTC CAG AGC TTG TAC CCC 2310Cys Pro Ala Asp Val Val Ala Thr Thr Phe Phe Gln Ser Leu Tyr Pro 745 750 755GGG TGC ACA ACC ACC TCA GGG TGC GTC GCA TCC ATC AGC CAC GTC GCC 2358Gly Cys Thr Thr Thr Ser Gly Cys Val Ala Ser Ile Ser His Val Ala 760 765 770CCA GAC TAC CGC AAC AGC CAG GCG CAA ACG CTC TGC TTC ACG CAG GAG 2406Pro Asp Tyr Arg Asn Ser Gln Ala Gln Thr Leu Cys Phe Thr Gln Glu775 780 785 790GAA AAG TCG CGC CAC GGG GCT GAG GGC GCG ATG ACT GTG CAC GAA GCG 2454Glu Lys Ser Arg His Gly Ala Glu Gly Ala Met Thr Val His Glu Ala 795 800 805CAG GGA CGC ACT TTT GCG TCT GTC ATT CTG CAT TAC AAC GGC TCC ACA 2502Gln Gly Arg Thr Phe Ala Ser Val Ile Leu His Tyr Asn Gly Ser Thr 810 815 820GCA GAG CAG AAG CTC CTC GCT GAG AAG TCG CAC CTT CTA GTC GGC ATC 2550Ala Glu Gln Lys Leu Leu Ala Glu Lys Ser His Leu Leu Val Gly Ile 825 830 835ACG CGC CAC ACC AAC CAC CTG TAC ATC CGC GAC CCG ACA GGT GAC ATT 2598Thr Arg His Thr Asn His Leu Tyr Ile Arg Asp Pro Thr Gly Asp Ile 840 845 850GAG AGA CAA CTC AAC CAT AGC GCG AAA GCC GAG GTG TTT ACA GAC ATC 2646Glu Arg Gln Leu Asn His Ser Ala Lys Ala Glu Val Phe Thr Asp Ile855 860 865 870CCT GCA CCC CTG GAG ATC ACG ACT GTC AAA CCG AGT GAA GAG GTG CAG 2694Pro Ala Pro Leu Glu Ile Thr Thr Val Lys Pro Ser Glu Glu Val Gln 875 880 885CGC AAC GAA GTG ATG GCA ACG ATA CCC CCG CAG AGT GCC ACG CCG CAC 2742Arg Asn Glu Val Met Ala Thr Ile Pro Pro Gln Ser Ala Thr Pro His 890 895 900GGA GCA ATC CAT CTG CTC CGC AAG AAC TTC GGG GAC CAA CCC GAC TGT 2790Gly Ala Ile His Leu Leu Arg Lys Asn Phe Gly Asp Gln Pro Asp Cys 905 910 915GGC TGT GTC GCT TTG GCG AAG ACC GGC TAC GAG GTG TTT GGC GGT CGT 2838Gly Cys Val Ala Leu Ala Lys Thr Gly Tyr Glu Val Phe Gly Gly Arg 920 925 930GCC AAA ATC AAC GTA GAG CTT GCC GAA CCC GAC GCG ACC CCG AAG CCG 2886Ala Lys Ile Asn Val Glu Leu Ala Glu Pro Asp Ala Thr Pro Lys Pro935 940 945 950CAT AGG GCG TTC CAG GAA GGG GTA CAG TGG GTC AAG GTC ACC AAC GCG 2934His Arg Ala Phe Gln Glu Gly Val Gln Trp Val Lys Val Thr Asn Ala 955 960 965TCT AAC AAA CAC CAG GCG CTC CAG ACG CTG TTG TCC CGC TAC ACC AAG 2982Ser Asn Lys His Gln Ala Leu Gln Thr Leu Leu Ser Arg Tyr Thr Lys 970 975 980CGA AGC GCT GAC CTG CCG CTA CAC GAA GCT AAG GAG GAC GTC AAA CGC 3030Arg Ser Ala Asp Leu Pro Leu His Glu Ala Lys Glu Asp Val Lys Arg 985 990 995ATG CTA AAC TCG CTT GAC CGA CAT TGG GAC TGG ACT GTC ACT GAA GAC 3078Met Leu Asn Ser Leu Asp Arg His Trp Asp Trp Thr Val Thr Glu Asp 1000 1005 1010GCC CGT GAC CGA GCT GTC TTC GAG ACC CAG CTC AAG TTC ACC CAA CGC 3126Ala Arg Asp Arg Ala Val Phe Glu Thr Gln Leu Lys Phe Thr Gln Arg1015 1020 1025 1030GGC GGC ACC GTC GAA GAC CTG CTG GAG CCA GAC GAC CCC TAC ATC CGT 3174Gly Gly Thr Val Glu Asp Leu Leu Glu Pro Asp Asp Pro Tyr Ile Arg 1035 1040 1045GAC ATA GAC TTC CTT ATG AAG ACT CAG CAG AAA GTG TCG CCC AAG CCG 3222Asp Ile Asp Phe Leu Met Lys Thr Gln Gln Lys Val Ser Pro Lys Pro 1050 1055 1060ATC AAT ACG GGC AAG GTC GGG CAG GGG ATC GCC GCT CAC TCA AAG TCT 3270Ile Asn Thr Gly Lys Val Gly Gln Gly Ile Ala Ala His Ser Lys Ser 1065 1070 1075CTC AAC TTC GTC CTC GCC GCT TGG ATA CGC ATA CTC GAG GAG ATA CTC 3318Leu Asn Phe Val Leu Ala Ala Trp Ile Arg Ile Leu Glu Glu Ile Leu 1080 1085 1090CGT ACC GGG AGC CGC ACG GTC CGG TAC AGC AAC GGT CTC CCC GAC GAA 3366Arg Thr Gly Ser Arg Thr Val Arg Tyr Ser Asn Gly Leu Pro Asp Glu1095 1100 1105 1110GAA GAG GCC ATG CTG CTC GAA GCG AAG ATC AAT CAA GTC CCA CAC GCC 3414Glu Glu Ala Met Leu Leu Glu Ala Lys Ile Asn Gln Val Pro His Ala 1115 1120 1125ACG TTC GTC TCG GCG GAC TGG ACC GAG TTT GAC ACC GCC CAC AAT AAC 3462Thr Phe Val Ser Ala Asp Trp Thr Glu Phe Asp Thr Ala His Asn Asn 1130 1135 1140ACG AGT GAG CTG CTC TTC GCC GCC CTT TTA GAG CGC ATC GGC ACG CCT 3510Thr Ser Glu Leu Leu Phe Ala Ala Leu Leu Glu Arg Ile Gly Thr Pro 1145 1150 1155GCA GCT GCC GTT AAT CTA TTC AGA GAA CGG TGT GGG AAA CGC ACC TTG 3558Ala Ala Ala Val Asn Leu Phe Arg Glu Arg Cys Gly Lys Arg Thr Leu 1160 1165 1170CGA GCG AAG GGT CTA GGC TCC GTT GAA GTC GAC GGT CTG CTC GAC TCC 3606Arg Ala Lys Gly Leu Gly Ser Val Glu Val Asp Gly Leu Leu Asp Ser1175 1180 1185 1190GGC GCA GCT TGG ACG CCT TGC CGC AAC ACC ATC TTC TCT GCC GCC GTC 3654Gly Ala Ala Trp Thr Pro Cys Arg Asn Thr Ile Phe Ser Ala Ala Val 1195 1200 1205ATG CTC ACG CTC TTC CGC GGC GTC AAG TTC GCA GCT TTC AAA GGC GAC 3702Met Leu Thr Leu Phe Arg Gly Val Lys Phe Ala Ala Phe Lys Gly Asp 1210 1215 1220GAC TCG CTC CTC TGT GGT AGC CAT TAC CTC CGT TTC GAC GCT AGC CGC 3750Asp Ser Leu Leu Cys Gly Ser His Tyr Leu Arg Phe Asp Ala Ser Arg 1225 1230 1235CTT CAC ATG GGC GAA CGT TAC AAG ACC AAA CAT TTG AAG GTC GAG GTG 3798Leu His Met Gly Glu Arg Tyr Lys Thr Lys His Leu Lys Val Glu Val 1240 1245 1250CAG AAA ATC GTG CCG TAC ATC GGA CTC CTC GTC TCC GCT GAG CAG GTC 3846Gln Lys Ile Val Pro Tyr Ile Gly Leu Leu Val Ser Ala Glu Gln Val1255 1260 1265 1270GTC CTC GAC CCT GTC AGG AGC GCT CTC AAG ATA TTT GGG CGC TGC TAC 3894Val Leu Asp Pro Val Arg Ser Ala Leu Lys Ile Phe Gly Arg Cys Tyr 1275 1280 1285ACA AGC GAA CTC CTT TAC TCC AAG TAC GTG GAG GCT GTG AGA GAC ATC 3942Thr Ser Glu Leu Leu Tyr Ser Lys Tyr Val Glu Ala Val Arg Asp Ile 1290 1295 1300ACC AAG GGC TGG AGT GAC GCC CGC TAC CAC AGC CTC CTG TGC CAC ATG 3990Thr Lys Gly Trp Ser Asp Ala Arg Tyr His Ser Leu Leu Cys His Met 1305 1310 1315TCA GCA TGC TAC TAC AAT TAC GCG CCG GAG TCT GCG GCG TAC ATC ATC 4038Ser Ala Cys Tyr Tyr Asn Tyr Ala Pro Glu Ser Ala Ala Tyr Ile Ile 1320 1325 1330GAC GCT GTT GTT CGC TTT GGG CGC GGC GAC TTC CCG TTT GAA CAA CTG 4086Asp Ala Val Val Arg Phe Gly Arg Gly Asp Phe Pro Phe Glu Gln Leu1335 1340 1345 1350CGC GTG GTG CGT GCC CAT GTG CAG GCA CCC GAC GCT TAC AGC AGC ACG 4134Arg Val Val Arg Ala His Val Gln Ala Pro Asp Ala Tyr Ser Ser Thr 1355 1360 1365TAT CCG GCT AAC GTG CGC GCA TCG TGC CTT GAC CAC GTC TTC GAG CCC 4182Tyr Pro Ala Asn Val Arg Ala Ser Cys Leu Asp His Val Phe Glu Pro 1370 1375 1380CGC CAG GCC GCC GCC CCG GCA GGT TTC GTT GCG ACA TGT GCG AAG CCG 4230Arg Gln Ala Ala Ala Pro Ala Gly Phe Val Ala Thr Cys Ala Lys Pro 1385 1390 1395GAA ACG CCT TCT TCA CTT ACC GCG AAA GCT GGT GTT TCT GCG ACT ACA 4278Glu Thr Pro Ser Ser Leu Thr Ala Lys Ala Gly Val Ser Ala Thr Thr 1400 1405 1410AGC CAC GTT GCG ACT GGG ACT GCG CCC CCG GAG TCT CCA TGG GAT GCA 4326Ser His Val Ala Thr Gly Thr Ala Pro Pro Glu Ser Pro Trp Asp Ala1415 1420 1425 1430CCT GCA GCC AAC AGC TTT TCG GAG TTA TTG ACA CCG GAG ACC CCG TCC 4374Pro Ala Ala Asn Ser Phe Ser Glu Leu Leu Thr Pro Glu Thr Pro Ser 1435 1440 1445ACA TCA TCC TCG CCG TCA TCG TCT TCA TCG GAC TCC TCT ACA TCG TGT 4422Thr Ser Ser Ser Pro Ser Ser Ser Ser Ser Asp Ser Ser Thr Ser Cys 1450 1455 1460GGA AGG TCG CTC AGT GGT GGA GAC ACC GCA AGG ACC ACA GAA GAC TTG 4470Gly Arg Ser Leu Ser Gly Gly Asp Thr Ala Arg Thr Thr Glu Asp Leu 1465 1470 1475AAC AGC AGA AAG CCG CCT TCG CAA GAC AGG CAA TCA CGC TCG TCT GAA 4518Asn Ser Arg Lys Pro Pro Ser Gln Asp Arg Gln Ser Arg Ser Ser Glu 1480 1485 1490TGT CTG GAC AGA AGC GGA GAA AGG ACA GGC AGT TCG TTA ACT GCC CCC 4566Cys Leu Asp Arg Ser Gly Glu Arg Thr Gly Ser Ser Leu Thr Ala Pro1495 1500 1505 1510ACT GCT CCG AGC CCC TCA TTC TCA TTT TCG GAA AGA GCT CGA CTG GCG 4614Thr Ala Pro Ser Pro Ser Phe Ser Phe Ser Glu Arg Ala Arg Leu Ala 1515 1520 1525ACC GGG CCG ACT GTC GCC GCT GCG ACA TCA CCT TCG GCA ACC CCA TCC 4662Thr Gly Pro Thr Val Ala Ala Ala Thr Ser Pro Ser Ala Thr Pro Ser 1530 1535 1540TGC GCC ACG GAC CAG GTT GCC GCG AGG ACC ACG CCG GAC TTT GCG CCT 4710Cys Ala Thr Asp Gln Val Ala Ala Arg Thr Thr Pro Asp Phe Ala Pro 1545 1550 1555TTC CTG GGT TCC CAG TCT GCC CGT GCT GTC TCG AAG CCG TAC CGG CCC 4758Phe Leu Gly Ser Gln Ser Ala Arg Ala Val Ser Lys Pro Tyr Arg Pro 1560 1565 1570CCC ACG ACT GCC CGT TGG AAA GAA GTC ACC CCG CTC CAC GCG TGG AAG 4806Pro Thr Thr Ala Arg Trp Lys Glu Val Thr Pro Leu His Ala Trp Lys1575 1580 1585 1590GGC GTG ACC GGA GAC CGA CCG GAA GTC AGG GAG GAC CCG GAG ACA GCG 4854Gly Val Thr Gly Asp Arg Pro Glu Val Arg Glu Asp Pro Glu Thr Ala 1595 1600 1605GCG GTC GTC CAG GCT CTG ATC AGC GGC CGT TAT CCT CAG AAG ACG AAG 4902Ala Val Val Gln Ala Leu Ile Ser Gly Arg Tyr Pro Gln Lys Thr Lys 1610 1615 1620CTT TCC TCC GAC GCA TCC AAA GGC TAC TCA AGA ACT AAG GGA TGC TCA 4950Leu Ser Ser Asp Ala Ser Lys Gly Tyr Ser Arg Thr Lys Gly Cys Ser 1625 1630 1635CAA TCC ACC TCT TTT CCT GCC CCG AGT GCG GAT TAC CAG GCC CGC GAC 4998Gln Ser Thr Ser Phe Pro Ala Pro Ser Ala Asp Tyr Gln Ala Arg Asp 1640 1645 1650TGC CAG ACA GTC CGA GTC TGC CGC GCC GCT GCA GAG ATG GCG CGC TCA 5046Cys Gln Thr Val Arg Val Cys Arg Ala Ala Ala Glu Met Ala Arg Ser1655 1660 1665 1670TGT ATT CAC GAG CCG TTG GCT TCA TCT GCC GCC AGT GCC GAC TTG AAG 5094Cys Ile His Glu Pro Leu Ala Ser Ser Ala Ala Ser Ala Asp Leu Lys 1675 1680 1685CGC ATA CGC TCT ACC TCG GAC TCT GTT CCC GAT GTA AAG ATC AGC AAG 5142Arg Ile Arg Ser Thr Ser Asp Ser Val Pro Asp Val Lys Ile Ser Lys 1690 1695 1700AGC GCA TGAAGGAACA AAATTAGTTT CCTTGTTCGT AAACAAGGTG GTCCCTCCCA 5198Ser AlaTTGAGGTAAA GACTCTGGTG AGTCCTCAAC GTTACTCGTT GAGTCTGCTG CGGTTCGA 5258CCATTCCCAA GCAGCAAAGG GTGCGCAACT AGTACGGCGC CCCCTGGGAT ACCA 5312 1704 amino acids amino acid linear protein unknown 40Met Tyr Ala Lys Ala Thr Asp Val Ala Arg Val Tyr Ala Ala Ala Asp 1 5 10 15Val Ala Tyr Ala Asn Val Leu Gln Gln Arg Ala Val Lys Leu Asp Phe 20 25 30Ala Pro Pro Leu Lys Ala Leu Glu Thr Leu His Arg Leu Tyr Tyr Pro 35 40 45Leu Arg Phe Lys Gly Gly Thr Leu Pro Pro Thr Gln His Pro Ile Leu 50 55 60Ala Gly His Gln Arg Val Ala Glu Glu Val Leu His Asn Phe Ala Arg 65 70 75 80Gly Arg Ser Thr Val Leu Glu Ile Gly Pro Ser Leu His Ser Ala Leu 85 90 95Lys Leu His Gly Ala Pro Asn Ala Pro Val Ala Asp Tyr His Gly Cys 100 105 110Thr Lys Tyr Gly Thr Arg Asp Gly Ser Arg His Ile Thr Ala Leu Glu 115 120 125Ser Arg Ser Val Ala Thr Gly Arg Pro Glu Phe Lys Ala Asp Ala Ser 130 135 140Leu Leu Ala Asn Gly Ile Ala Ser Arg Thr Phe Cys Val Asp Gly Val145 150 155 160Gly Ser Cys Ala Phe Lys Ser Arg Val Gly Ile Ala Asn His Ser Leu 165 170 175Tyr Asp Val Thr Leu Glu Glu Leu Ala Asn Ala Phe Glu Asn His Gly 180 185 190Leu His Met Val Arg Ala Phe Met His Met Pro Glu Glu Leu Leu Tyr 195 200 205Met Asp Asn Val Val Asn Ala Glu Leu Gly Tyr Arg Phe His Val Ile 210 215 220Glu Glu Pro Met Ala Val Lys Asp Cys Ala Phe Gln Gly Gly Asp Leu225 230 235 240Arg Leu His Phe Pro Glu Leu Asp Phe Ile Asn Glu Ser Gln Glu Arg 245 250 255Arg Ile Glu Arg Leu Ala Ala Arg Gly Ser Tyr Ser Arg Arg Ala Val 260 265 270Ile Phe Ser Gly Asp Asp Asp Trp Gly Asp Ala Tyr Leu His Asp Phe 275 280 285His Thr Trp Leu Ala Tyr Leu Leu Val Arg Asn Tyr Pro Thr Pro Phe 290 295 300Gly Phe Ser Leu His Ile Glu Val Gln Arg Arg His Gly Ser Ser Ile305 310 315 320Glu Leu Arg Ile Thr Arg Ala Pro Pro Gly Asp Arg Met Leu Ala Val 325 330 335Val Pro Arg Thr Ser Gln Gly Leu Cys Arg Ile Pro Asn Ile Phe Tyr 340 345 350Tyr Ala Asp Ala Ser Gly Thr Glu His Lys Thr Ile Leu Thr Ser Gln 355 360 365His Lys Val Asn Met Leu Leu Asn Phe Met Gln Thr Arg Pro Glu Lys 370 375 380Glu Leu Val Asp Met Thr Val Leu Met Ser Phe Ala Arg Ala Arg Leu385 390 395 400Arg Ala Ile Val Val Ala Ser Glu Val Thr Glu Ser Ser Trp Asn Ile 405 410 415Ser Pro Ala Asp Leu Val Arg Thr Val Val Ser Leu Tyr Val Leu His 420 425 430Ile Ile Glu Arg Arg Arg Ala Ala Val Ala Val Lys Thr Ala Lys Asp 435 440 445Asp Val Phe Gly Glu Thr Ser Phe Trp Glu Ser Leu Lys His Val Leu 450 455 460Gly Ser Cys Cys Gly Leu Arg Asn Leu Lys Gly Thr Asp Val Val Phe465 470 475 480Thr Lys Arg Val Val Asp Lys Tyr Arg Val His Ser Leu Gly Asp Ile 485 490 495Ile Cys Asp Val Arg Leu Ser Pro Glu Gln Val Gly Phe Leu Pro Ser 500 505 510Arg Val Pro Pro Ala Arg Val Phe His Asp Arg Glu Glu Leu Glu Val 515 520 525Leu Arg Glu Ala Gly Cys Tyr Asn Glu Arg Pro Val Pro Ser Thr Pro 530 535 540Pro Val Glu Glu Pro Gln Gly Phe Asp Ala Asp Leu Trp His Ala Thr545 550 555 560Ala Ala Ser Leu Pro Glu Tyr Arg Ala Thr Leu Gln Ala Gly Leu Asn 565 570 575Thr Asp Val Lys Gln Leu Lys Ile Thr Leu Glu Asn Ala Leu Lys Thr 580 585 590Ile Asp Gly Leu Thr Leu Ser Pro Val Arg Gly Leu Glu Met Tyr Glu 595 600 605Gly Pro Pro Gly Ser Gly Lys Thr Gly Thr Leu Ile Ala Ala Leu Glu 610 615 620Ala Ala Gly Gly Lys Ala Leu Tyr Val Ala Pro Thr Arg Glu Leu Arg625 630 635 640Glu Ala Met Asp Arg Arg Ile Lys Pro Pro Ser Ala Ser Ala Thr Gln 645 650 655His Val Ala Leu Ala Ile Leu Arg Arg Ala Thr Ala Glu Gly Ala Pro 660 665 670Phe Ala Thr Val Val Ile Asp Glu Cys Phe Met Phe Pro Leu Val Tyr 675 680 685Val Ala Ile Val His Ala Leu Ser Pro Ser Ser Arg Ile Val Leu Val 690 695 700Gly Asp Val His Gln Ile Gly Phe Ile Asp Phe Gln Gly Thr Ser Ala705 710 715 720Asn Met Pro Leu Val Arg Asp Val Val Lys Gln Cys Arg Arg Arg Thr 725 730 735Phe Asn Gln Thr Lys Arg Cys Pro Ala Asp Val Val Ala Thr Thr Phe 740 745 750Phe Gln Ser Leu Tyr Pro Gly Cys Thr Thr Thr Ser Gly Cys Val Ala 755 760 765Ser Ile Ser His Val Ala Pro Asp Tyr Arg Asn Ser Gln Ala Gln Thr 770 775 780Leu Cys Phe Thr Gln Glu Glu Lys Ser Arg His Gly Ala Glu Gly Ala785 790 795 800Met Thr Val His Glu Ala Gln Gly Arg Thr Phe Ala Ser Val Ile Leu 805 810 815His Tyr Asn Gly Ser Thr Ala Glu Gln Lys Leu Leu Ala Glu Lys Ser 820 825 830His Leu Leu Val Gly Ile Thr Arg His Thr Asn His Leu Tyr Ile Arg 835 840 845Asp Pro Thr Gly Asp Ile Glu Arg Gln Leu Asn His Ser Ala Lys Ala 850 855 860Glu Val Phe Thr Asp Ile Pro Ala Pro Leu Glu Ile Thr Thr Val Lys865 870 875 880Pro Ser Glu Glu Val Gln Arg Asn Glu Val Met Ala Thr Ile Pro Pro 885 890 895Gln Ser Ala Thr Pro His Gly Ala Ile His Leu Leu Arg Lys Asn Phe 900 905 910Gly Asp Gln Pro Asp Cys Gly Cys Val Ala Leu Ala Lys Thr Gly Tyr 915 920 925Glu Val Phe Gly Gly Arg Ala Lys Ile Asn Val Glu Leu Ala Glu Pro 930 935 940Asp Ala Thr Pro Lys Pro His Arg Ala Phe Gln Glu Gly Val Gln Trp945 950 955 960Val Lys Val Thr Asn Ala Ser Asn Lys His Gln Ala Leu Gln Thr Leu 965 970 975Leu Ser Arg Tyr Thr Lys Arg Ser Ala Asp Leu Pro Leu His Glu Ala 980 985 990Lys Glu Asp Val Lys Arg Met Leu Asn Ser Leu Asp Arg His Trp Asp 995 1000 1005Trp Thr Val Thr Glu Asp Ala Arg Asp Arg Ala Val Phe Glu Thr Gln 1010 1015 1020Leu Lys Phe Thr Gln Arg Gly Gly Thr Val Glu Asp Leu Leu Glu Pro1025 1030 1035 1040Asp Asp Pro Tyr Ile Arg Asp Ile Asp Phe Leu Met Lys Thr Gln Gln 1045 1050 1055Lys Val Ser Pro Lys Pro Ile Asn Thr Gly Lys Val Gly Gln Gly Ile 1060 1065 1070Ala Ala His Ser Lys Ser Leu Asn Phe Val Leu Ala Ala Trp Ile Arg 1075 1080 1085Ile Leu Glu Glu Ile Leu Arg Thr Gly Ser Arg Thr Val Arg Tyr Ser 1090 1095 1100Asn Gly Leu Pro Asp Glu Glu Glu Ala Met Leu Leu Glu Ala Lys Ile1105 1110 1115 1120Asn Gln Val Pro His Ala Thr Phe Val Ser Ala Asp Trp Thr Glu Phe 1125 1130 1135Asp Thr Ala His Asn Asn Thr Ser Glu Leu Leu Phe Ala Ala Leu Leu 1140 1145 1150Glu Arg Ile Gly Thr Pro Ala Ala Ala Val Asn Leu Phe Arg Glu Arg 1155 1160 1165Cys Gly Lys Arg Thr Leu Arg Ala Lys Gly Leu Gly Ser Val Glu Val 1170 1175 1180Asp Gly Leu Leu Asp Ser Gly Ala Ala Trp Thr Pro Cys Arg Asn Thr1185 1190 1195 1200Ile Phe Ser Ala Ala Val Met Leu Thr Leu Phe Arg Gly Val Lys Phe 1205 1210 1215Ala Ala Phe Lys Gly Asp Asp Ser Leu Leu Cys Gly Ser His Tyr Leu 1220 1225 1230Arg Phe Asp Ala Ser Arg Leu His Met Gly Glu Arg Tyr Lys Thr Lys 1235 1240 1245His Leu Lys Val Glu Val Gln Lys Ile Val Pro Tyr Ile Gly Leu Leu 1250 1255 1260Val Ser Ala Glu Gln Val Val Leu Asp Pro Val Arg Ser Ala Leu Lys1265 1270 1275 1280Ile Phe Gly Arg Cys Tyr Thr Ser Glu Leu Leu Tyr Ser Lys Tyr Val 1285 1290 1295Glu Ala Val Arg Asp Ile Thr Lys Gly Trp Ser Asp Ala Arg Tyr His 1300 1305 1310Ser Leu Leu Cys His Met Ser Ala Cys Tyr Tyr Asn Tyr Ala Pro Glu 1315 1320 1325Ser Ala Ala Tyr Ile Ile Asp Ala Val Val Arg Phe Gly Arg Gly Asp 1330 1335 1340Phe Pro Phe Glu Gln Leu Arg Val Val Arg Ala His Val Gln Ala Pro1345 1350 1355 1360Asp Ala Tyr Ser Ser Thr Tyr Pro Ala Asn Val Arg Ala Ser Cys Leu 1365 1370 1375Asp His Val Phe Glu Pro Arg Gln Ala Ala Ala Pro Ala Gly Phe Val 1380 1385 1390Ala Thr Cys Ala Lys Pro Glu Thr Pro Ser Ser Leu Thr Ala Lys Ala 1395 1400 1405Gly Val Ser Ala Thr Thr Ser His Val Ala Thr Gly Thr Ala Pro Pro 1410 1415 1420Glu Ser Pro Trp Asp Ala Pro Ala Ala Asn Ser Phe Ser Glu Leu Leu1425 1430 1435 1440Thr Pro Glu Thr Pro Ser Thr Ser Ser Ser Pro Ser Ser Ser Ser Ser 1445 1450 1455Asp Ser Ser Thr Ser Cys Gly Arg Ser Leu Ser Gly Gly Asp Thr Ala 1460 1465 1470Arg Thr Thr Glu Asp Leu Asn Ser Arg Lys Pro Pro Ser Gln Asp Arg 1475 1480 1485Gln Ser Arg Ser Ser Glu Cys Leu Asp Arg Ser Gly Glu Arg Thr Gly 1490 1495 1500Ser Ser Leu Thr Ala Pro Thr Ala Pro Ser Pro Ser Phe Ser Phe Ser1505 1510 1515 1520Glu Arg Ala Arg Leu Ala Thr Gly Pro Thr Val Ala Ala Ala Thr Ser 1525 1530 1535Pro Ser Ala Thr Pro Ser Cys Ala Thr Asp Gln Val Ala Ala Arg Thr 1540 1545 1550Thr Pro Asp Phe Ala Pro Phe Leu Gly Ser Gln Ser Ala Arg Ala Val 1555 1560 1565Ser Lys Pro Tyr Arg Pro Pro Thr Thr Ala Arg Trp Lys Glu Val Thr 1570 1575 1580Pro Leu His Ala Trp Lys Gly Val Thr Gly Asp Arg Pro Glu Val Arg1585 1590 1595 1600Glu Asp Pro Glu Thr Ala Ala Val Val Gln Ala Leu Ile Ser Gly Arg 1605 1610 1615Tyr Pro Gln Lys Thr Lys Leu Ser Ser Asp Ala Ser Lys Gly Tyr Ser 1620 1625 1630Arg Thr Lys Gly Cys Ser Gln Ser Thr Ser Phe Pro Ala Pro Ser Ala 1635 1640 1645Asp Tyr Gln Ala Arg Asp Cys Gln Thr Val Arg Val Cys Arg Ala Ala 1650 1655 1660Ala Glu Met Ala Arg Ser Cys Ile His Glu Pro Leu Ala Ser Ser Ala1665 1670 1675 1680Ala Ser Ala Asp Leu Lys Arg Ile Arg Ser Thr Ser Asp Ser Val Pro 1685 1690 1695Asp Val Lys Ile Ser Lys Ser Ala 1700 5312 base pairs nucleic acid unknown unknown DNA unknown CDS 4218..4514 41GTTCTGCCTC CCCCGGACGG TAAATATAGG GGAACAATGT ACGCGAAAGC GACAGACGTG 60GCGCGTGTCT ACGCCGCGGC AGATGTCGCC TACGCGAACG TACTGCAGCA GAGAGCAGT 120AAGTTGGACT TCGCCCCGCC ACTGAAGGCA CTAGAAACCC TCCACAGACT GTACTATCC 180CTGCGCTTCA AAGGGGGCAC TTTACCCCCG ACACAACACC CGATCCTGGC CGGGCACCA 240CGTGTCGCAG AAGAGGTTCT GCACAATTTC GCCAGGGGAC GTAGCACAGT GCTCGAGAT 300GGGCCGTCTC TGCACAGCGC ACTTAAGCTA CATGGGGCAC CGAACGCCCC CGTCGCAGA 360TATCACGGGT GCACCAAGTA CGGCACCCGC GACGGCTCGC GACACATTAC GGCCTTAGA 420TCTAGATCCG TCGCCACAGG CCGGCCCGAG TTCAAGGCCG ACGCCTCACT GCTCGCCAA 480GGCATTGCCT CCCGCACCTT CTGCGTCGAC GGAGTCGGCT CTTGCGCGTT CAAATCGCG 540GTTGGAATTG CCAATCACTC CCTCTATGAC GTGACCCTAG AGGAGCTGGC CAATGCGTT 600GAGAACCACG GACTTCACAT GGTCCGCGCG TTCATGCACA TGCCAGAAGA GCTGCTCTA 660ATGGACAACG TGGTTAATGC CGAGCTCGGC TACCGCTTCC ACGTTATTGA AGAGCCTAT 720GCTGTGAAGG ACTGCGCATT CCAGGGGGGG GACCTCCGTC TCCACTTCCC TGAGTTGGA 780TTCATCAACG AGAGCCAAGA GCGGCGCATC GAGAGGCTGG CCGCCCGCGG CTCCTACTC 840AGACGCGCCG TCATTTTCTC CGGCGACGAC GACTGGGGTG ATGCGTACTT ACACGACTT 900CACACATGGC TCGCCTACCT ACTGGTGAGG AACTACCCCA CTCCGTTTGG TTTCTCACT 960CATATAGAAG TCCAGAGGCG CCACGGCTCC AGCATTGAGC TGCGCATCAC TCGCGCGC 1020CCTGGAGACC GCATGCTGGC CGTCGTCCCA AGGACGTCCC AAGGCCTCTG CAGAATCC 1080AACATCTTTT ATTACGCCGA CGCGTCGGGC ACTGAGCATA AGACCATCCT TACGTCAC 1140CACAAAGTCA ACATGCTGCT CAATTTTATG CAAACGCGTC CTGAGAAGGA ACTAGTCG 1200ATGACCGTCT TGATGTCGTT CGCGCGCGCT AGGCTGCGCG CGATCGTGGT CGCCTCAG 1260GTCACCGAGA GCTCCTGGAA CATCTCACCG GCTGACCTGG TCCGCACTGT CGTGTCTC 1320TACGTCCTCC ACATCATCGA GCGCCGAAGG GCTGCGGTCG CTGTCAAGAC CGCCAAGG 1380GACGTCTTTG GAGAGACTTC GTTCTGGGAG AGTCTCAAGC ACGTCTTGGG CTCCTGTT 1440GGTCTGCGCA ACCTCAAAGG CACCGACGTC GTCTTTACTA AGCGCGTCGT CGATAAGT 1500CGAGTCCACT CGCTCGGAGA CATAATCTGC GACGTCCGCC TGTCCCCTGA ACAGGTCG 1560TTCCTGCCGT CCCGCGTACC ACCTGCCCGC GTCTTTCACG ACAGGGAAGA GCTTGAGG 1620CTTCGCGAAG CTGGCTGCTA CAACGAACGT CCGGTACCTT CCACTCCTCC TGTGGAGG 1680CCCCAAGGTT TCGACGCCGA CTTGTGGCAC GCGACCGCGG CCTCACTCCC CGAGTACC 1740GCCACCTTGC AGGCAGGTCT CAACACCGAC GTCAAGCAGC TCAAGATCAC CCTCGAGA 1800GCCCTCAAGA CCATCGACGG GCTCACCCTC TCCCCAGTCA GAGGCCTCGA GATGTACG 1860GGCCCGCCAG GCAGCGGCAA GACGGGCACC CTCATCGCCG CCCTTGAGGC CGCGGGCG 1920AAAGCACTTT ACGTGGCACC CACCAGAGAA CTGAGAGAGG CTATGGACCG GCGGATCA 1980CCGCCGTCCG CCTCGGCTAC GCAACATGTC GCCCTTGCGA TTCTCCGTCG TGCCACCG 2040GAGGGCGCCC CTTTCGCTAC CGTGGTTATC GACGAGTGCT TCATGTTCCC GCTCGTGT 2100GTCGCGATCG TGCACGCCTT GTCCCCGAGC TCACGAATAG TCCTTGTAGG GGACGTCC 2160CAAATCGGGT TTATAGACTT CCAAGGCACA AGCGCGAACA TGCCGCTCGT TCGCGACG 2220GTTAAGCAGT GCCGTCGGCG CACTTTCAAC CAAACCAAGC GCTGTCCGGC CGACGTCG 2280GCCACCACGT TTTTCCAGAG CTTGTACCCC GGGTGCACAA CCACCTCAGG GTGCGTCG 2340TCCATCAGCC ACGTCGCCCC AGACTACCGC AACAGCCAGG CGCAAACGCT CTGCTTCA 2400CAGGAGGAAA AGTCGCGCCA CGGGGCTGAG GGCGCGATGA CTGTGCACGA AGCGCAGG 2460CGCACTTTTG CGTCTGTCAT TCTGCATTAC AACGGCTCCA CAGCAGAGCA GAAGCTCC 2520GCTGAGAAGT CGCACCTTCT AGTCGGCATC ACGCGCCACA CCAACCACCT GTACATCC 2580GACCCGACAG GTGACATTGA GAGACAACTC AACCATAGCG CGAAAGCCGA GGTGTTTA 2640GACATCCCTG CACCCCTGGA GATCACGACT GTCAAACCGA GTGAAGAGGT GCAGCGCA 2700GAAGTGATGG CAACGATACC CCCGCAGAGT GCCACGCCGC ACGGAGCAAT CCATCTGC 2760CGCAAGAACT TCGGGGACCA ACCCGACTGT GGCTGTGTCG CTTTGGCGAA GACCGGCT 2820GAGGTGTTTG GCGGTCGTGC CAAAATCAAC GTAGAGCTTG CCGAACCCGA CGCGACCC 2880AAGCCGCATA GGGCGTTCCA GGAAGGGGTA CAGTGGGTCA AGGTCACCAA CGCGTCTA 2940AAACACCAGG CGCTCCAGAC GCTGTTGTCC CGCTACACCA AGCGAAGCGC TGACCTGC 3000CTACACGAAG CTAAGGAGGA CGTCAAACGC ATGCTAAACT CGCTTGACCG ACATTGGG 3060TGGACTGTCA CTGAAGACGC CCGTGACCGA GCTGTCTTCG AGACCCAGCT CAAGTTCA 3120CAACGCGGCG GCACCGTCGA AGACCTGCTG GAGCCAGACG ACCCCTACAT CCGTGACA 3180GACTTCCTTA TGAAGACTCA GCAGAAAGTG TCGCCCAAGC CGATCAATAC GGGCAAGG 3240GGGCAGGGGA TCGCCGCTCA CTCAAAGTCT CTCAACTTCG TCCTCGCCGC TTGGATAC 3300ATACTCGAGG AGATACTCCG TACCGGGAGC CGCACGGTCC GGTACAGCAA CGGTCTCC 3360GACGAAGAAG AGGCCATGCT GCTCGAAGCG AAGATCAATC AAGTCCCACA CGCCACGT 3420GTCTCGGCGG ACTGGACCGA GTTTGACACC GCCCACAATA ACACGAGTGA GCTGCTCT 3480GCCGCCCTTT TAGAGCGCAT CGGCACGCCT GCAGCTGCCG TTAATCTATT CAGAGAAC 3540TGTGGGAAAC GCACCTTGCG AGCGAAGGGT CTAGGCTCCG TTGAAGTCGA CGGTCTGC 3600GACTCCGGCG CAGCTTGGAC GCCTTGCCGC AACACCATCT TCTCTGCCGC CGTCATGC 3660ACGCTCTTCC GCGGCGTCAA GTTCGCAGCT TTCAAAGGCG ACGACTCGCT CCTCTGTG 3720AGCCATTACC TCCGTTTCGA CGCTAGCCGC CTTCACATGG GCGAACGTTA CAAGACCA 3780CATTTGAAGG TCGAGGTGCA GAAAATCGTG CCGTACATCG GACTCCTCGT CTCCGCTG 3840CAGGTCGTCC TCGACCCTGT CAGGAGCGCT CTCAAGATAT TTGGGCGCTG CTACACAA 3900GAACTCCTTT ACTCCAAGTA CGTGGAGGCT GTGAGAGACA TCACCAAGGG CTGGAGTG 3960GCCCGCTACC ACAGCCTCCT GTGCCACATG TCAGCATGCT ACTACAATTA CGCGCCGG 4020TCTGCGGCGT ACATCATCGA CGCTGTTGTT CGCTTTGGGC GCGGCGACTT CCCGTTTG 4080CAACTGCGCG TGGTGCGTGC CCATGTGCAG GCACCCGACG CTTACAGCAG CACGTATC 4140GCTAACGTGC GCGCATCGTG CCTTGACCAC GTCTTCGAGC CCCGCCAGGC CGCCGCCC 4200GCAGGTTTCG TTGCGAC ATG TGC GAA GCC GGA AAC GCC TTC TTC ACT TAC 4250 Met Cys Glu Ala Gly Asn Ala Phe Phe Thr Tyr 1 5 10CGC GAA AGC TGG TGT TTC TGC GAC TAC AAG CCA CGT TGC GAC TGG GAC 4298Arg Glu Ser Trp Cys Phe Cys Asp Tyr Lys Pro Arg Cys Asp Trp Asp 15 20 25TGC GCC CCC GGA GTC TCC ATG GGA TGC ACC TGC AGC CAA CAG CTT TTC 4346Cys Ala Pro Gly Val Ser Met Gly Cys Thr Cys Ser Gln Gln Leu Phe 30 35 40GGA GTT ATT GAC ACC GGA GAC CCC GTC CAC ATC ATC CTC GCC GTC ATC 4394Gly Val Ile Asp Thr Gly Asp Pro Val His Ile Ile Leu Ala Val Ile 45 50 55GTC TTC ATC GGA CTC CTC TAC ATC GTG TGG AAG GTC GCT CAG TGG TGG 4442Val Phe Ile Gly Leu Leu Tyr Ile Val Trp Lys Val Ala Gln Trp Trp 60 65 70 75AGA CAC CGC AAG GAC CAC AGA AGA CTT GAA CAG CAG AAA GCC GCC TTC 4490Arg His Arg Lys Asp His Arg Arg Leu Glu Gln Gln Lys Ala Ala Phe 80 85 90GCA AGA CAG GCA ATC ACG CTC GTC TGAATGTCTG GACAGAAGCG GAGAAAGGA 4544Ala Arg Gln Ala Ile Thr Leu Val 95AGGCAGTTCG TTAACTGCCC CCACTGCTCC GAGCCCCTCA TTCTCATTTT CGGAAAGA 4604TCGACTGGCG ACCGGGCCGA CTGTCGCCGC TGCGACATCA CCTTCGGCAA CCCCATCC 4664CGCCACGGAC CAGGTTGCCG CGAGGACCAC GCCGGACTTT GCGCCTTTCC TGGGTTCC 4724GTCTGCCCGT GCTGTCTCGA AGCCGTACCG GCCCCCCACG ACTGCCCGTT GGAAAGAA 4784CACCCCGCTC CACGCGTGGA AGGGCGTGAC CGGAGACCGA CCGGAAGTCA GGGAGGAC 4844GGAGACAGCG GCGGTCGTCC AGGCTCTGAT CAGCGGCCGT TATCCTCAGA AGACGAAG 4904TTCCTCCGAC GCATCCAAAG GCTACTCAAG AACTAAGGGA TGCTCACAAT CCACCTCT 4964TCCTGCCCCG AGTGCGGATT ACCAGGCCCG CGACTGCCAG ACAGTCCGAG TCTGCCGC 5024CGCTGCAGAG ATGGCGCGCT CATGTATTCA CGAGCCGTTG GCTTCATCTG CCGCCAGT 5084CGACTTGAAG CGCATACGCT CTACCTCGGA CTCTGTTCCC GATGTAAAGA TCAGCAAG 5144CGCATGAAGG AACAAAATTA GTTTCCTTGT TCGTAAACAA GGTGGTCCCT CCCATTGA 5204TAAAGACTCT GGTGAGTCCT CAACGTTACT CGTTGAGTCT GCTGCGGTTC GATTCCAT 5264CCAAGCAGCA AAGGGTGCGC AACTAGTACG GCGCCCCCTG GGATACCA 5312 99 amino acids amino acid linear protein unknown 42Met Cys Glu Ala Gly Asn Ala Phe Phe Thr Tyr Arg Glu Ser Trp Cys 1 5 10 15Phe Cys Asp Tyr Lys Pro Arg Cys Asp Trp Asp Cys Ala Pro Gly Val 20 25 30Ser Met Gly Cys Thr Cys Ser Gln Gln Leu Phe Gly Val Ile Asp Thr 35 40 45Gly Asp Pro Val His Ile Ile Leu Ala Val Ile Val Phe Ile Gly Leu 50 55 60Leu Tyr Ile Val Trp Lys Val Ala Gln Trp Trp Arg His Arg Lys Asp 65 70 75 80His Arg Arg Leu Glu Gln Gln Lys Ala Ala Phe Ala Arg Gln Ala Ile 85 90 95Thr Leu Val 5312 base pairs nucleic acid unknown unknown DNA unknown CDS 4518..4937 43GTTCTGCCTC CCCCGGACGG TAAATATAGG GGAACAATGT ACGCGAAAGC GACAGACGTG 60GCGCGTGTCT ACGCCGCGGC AGATGTCGCC TACGCGAACG TACTGCAGCA GAGAGCAGT 120AAGTTGGACT TCGCCCCGCC ACTGAAGGCA CTAGAAACCC TCCACAGACT GTACTATCC 180CTGCGCTTCA AAGGGGGCAC TTTACCCCCG ACACAACACC CGATCCTGGC CGGGCACCA 240CGTGTCGCAG AAGAGGTTCT GCACAATTTC GCCAGGGGAC GTAGCACAGT GCTCGAGAT 300GGGCCGTCTC TGCACAGCGC ACTTAAGCTA CATGGGGCAC CGAACGCCCC CGTCGCAGA 360TATCACGGGT GCACCAAGTA CGGCACCCGC GACGGCTCGC GACACATTAC GGCCTTAGA 420TCTAGATCCG TCGCCACAGG CCGGCCCGAG TTCAAGGCCG ACGCCTCACT GCTCGCCAA 480GGCATTGCCT CCCGCACCTT CTGCGTCGAC GGAGTCGGCT CTTGCGCGTT CAAATCGCG 540GTTGGAATTG CCAATCACTC CCTCTATGAC GTGACCCTAG AGGAGCTGGC CAATGCGTT 600GAGAACCACG GACTTCACAT GGTCCGCGCG TTCATGCACA TGCCAGAAGA GCTGCTCTA 660ATGGACAACG TGGTTAATGC CGAGCTCGGC TACCGCTTCC ACGTTATTGA AGAGCCTAT 720GCTGTGAAGG ACTGCGCATT CCAGGGGGGG GACCTCCGTC TCCACTTCCC TGAGTTGGA 780TTCATCAACG AGAGCCAAGA GCGGCGCATC GAGAGGCTGG CCGCCCGCGG CTCCTACTC 840AGACGCGCCG TCATTTTCTC CGGCGACGAC GACTGGGGTG ATGCGTACTT ACACGACTT 900CACACATGGC TCGCCTACCT ACTGGTGAGG AACTACCCCA CTCCGTTTGG TTTCTCACT 960CATATAGAAG TCCAGAGGCG CCACGGCTCC AGCATTGAGC TGCGCATCAC TCGCGCGC 1020CCTGGAGACC GCATGCTGGC CGTCGTCCCA AGGACGTCCC AAGGCCTCTG CAGAATCC 1080AACATCTTTT ATTACGCCGA CGCGTCGGGC ACTGAGCATA AGACCATCCT TACGTCAC 1140CACAAAGTCA ACATGCTGCT CAATTTTATG CAAACGCGTC CTGAGAAGGA ACTAGTCG 1200ATGACCGTCT TGATGTCGTT CGCGCGCGCT AGGCTGCGCG CGATCGTGGT CGCCTCAG 1260GTCACCGAGA GCTCCTGGAA CATCTCACCG GCTGACCTGG TCCGCACTGT CGTGTCTC 1320TACGTCCTCC ACATCATCGA GCGCCGAAGG GCTGCGGTCG CTGTCAAGAC CGCCAAGG 1380GACGTCTTTG GAGAGACTTC GTTCTGGGAG AGTCTCAAGC ACGTCTTGGG CTCCTGTT 1440GGTCTGCGCA ACCTCAAAGG CACCGACGTC GTCTTTACTA AGCGCGTCGT CGATAAGT 1500CGAGTCCACT CGCTCGGAGA CATAATCTGC GACGTCCGCC TGTCCCCTGA ACAGGTCG 1560TTCCTGCCGT CCCGCGTACC ACCTGCCCGC GTCTTTCACG ACAGGGAAGA GCTTGAGG 1620CTTCGCGAAG CTGGCTGCTA CAACGAACGT CCGGTACCTT CCACTCCTCC TGTGGAGG 1680CCCCAAGGTT TCGACGCCGA CTTGTGGCAC GCGACCGCGG CCTCACTCCC CGAGTACC 1740GCCACCTTGC AGGCAGGTCT CAACACCGAC GTCAAGCAGC TCAAGATCAC CCTCGAGA 1800GCCCTCAAGA CCATCGACGG GCTCACCCTC TCCCCAGTCA GAGGCCTCGA GATGTACG 1860GGCCCGCCAG GCAGCGGCAA GACGGGCACC CTCATCGCCG CCCTTGAGGC CGCGGGCG 1920AAAGCACTTT ACGTGGCACC CACCAGAGAA CTGAGAGAGG CTATGGACCG GCGGATCA 1980CCGCCGTCCG CCTCGGCTAC GCAACATGTC GCCCTTGCGA TTCTCCGTCG TGCCACCG 2040GAGGGCGCCC CTTTCGCTAC CGTGGTTATC GACGAGTGCT TCATGTTCCC GCTCGTGT 2100GTCGCGATCG TGCACGCCTT GTCCCCGAGC TCACGAATAG TCCTTGTAGG GGACGTCC 2160CAAATCGGGT TTATAGACTT CCAAGGCACA AGCGCGAACA TGCCGCTCGT TCGCGACG 2220GTTAAGCAGT GCCGTCGGCG CACTTTCAAC CAAACCAAGC GCTGTCCGGC CGACGTCG 2280GCCACCACGT TTTTCCAGAG CTTGTACCCC GGGTGCACAA CCACCTCAGG GTGCGTCG 2340TCCATCAGCC ACGTCGCCCC AGACTACCGC AACAGCCAGG CGCAAACGCT CTGCTTCA 2400CAGGAGGAAA AGTCGCGCCA CGGGGCTGAG GGCGCGATGA CTGTGCACGA AGCGCAGG 2460CGCACTTTTG CGTCTGTCAT TCTGCATTAC AACGGCTCCA CAGCAGAGCA GAAGCTCC 2520GCTGAGAAGT CGCACCTTCT AGTCGGCATC ACGCGCCACA CCAACCACCT GTACATCC 2580GACCCGACAG GTGACATTGA GAGACAACTC AACCATAGCG CGAAAGCCGA GGTGTTTA 2640GACATCCCTG CACCCCTGGA GATCACGACT GTCAAACCGA GTGAAGAGGT GCAGCGCA 2700GAAGTGATGG CAACGATACC CCCGCAGAGT GCCACGCCGC ACGGAGCAAT CCATCTGC 2760CGCAAGAACT TCGGGGACCA ACCCGACTGT GGCTGTGTCG CTTTGGCGAA GACCGGCT 2820GAGGTGTTTG GCGGTCGTGC CAAAATCAAC GTAGAGCTTG CCGAACCCGA CGCGACCC 2880AAGCCGCATA GGGCGTTCCA GGAAGGGGTA CAGTGGGTCA AGGTCACCAA CGCGTCTA 2940AAACACCAGG CGCTCCAGAC GCTGTTGTCC CGCTACACCA AGCGAAGCGC TGACCTGC 3000CTACACGAAG CTAAGGAGGA CGTCAAACGC ATGCTAAACT CGCTTGACCG ACATTGGG 3060TGGACTGTCA CTGAAGACGC CCGTGACCGA GCTGTCTTCG AGACCCAGCT CAAGTTCA 3120CAACGCGGCG GCACCGTCGA AGACCTGCTG GAGCCAGACG ACCCCTACAT CCGTGACA 3180GACTTCCTTA TGAAGACTCA GCAGAAAGTG TCGCCCAAGC CGATCAATAC GGGCAAGG 3240GGGCAGGGGA TCGCCGCTCA CTCAAAGTCT CTCAACTTCG TCCTCGCCGC TTGGATAC 3300ATACTCGAGG AGATACTCCG TACCGGGAGC CGCACGGTCC GGTACAGCAA CGGTCTCC 3360GACGAAGAAG AGGCCATGCT GCTCGAAGCG AAGATCAATC AAGTCCCACA CGCCACGT 3420GTCTCGGCGG ACTGGACCGA GTTTGACACC GCCCACAATA ACACGAGTGA GCTGCTCT 3480GCCGCCCTTT TAGAGCGCAT CGGCACGCCT GCAGCTGCCG TTAATCTATT CAGAGAAC 3540TGTGGGAAAC GCACCTTGCG AGCGAAGGGT CTAGGCTCCG TTGAAGTCGA CGGTCTGC 3600GACTCCGGCG CAGCTTGGAC GCCTTGCCGC AACACCATCT TCTCTGCCGC CGTCATGC 3660ACGCTCTTCC GCGGCGTCAA GTTCGCAGCT TTCAAAGGCG ACGACTCGCT CCTCTGTG 3720AGCCATTACC TCCGTTTCGA CGCTAGCCGC CTTCACATGG GCGAACGTTA CAAGACCA 3780CATTTGAAGG TCGAGGTGCA GAAAATCGTG CCGTACATCG GACTCCTCGT CTCCGCTG 3840CAGGTCGTCC TCGACCCTGT CAGGAGCGCT CTCAAGATAT TTGGGCGCTG CTACACAA 3900GAACTCCTTT ACTCCAAGTA CGTGGAGGCT GTGAGAGACA TCACCAAGGG CTGGAGTG 3960GCCCGCTACC ACAGCCTCCT GTGCCACATG TCAGCATGCT ACTACAATTA CGCGCCGG 4020TCTGCGGCGT ACATCATCGA CGCTGTTGTT CGCTTTGGGC GCGGCGACTT CCCGTTTG 4080CAACTGCGCG TGGTGCGTGC CCATGTGCAG GCACCCGACG CTTACAGCAG CACGTATC 4140GCTAACGTGC GCGCATCGTG CCTTGACCAC GTCTTCGAGC CCCGCCAGGC CGCCGCCC 4200GCAGGTTTCG TTGCGACATG TGCGAAGCCG GAAACGCCTT CTTCACTTAC CGCGAAAG 4260GGTGTTTCTG CGACTACAAG CCACGTTGCG ACTGGGACTG CGCCCCCGGA GTCTCCAT 4320GATGCACCTG CAGCCAACAG CTTTTCGGAG TTATTGACAC CGGAGACCCC GTCCACAT 4380TCCTCGCCGT CATCGTCTTC ATCGGACTCC TCTACATCGT GTGGAAGGTC GCTCAGTG 4440GGAGACACCG CAAGGACCAC AGAAGACTTG AACAGCAGAA AGCCGCCTTC GCAAGACA 4500CAATCACGCT CGTCTGA ATG TCT GGA CAG AAG CGG AGA AAG GAC AGG CAG 4550 Met Ser Gly Gln Lys Arg Arg Lys Asp Arg Gln 1 5 10TTC GTT AAC TGC CCC CAC TGC TCC GAG CCC CTC ATT CTC ATT TTC GGA 4598Phe Val Asn Cys Pro His Cys Ser Glu Pro Leu Ile Leu Ile Phe Gly 15 20 25AAG AGC TCG ACT GGC GAC CGG GCC GAC TGT CGC CGC TGC GAC ATC ACC 4646Lys Ser Ser Thr Gly Asp Arg Ala Asp Cys Arg Arg Cys Asp Ile Thr 30 35 40TTC GGC AAC CCC ATC CTG CGC CAC GGA CCA GGT TGC CGC GAG GAC CAC 4694Phe Gly Asn Pro Ile Leu Arg His Gly Pro Gly Cys Arg Glu Asp His 45 50 55GCC GGA CTT TGC GCC TTT CCT GGG TTC CCA GTC TGC CCG TGC TGT CTC 4742Ala Gly Leu Cys Ala Phe Pro Gly Phe Pro Val Cys Pro Cys Cys Leu 60 65 70 75GAA GCC GTA CCG GCC CCC CAC GAC TGC CCG TTG GAA AGA AGT CAC CCC 4790Glu Ala Val Pro Ala Pro His Asp Cys Pro Leu Glu Arg Ser His Pro 80 85 90GCT CCA CGC GTG GAA GGG CGT GAC CGG AGA CCG ACC GGA AGT CAG GGA 4838Ala Pro Arg Val Glu Gly Arg Asp Arg Arg Pro Thr Gly Ser Gln Gly 95 100 105GGA CCC GGA GAC AGC GGC GGT CGT CCA GGC TCT GAT CAG CGG CCG TTA 4886Gly Pro Gly Asp Ser Gly Gly Arg Pro Gly Ser Asp Gln Arg Pro Leu 110 115 120TCC TCA GAA GAC GAA GCT TTC CTC CGA CGC ATC CAA AGG CTA CTC AAG 4934Ser Ser Glu Asp Glu Ala Phe Leu Arg Arg Ile Gln Arg Leu Leu Lys 125 130 135AAC TAAGGGATGC TCACAATCCA CCTCTTTTCC TGCCCCGAGT GCGGATTACC 4987Asn140AGGCCCGCGA CTGCCAGACA GTCCGAGTCT GCCGCGCCGC TGCAGAGATG GCGCGCTC 5047GTATTCACGA GCCGTTGGCT TCATCTGCCG CCAGTGCCGA CTTGAAGCGC ATACGCTC 5107CCTCGGACTC TGTTCCCGAT GTAAAGATCA GCAAGAGCGC ATGAAGGAAC AAAATTAG 5167TCCTTGTTCG TAAACAAGGT GGTCCCTCCC ATTGAGGTAA AGACTCTGGT GAGTCCTC 5227CGTTACTCGT TGAGTCTGCT GCGGTTCGAT TCCATTCCCA AGCAGCAAAG GGTGCGCA 5287TAGTACGGCG CCCCCTGGGA TACCA 5312 140 amino acids amino acid linear protein unknown 44Met Ser Gly Gln Lys Arg Arg Lys Asp Arg Gln Phe Val Asn Cys Pro 1 5 10 15His Cys Ser Glu Pro Leu Ile Leu Ile Phe Gly Lys Ser Ser Thr Gly 20 25 30Asp Arg Ala Asp Cys Arg Arg Cys Asp Ile Thr Phe Gly Asn Pro Ile 35 40 45Leu Arg His Gly Pro Gly Cys Arg Glu Asp His Ala Gly Leu Cys Ala 50 55 60Phe Pro Gly Phe Pro Val Cys Pro Cys Cys Leu Glu Ala Val Pro Ala 65 70 75 80Pro His Asp Cys Pro Leu Glu Arg Ser His Pro Ala Pro Arg Val Glu 85 90 95Gly Arg Asp Arg Arg Pro Thr Gly Ser Gln Gly Gly Pro Gly Asp Ser 100 105 110Gly Gly Arg Pro Gly Ser Asp Gln Arg Pro Leu Ser Ser Glu Asp Glu 115 120 125Ala Phe Leu Arg Arg Ile Gln Arg Leu Leu Lys Asn 130 135 140 5368 base pairs nucleic acid unknown unknown DNA unknown CDS 4944..5162 45GTTCTGCCTC CCCCGGACGG TAAATATAGG GGAACAATGT ACGCGAAAGC GACAGACGTG 60GCGCGTGTCT ACGCCGCGGC AGATGTCGCC TACGCGAACG TACTGCAGCA GAGAGCAGT 120AAGTTGGACT TCGCCCCGCC ACTGAAGGCA CTAGAAACCC TCCACAGACT GTACTATCC 180CTGCGCTTCA AAGGGGGCAC TTTACCCCCG ACACAACACC CGATCCTGGC CGGGCACCA 240CGTGTCGCAG AAGAGGTTCT GCACAATTTC GCCAGGGGAC GTAGCACAGT GCTCGAGAT 300GGGCCGTCTC TGCACAGCGC ACTTAAGCTA CATGGGGCAC CGAACGCCCC CGTCGCAGA 360TATCACGGGT GCACCAAGTA CGGCACCCGC GACGGCTCGC GACACATTAC GGCCTTAGA 420TCTAGATCCG TCGCCACAGG CCGGCCCGAG TTCAAGGCCG ACGCCTCACT GCTCGCCAA 480GGCATTGCCT CCCGCACCTT CTGCGTCGAC GGAGTCGGCT CTTGCGCGTT CAAATCGCG 540GTTGGAATTG CCAATCACTC CCTCTATGAC GTGACCCTAG AGGAGCTGGC CAATGCGTT 600GAGAACCACG GACTTCACAT GGTCCGCGCG TTCATGCACA TGCCAGAAGA GCTGCTCTA 660ATGGACAACG TGGTTAATGC CGAGCTCGGC TACCGCTTCC ACGTTATTGA AGAGCCTAT 720GCTGTGAAGG ACTGCGCATT CCAGGGGGGG GACCTCCGTC TCCACTTCCC TGAGTTGGA 780TTCATCAACG AGAGCCAAGA GCGGCGCATC GAGAGGCTGG CCGCCCGCGG CTCCTACTC 840AGACGCGCCG TCATTTTCTC CGGCGACGAC GACTGGGGTG ATGCGTACTT ACACGACTT 900CACACATGGC TCGCCTACCT ACTGGTGAGG AACTACCCCA CTCCGTTTGG TTTCTCACT 960CATATAGAAG TCCAGAGGCG CCACGGCTCC AGCATTGAGC TGCGCATCAC TCGCGCGC 1020CCTGGAGACC GCATGCTGGC CGTCGTCCCA AGGACGTCCC AAGGCCTCTG CAGAATCC 1080AACATCTTTT ATTACGCCGA CGCGTCGGGC ACTGAGCATA AGACCATCCT TACGTCAC 1140CACAAAGTCA ACATGCTGCT CAATTTTATG CAAACGCGTC CTGAGAAGGA ACTAGTCG 1200ATGACCGTCT TGATGTCGTT CGCGCGCGCT AGGCTGCGCG CGATCGTGGT CGCCTCAG 1260GTCACCGAGA GCTCCTGGAA CATCTCACCG GCTGACCTGG TCCGCACTGT CGTGTCTC 1320TACGTCCTCC ACATCATCGA GCGCCGAAGG GCTGCGGTCG CTGTCAAGAC CGCCAAGG 1380GACGTCTTTG GAGAGACTTC GTTCTGGGAG AGTCTCAAGC ACGTCTTGGG CTCCTGTT 1440GGTCTGCGCA ACCTCAAAGG CACCGACGTC GTCTTTACTA AGCGCGTCGT CGATAAGT 1500CGAGTCCACT CGCTCGGAGA CATAATCTGC GACGTCCGCC TGTCCCCTGA ACAGGTCG 1560TTCCTGCCGT CCCGCGTACC ACCTGCCCGC GTCTTTCACG ACAGGGAAGA GCTTGAGG 1620CTTCGCGAAG CTGGCTGCTA CAACGAACGT CCGGTACCTT CCACTCCTCC TGTGGAGG 1680CCCCAAGGTT TCGACGCCGA CTTGTGGCAC GCGACCGCGG CCTCACTCCC CGAGTACC 1740GCCACCTTGC AGGCAGGTCT CAACACCGAC GTCAAGCAGC TCAAGATCAC CCTCGAGA 1800GCCCTCAAGA CCATCGACGG GCTCACCCTC TCCCCAGTCA GAGGCCTCGA GATGTACG 1860GGCCCGCCAG GCAGCGGCAA GACGGGCACC CTCATCGCCG CCCTTGAGGC CGCGGGCG 1920AAAGCACTTT ACGTGGCACC CACCAGAGAA CTGAGAGAGG CTATGGACCG GCGGATCA 1980CCGCCGTCCG CCTCGGCTAC GCAACATGTC GCCCTTGCGA TTCTCCGTCG TGCCACCG 2040GAGGGCGCCC CTTTCGCTAC CGTGGTTATC GACGAGTGCT TCATGTTCCC GCTCGTGT 2100GTCGCGATCG TGCACGCCTT GTCCCCGAGC TCACGAATAG TCCTTGTAGG GGACGTCC 2160CAAATCGGGT TTATAGACTT CCAAGGCACA AGCGCGAACA TGCCGCTCGT TCGCGACG 2220GTTAAGCAGT GCCGTCGGCG CACTTTCAAC CAAACCAAGC GCTGTCCGGC CGACGTCG 2280GCCACCACGT TTTTCCAGAG CTTGTACCCC GGGTGCACAA CCACCTCAGG GTGCGTCG 2340TCCATCAGCC ACGTCGCCCC AGACTACCGC AACAGCCAGG CGCAAACGCT CTGCTTCA 2400CAGGAGGAAA AGTCGCGCCA CGGGGCTGAG GGCGCGATGA CTGTGCACGA AGCGCAGG 2460CGCACTTTTG CGTCTGTCAT TCTGCATTAC AACGGCTCCA CAGCAGAGCA GAAGCTCC 2520GCTGAGAAGT CGCACCTTCT AGTCGGCATC ACGCGCCACA CCAACCACCT GTACATCC 2580GACCCGACAG GTGACATTGA GAGACAACTC AACCATAGCG CGAAAGCCGA GGTGTTTA 2640GACATCCCTG CACCCCTGGA GATCACGACT GTCAAACCGA GTGAAGAGGT GCAGCGCA 2700GAAGTGATGG CAACGATACC CCCGCAGAGT GCCACGCCGC ACGGAGCAAT CCATCTGC 2760CGCAAGAACT TCGGGGACCA ACCCGACTGT GGCTGTGTCG CTTTGGCGAA GACCGGCT 2820GAGGTGTTTG GCGGTCGTGC CAAAATCAAC GTAGAGCTTG CCGAACCCGA CGCGACCC 2880AAGCCGCATA GGGCGTTCCA GGAAGGGGTA CAGTGGGTCA AGGTCACCAA CGCGTCTA 2940AAACACCAGG CGCTCCAGAC GCTGTTGTCC CGCTACACCA AGCGAAGCGC TGACCTGC 3000CTACACGAAG CTAAGGAGGA CGTCAAACGC ATGCTAAACT CGCTTGACCG ACATTGGG 3060TGGACTGTCA CTGAAGACGC CCGTGACCGA GCTGTCTTCG AGACCCAGCT CAAGTTCA 3120CAACGCGGCG GCACCGTCGA AGACCTGCTG GAGCCAGACG ACCCCTACAT CCGTGACA 3180GACTTCCTTA TGAAGACTCA GCAGAAAGTG TCGCCCAAGC CGATCAATAC GGGCAAGG 3240GGGCAGGGGA TCGCCGCTCA CTCAAAGTCT CTCAACTTCG TCCTCGCCGC TTGGATAC 3300ATACTCGAGG AGATACTCCG TACCGGGAGC CGCACGGTCC GGTACAGCAA CGGTCTCC 3360GACGAAGAAG AGGCCATGCT GCTCGAAGCG AAGATCAATC AAGTCCCACA CGCCACGT 3420GTCTCGGCGG ACTGGACCGA GTTTGACACC GCCCACAATA ACACGAGTGA GCTGCTCT 3480GCCGCCCTTT TAGAGCGCAT CGGCACGCCT GCAGCTGCCG TTAATCTATT CAGAGAAC 3540TGTGGGAAAC GCACCTTGCG AGCGAAGGGT CTAGGCTCCG TTGAAGTCGA CGGTCTGC 3600GACTCCGGCG CAGCTTGGAC GCCTTGCCGC AACACCATCT TCTCTGCCGC CGTCATGC 3660ACGCTCTTCC GCGGCGTCAA GTTCGCAGCT TTCAAAGGCG ACGACTCGCT CCTCTGTG 3720AGCCATTACC TCCGTTTCGA CGCTAGCCGC CTTCACATGG GCGAACGTTA CAAGACCA 3780CATTTGAAGG TCGAGGTGCA GAAAATCGTG CCGTACATCG GACTCCTCGT CTCCGCTG 3840CAGGTCGTCC TCGACCCTGT CAGGAGCGCT CTCAAGATAT TTGGGCGCTG CTACACAA 3900GAACTCCTTT ACTCCAAGTA CGTGGAGGCT GTGAGAGACA TCACCAAGGG CTGGAGTG 3960GCCCGCTACC ACAGCCTCCT GTGCCACATG TCAGCATGCT ACTACAATTA CGCGCCGG 4020TCTGCGGCGT ACATCATCGA CGCTGTTGTT CGCTTTGGGC GCGGCGACTT CCCGTTTG 4080CAACTGCGCG TGGTGCGTGC CCATGTGCAG GCACCCGACG CTTACAGCAG CACGTATC 4140GCTAACGTGC GCGCATCGTG CCTTGACCAC GTCTTCGAGC CCCGCCAGGC CGCCGCCC 4200GCAGGTTTCG TTGCGACATG TGCGAAGCCG GAAACGCCTT CTTCACTTAC CGCGAAAG 4260GGTGTTTCTG CGACTACAAG CCACGTTGCG ACTGGGACTG CGCCCCCGGA GTCTCCAT 4320GATGCACCTG CAGCCAACAG CTTTTCGGAG TTATTGACAC CGGAGACCCC GTCCACAT 4380TCCTCGCCGT CATCGTCTTC ATCGGACTCC TCTACATCGT GTGGAAGGTC GCTCAGTG 4440GGAGACACCG CAAGGACCAC AGAAGACTTG AACAGCAGAA AGCCGCCTTC GCAAGACA 4500CAATCACGCT CGTCTGAATG TCTGGACAGA AGCGGAGAAA GGACAGGCAG TTCGTTAA 4560GCCCCCACTG CTCCGAGCCC CTCATTCTCA TTTTCGGAAA GAGCTCGACT GGCGACCG 4620CCGACTGTCG CCGCTGCGAC ATCACCTTCG GCAACCCCAT CCTGCGCCAC GGACCAGG 4680GCCGCGAGGA CCACGCCGGA CTTTGCGCCT TTCCTGGGTT CCCAGTCTGC CCGTGCTG 4740TCGAAGCCGT ACCGGCCCCC CACGACTGCC CGTTGGAAAG AAGTCACCCC GCTCCACG 4800TGGAAGGGCG TGACCGGAGA CCGACCGGAA GTCAGGGAGG ACCCGGAGAC AGCGGCGG 4860GTCCAGGCTC TGATCAGCGG CCGTTATCCT CAGAAGACGA AGCTTTCCTC CGACGCAT 4920AAAGGCTACT CAAGAACTAA GGG ATG CTC ACA ATC CAC CTC TTT TCC TGC 4970 Met Leu Thr Ile His Leu Phe Ser Cys 1 5TTC CCC GAG TGC GGA TTA CCA GGC CCG CGA CTG CCA GAC AGT CCG AGT 5018Phe Pro Glu Cys Gly Leu Pro Gly Pro Arg Leu Pro Asp Ser Pro Ser 10 15 20 25CTG GCG GAG ACC GCG CCG CTG CAG AGA TGG CGC GCT CAT GTA TTC ACG 5066Leu Ala Glu Thr Ala Pro Leu Gln Arg Trp Arg Ala His Val Phe Thr 30 35 40AGC CGT TGG CAG AGC AGA GAA TTA GAA AGT TCA TCT GCC GCC AGT GCC 5114Ser Arg Trp Gln Ser Arg Glu Leu Glu Ser Ser Ser Ala Ala Ser Ala 45 50 55GAC TTG AAG CGC ATA CGC TCT ACC TCG GAC AGG CAG GAA TTG CTC TGT 5162Asp Leu Lys Arg Ile Arg Ser Thr Ser Asp Arg Gln Glu Leu Leu Cys 60 65 70TCCCGATGTA AAGATCAGCA AGAGCGCATG AAGGAACAAA ATCAGCAGGG AGTGGATA 5222TTCCTTGTTC GTAAACAAGG TGGTCCCTCC CATTGAGGTA AAGACTCTGG TGAGTCCT 5282ACGTTACTCG TTGAGTCTGC TGCGGTTCGA TTCCATTCCC AAGCAGCAAA GGGTGCGC 5342CTAGTACGGC GCCCCCTGGG ATACCA 5368 73 amino acids amino acid linear protein unknown 46Met Leu Thr Ile His Leu Phe Ser Cys Phe Pro Glu Cys Gly Leu Pro 1 5 10 15Gly Pro Arg Leu Pro Asp Ser Pro Ser Leu Ala Glu Thr Ala Pro Leu 20 25 30Gln Arg Trp Arg Ala His Val Phe Thr Ser Arg Trp Gln Ser Arg Glu 35 40 45Leu Glu Ser Ser Ser Ala Ala Ser Ala Asp Leu Lys Arg Ile Arg Ser 50 55 60Thr Ser Asp Arg Gln Glu Leu Leu Cys 65 70 2478 base pairs nucleic acid unknown unknown DNA unknown CDS 283..753 47GTTTTTCTTT CTTTACCAAG TGTGGTAAAA TTTAAACAAA GAAGAAAACC AGGACCGTAA 60CCCGGCCCTT ACACACCTCG AGTCCGTGAC CACCGGATTA TACGTCGCCC ACCACACGG 120GCCTTTTCCG ACCACTCTCG AGAGTCGTTG GGAGTTTCGT CCGTGACCAC CCGGTTGGC 180GTCGACAGAC GCTTCCGGAC CACTAGAACC TCCTCGAGCG ACGCACACAC AGCACACAC 240CCGCCTTAGC TGCACCTACG GCAGCGTTGA TAGCGCGGAT TT ATG AGC GAG CAC 294 Met Ser Glu His 1ACC ATC GCC CAC TCC ATC ACA TTA CCA CCC GGT TAC ACC CTT GCC CTA 342Thr Ile Ala His Ser Ile Thr Leu Pro Pro Gly Tyr Thr Leu Ala Leu 5 10 15 20ATA CCC CCT GAA CCT GAA GCA GGA TGG GAG ATG CTG GAG TGG CGT CAC 390Ile Pro Pro Glu Pro Glu Ala Gly Trp Glu Met Leu Glu Trp Arg His 25 30 35AGC GAC CTC ACA ACC GTC GCG GAA CCC GTA ACG TTC GGG TCA GCG CCA 438Ser Asp Leu Thr Thr Val Ala Glu Pro Val Thr Phe Gly Ser Ala Pro 40 45 50ACA CCG TCA CCG TCA ATG GTA GAA GAA ACC AAC GGC GTC GGA CCG GAA 486Thr Pro Ser Pro Ser Met Val Glu Glu Thr Asn Gly Val Gly Pro Glu 55 60 65GGC AAG TTT CTC CCC CTG ACA ATT TCA CCG CTG CTG CAC AAG ACC TCG 534Gly Lys Phe Leu Pro Leu Thr Ile Ser Pro Leu Leu His Lys Thr Ser 70 75 80CGC AAA GCC TTG ACG CCA ACA CCG TCA CTT TCC CCG CTA ACA TCT CTA 582Arg Lys Ala Leu Thr Pro Thr Pro Ser Leu Ser Pro Leu Thr Ser Leu 85 90 95 100GCA TGC CCG AAT TCC GGA ATT GGG CCA AGG GAA AGA TCG ACC TCG ACT 630Ala Cys Pro Asn Ser Gly Ile Gly Pro Arg Glu Arg Ser Thr Ser Thr 105 110 115CCG ATT CCA TCG GCT GGT ACT TCA AGT ACC TTG ACC CAG CGG GTG CTA 678Pro Ile Pro Ser Ala Gly Thr Ser Ser Thr Leu Thr Gln Arg Val Leu 120 125 130CAG AGT CTG CGC GCG CCG TCG GCG AGT ACT CGA AGA TCC CTG ACG GCC 726Gln Ser Leu Arg Ala Pro Ser Ala Ser Thr Arg Arg Ser Leu Thr Ala 135 140 145TCG TCA AGT TCT CCG TCG ACG CAG AGA TAAGAGAGAT CTATAACGAG 773Ser Ser Ser Ser Pro Ser Thr Gln Arg 150 155GAGTGCCCCG TCGTCACTGA CGTGTCCGTC CCCCTCGACG GCCGCCAGTG GAGCCTCTC 833ATTTTCTCCT TTCCGATGTT CAGAACCGCC TACGTCGCCG TAGCGAACGT CGAGAACAA 893GAGATGTCGC TCGACGTTGT CAACGACCTC ATCGAGTGGC TCAACAATCT CGCCGACTG 953CGTTATGTCG TTGACTCTGA ACAGTGGATT AACTTCACCA ATGACACCAC GTACTACG 1013CGCATCCGCG TTCTACGTCC AACCTACGAC GTTCCAGACC CCACAGAGGG CCTTGTTC 1073ACAGTCTCAG ACTACCGCCT CACTTATAAG GCGATAACAT GTGAAGCCAA CATGCCAA 1133CTCGTCGACC AAGGCTTTTG GATCGGCGGC CAGTACGCTC TCACCCCGAC TAGCCTAC 1193CAGTACGACG TCAGCGAGGC CTACGCTCTG CACACTTTGA CCTTCGCCAG ACCATCCA 1253GCCGCTGCAC TCGCGTTTGT GTGGGCAGGT TTGCCACAGG GTGGCACTGC GCCTGCAG 1313ACTCCAGCCT GGGAGCAGGC ATCCTCGGGT GGCTACCTCA CCTGGCGCCA CAACGGTA 1373ACTTTCCCAG CTGGCTCCGT TAGCTACGTT CTCCCTGAGG GTTTCGCCCT TGAGCGCT 1433GACCCGAACG ACGGCTCTTG GACCGACTTC GCTTCCGCAG GAGACACCGT CACTTTCC 1493CAGGTCGCCG TCGACGAGGT CGTTGTGACC AACAACCCCG CCGGCGGCGG CAGCGCCC 1553ACCTTCACCG TGAGAGTGCC CCCTTCAAAC GCTTACACCA ACACCGTGTT TAGGAACA 1613CTCTTAGAGA CTCGACCCTC CTCTCGTAGG CTCGAACTCC CTATGCCACC TGCTGACT 1673GGACAGACGG TCGCCAACAA CCCGAAGATC GAGCAGTCGC TTCTTAAAGA AACACTTG 1733TGCTATTTGG TCCACTCCAA AATGCGAAAC CCCGTTTTCC AGCTCACGCC AGCCAGCT 1793TTTGGCGCCG TTTCCTTCAA CAATCCGGGT TATGAGCGCA CACGCGACCT CCCGGACT 1853ACTGGCATCC GTGACTCATT CGACCAGAAC ATGTCCACCG CTGTGGCCCA CTTCCGCT 1913CTCTCCCACT CCTGCAGTAT CGTCACTAAG ACCTACCAGG GTTGGGAAGG CGTCACGA 1973GTCAACACGC CTTTCGGCCA ATTCGCGCAC GCGGGCCTCC TCAAGAATGA GGAGATCC 2033TGCCTCGCCG ACGACCTGGC CACCCGTCTC ACAGGTGTCT ACCCCGCCAC TGACAACT 2093GCGGCCGCCG TTTCTGCCTT CGCCGCGAAC ATGCTGTCCT CCGTGCTGAA GTCGGAGG 2153ACGTCCTCCA TCATCAAGTC CGTTGGCGAG ACTGCCGTCG GCGCGGCTCA GTCCGGCC 2213GCGAAGCTAC CCGGACTGCT AATGAGTGTA CCAGGGAAGA TTGCCGCGCG TGTCCGCG 2273CGCCGAGCGC GCCGCCGCGC CGCTCGTGCC AATTAGTTTG CTCGCTCCTG TTTCGCCG 2333TCGTAAAACG GCGTGGTCCC GCACATTACG CGTACCCTAA AGACTCTGGT GAGTCCCC 2393CGTTACACGA CGGGTCTGCC GCGGTTCGAT TCCATTCCCA AGCGGCAAGA AGGACGTA 2453TAGCTCTGCG TCCCTCGGGA TACCA 2478 157 amino acids amino acid linear protein unknown 48Met Ser Glu His Thr Ile Ala His Ser Ile Thr Leu Pro Pro Gly Tyr 1 5 10 15Thr Leu Ala Leu Ile Pro Pro Glu Pro Glu Ala Gly Trp Glu Met Leu 20 25 30Glu Trp Arg His Ser Asp Leu Thr Thr Val Ala Glu Pro Val Thr Phe 35 40 45Gly Ser Ala Pro Thr Pro Ser Pro Ser Met Val Glu Glu Thr Asn Gly 50 55 60Val Gly Pro Glu Gly Lys Phe Leu Pro Leu Thr Ile Ser Pro Leu Leu 65 70 75 80His Lys Thr Ser Arg Lys Ala Leu Thr Pro Thr Pro Ser Leu Ser Pro 85 90 95Leu Thr Ser Leu Ala Cys Pro Asn Ser Gly Ile Gly Pro Arg Glu Arg 100 105 110Ser Thr Ser Thr Pro Ile Pro Ser Ala Gly Thr Ser Ser Thr Leu Thr 115 120 125Gln Arg Val Leu Gln Ser Leu Arg Ala Pro Ser Ala Ser Thr Arg Arg 130 135 140Ser Leu Thr Ala Ser Ser Ser Ser Pro Ser Thr Gln Arg145 150 155 2478 base pairs nucleic acid unknown unknown DNA unknown CDS 366..2306 49GTTTTTCTTT CTTTACCAAG TGTGGTAAAA TTTAAACAAA GAAGAAAACC AGGACCGTAA 60CCCGGCCCTT ACACACCTCG AGTCCGTGAC CACCGGATTA TACGTCGCCC ACCACACGG 120GCCTTTTCCG ACCACTCTCG AGAGTCGTTG GGAGTTTCGT CCGTGACCAC CCGGTTGGC 180GTCGACAGAC GCTTCCGGAC CACTAGAACC TCCTCGAGCG ACGCACACAC AGCACACAC 240CCGCCTTAGC TGCACCTACG GCAGCGTTGA TAGCGCGGAT TTATGAGCGA GCACACCAT 300GCCCACTCCA TCACATTACC ACCCGGTTAC ACCCTTGCCC TAATACCCCC TGAACCTGA 360GCAGG ATG GGA GAT GCT GGA GTG GCG TCA CAG CGA CCT CAC AAC CGT 407 Met Gly Asp Ala Gly Val Ala Ser Gln Arg Pro His Asn Arg 1 5 10CGC GGA ACC CGT AAC GTT CGG GTC AGC GCC AAC ACC GTC ACC GTC AAT 455Arg Gly Thr Arg Asn Val Arg Val Ser Ala Asn Thr Val Thr Val Asn 15 20 25 30GGT AGA AGA AAC CAA CGG CGT CGG ACC GGA AGG CAA GTT TCT CCC CCT 503Gly Arg Arg Asn Gln Arg Arg Arg Thr Gly Arg Gln Val Ser Pro Pro 35 40 45GAC AAT TTC ACC GCT GCT GCA CAA GAC CTC GCG CAA AGC CTT GAC GCC 551Asp Asn Phe Thr Ala Ala Ala Gln Asp Leu Ala Gln Ser Leu Asp Ala 50 55 60AAC ACC GTC ACT TTC CCC GCT AAC ATC TCT AGC ATG CCC GAA TTC CGG 599Asn Thr Val Thr Phe Pro Ala Asn Ile Ser Ser Met Pro Glu Phe Arg 65 70 75AAT TGG GCC AAG GGA AAG ATC GAC CTC GAC TCC GAT TCC ATC GGC TGG 647Asn Trp Ala Lys Gly Lys Ile Asp Leu Asp Ser Asp Ser Ile Gly Trp 80 85 90TAC TTC AAG TAC CTT GAC CCA GCG GGT GCT ACA GAG TCT GCG CGC GCC 695Tyr Phe Lys Tyr Leu Asp Pro Ala Gly Ala Thr Glu Ser Ala Arg Ala 95 100 105 110GTC GGC GAG TAC TCG AAG ATC CCT GAC GGC CTC GTC AAG TTC TCC GTC 743Val Gly Glu Tyr Ser Lys Ile Pro Asp Gly Leu Val Lys Phe Ser Val 115 120 125GAC GCA GAG ATA AGA GAG ATC TAT AAC GAG GAG TGC CCC GTC GTC ACT 791Asp Ala Glu Ile Arg Glu Ile Tyr Asn Glu Glu Cys Pro Val Val Thr 130 135 140GAC GTG TCC GTC CCC CTC GAC GGC CGC CAG TGG AGC CTC TCG ATT TTC 839Asp Val Ser Val Pro Leu Asp Gly Arg Gln Trp Ser Leu Ser Ile Phe 145 150 155TCC TTT CCG ATG TTC AGA ACC GCC TAC GTC GCC GTA GCG AAC GTC GAG 887Ser Phe Pro Met Phe Arg Thr Ala Tyr Val Ala Val Ala Asn Val Glu 160 165 170AAC AAG GAG ATG TCG CTC GAC GTT GTC AAC GAC CTC ATC GAG TGG CTC 935Asn Lys Glu Met Ser Leu Asp Val Val Asn Asp Leu Ile Glu Trp Leu175 180 185 190AAC AAT CTC GCC GAC TGG CGT TAT GTC GTT GAC TCT GAA CAG TGG ATT 983Asn Asn Leu Ala Asp Trp Arg Tyr Val Val Asp Ser Glu Gln Trp Ile 195 200 205AAC TTC ACC AAT GAC ACC ACG TAC TAC GTC CGC ATC CGC GTT CTA CGT 1031Asn Phe Thr Asn Asp Thr Thr Tyr Tyr Val Arg Ile Arg Val Leu Arg 210 215 220CCA ACC TAC GAC GTT CCA GAC CCC ACA GAG GGC CTT GTT CGC ACA GTC 1079Pro Thr Tyr Asp Val Pro Asp Pro Thr Glu Gly Leu Val Arg Thr Val 225 230 235TCA GAC TAC CGC CTC ACT TAT AAG GCG ATA ACA TGT GAA GCC AAC ATG 1127Ser Asp Tyr Arg Leu Thr Tyr Lys Ala Ile Thr Cys Glu Ala Asn Met 240 245 250CCA ACA CTC GTC GAC CAA GGC TTT TGG ATC GGC GGC CAG TAC GCT CTC 1175Pro Thr Leu Val Asp Gln Gly Phe Trp Ile Gly Gly Gln Tyr Ala Leu255 260 265 270ACC CCG ACT AGC CTA CCG CAG TAC GAC GTC AGC GAG GCC TAC GCT CTG 1223Thr Pro Thr Ser Leu Pro Gln Tyr Asp Val Ser Glu Ala Tyr Ala Leu 275 280 285CAC ACT TTG ACC TTC GCC AGA CCA TCC AGC GCC GCT GCA CTC GCG TTT 1271His Thr Leu Thr Phe Ala Arg Pro Ser Ser Ala Ala Ala Leu Ala Phe 290 295 300GTG TGG GCA GGT TTG CCA CAG GGT GGC ACT GCG CCT GCA GGC ACT CCA 1319Val Trp Ala Gly Leu Pro Gln Gly Gly Thr Ala Pro Ala Gly Thr Pro 305 310 315GCC TGG GAG CAG GCA TCC TCG GGT GGC TAC CTC ACC TGG CGC CAC AAC 1367Ala Trp Glu Gln Ala Ser Ser Gly Gly Tyr Leu Thr Trp Arg His Asn 320 325 330GGT ACT ACT TTC CCA GCT GGC TCC GTT AGC TAC GTT CTC CCT GAG GGT 1415Gly Thr Thr Phe Pro Ala Gly Ser Val Ser Tyr Val Leu Pro Glu Gly335 340 345 350TTC GCC CTT GAG CGC TAC GAC CCG AAC GAC GGC TCT TGG ACC GAC TTC 1463Phe Ala Leu Glu Arg Tyr Asp Pro Asn Asp Gly Ser Trp Thr Asp Phe 355 360 365GCT TCC GCA GGA GAC ACC GTC ACT TTC CGG CAG GTC GCC GTC GAC GAG 1511Ala Ser Ala Gly Asp Thr Val Thr Phe Arg Gln Val Ala Val Asp Glu 370 375 380GTC GTT GTG ACC AAC AAC CCC GCC GGC GGC GGC AGC GCC CCC ACC TTC 1559Val Val Val Thr Asn Asn Pro Ala Gly Gly Gly Ser Ala Pro Thr Phe 385 390 395ACC GTG AGA GTG CCC CCT TCA AAC GCT TAC ACC AAC ACC GTG TTT AGG 1607Thr Val Arg Val Pro Pro Ser Asn Ala Tyr Thr Asn Thr Val Phe Arg 400 405 410AAC ACG CTC TTA GAG ACT CGA CCC TCC TCT CGT AGG CTC GAA CTC CCT 1655Asn Thr Leu Leu Glu Thr Arg Pro Ser Ser Arg Arg Leu Glu Leu Pro415 420 425 430ATG CCA CCT GCT GAC TTT GGA CAG ACG GTC GCC AAC AAC CCG AAG ATC 1703Met Pro Pro Ala Asp Phe Gly Gln Thr Val Ala Asn Asn Pro Lys Ile 435 440 445GAG CAG TCG CTT CTT AAA GAA ACA CTT GGC TGC TAT TTG GTC CAC TCC 1751Glu Gln Ser Leu Leu Lys Glu Thr Leu Gly Cys Tyr Leu Val His Ser 450 455 460AAA ATG CGA AAC CCC GTT TTC CAG CTC ACG CCA GCC AGC TCC TTT GGC 1799Lys Met Arg Asn Pro Val Phe Gln Leu Thr Pro Ala Ser Ser Phe Gly 465 470 475GCC GTT TCC TTC AAC AAT CCG GGT TAT GAG CGC ACA CGC GAC CTC CCG 1847Ala Val Ser Phe Asn Asn Pro Gly Tyr Glu Arg Thr Arg Asp Leu Pro 480 485 490GAC TAC ACT GGC ATC CGT GAC TCA TTC GAC CAG AAC ATG TCC ACC GCT 1895Asp Tyr Thr Gly Ile Arg Asp Ser Phe Asp Gln Asn Met Ser Thr Ala495 500 505 510GTG GCC CAC TTC CGC TCA CTC TCC CAC TCC TGC AGT ATC GTC ACT AAG 1943Val Ala His Phe Arg Ser Leu Ser His Ser Cys Ser Ile Val Thr Lys 515 520 525ACC TAC CAG GGT TGG GAA GGC GTC ACG AAC GTC AAC ACG CCT TTC GGC 1991Thr Tyr Gln Gly Trp Glu Gly Val Thr Asn Val Asn Thr Pro Phe Gly 530 535 540CAA TTC GCG CAC GCG GGC CTC CTC AAG AAT GAG GAG ATC CTC TGC CTC 2039Gln Phe Ala His Ala Gly Leu Leu Lys Asn Glu Glu Ile Leu Cys Leu 545 550 555GCC GAC GAC CTG GCC ACC CGT CTC ACA GGT GTC TAC CCC GCC ACT GAC 2087Ala Asp Asp Leu Ala Thr Arg Leu Thr Gly Val Tyr Pro Ala Thr Asp 560 565 570AAC TTC GCG GCC GCC GTT TCT GCC TTC GCC GCG AAC ATG CTG TCC TCC 2135Asn Phe Ala Ala Ala Val Ser Ala Phe Ala Ala Asn Met Leu Ser Ser575 580 585 590GTG CTG AAG TCG GAG GCA ACG TCC TCC ATC ATC AAG TCC GTT GGC GAG 2183Val Leu Lys Ser Glu Ala Thr Ser Ser Ile Ile Lys Ser Val Gly Glu 595 600 605ACT GCC GTC GGC GCG GCT CAG TCC GGC CTC GCG AAG CTA CCC GGA CTG 2231Thr Ala Val Gly Ala Ala Gln Ser Gly Leu Ala Lys Leu Pro Gly Leu 610 615 620CTA ATG AGT GTA CCA GGG AAG ATT GCC GCG CGT GTC CGC GCG CGC CGA 2279Leu Met Ser Val Pro Gly Lys Ile Ala Ala Arg Val Arg Ala Arg Arg 625 630 635GCG CGC CGC CGC GCC GCT CGT GCC AAT TAGTTTGCTC GCTCCTGTTT 2326Ala Arg Arg Arg Ala Ala Arg Ala Asn 640 645CGCCGTTTCG TAAAACGGCG TGGTCCCGCA CATTACGCGT ACCCTAAAGA CTCTGGTG 2386TCCCCGTCGT TACACGACGG GTCTGCCGCG GTTCGATTCC ATTCCCAAGC GGCAAGAA 2446ACGTAGTTAG CTCTGCGTCC CTCGGGATAC CA 2478 647 amino acids amino acid linear protein unknown 50Met Gly Asp Ala Gly Val Ala Ser Gln Arg Pro His Asn Arg Arg Gly 1 5 10 15Thr Arg Asn Val Arg Val Ser Ala Asn Thr Val Thr Val Asn Gly Arg 20 25 30Arg Asn Gln Arg Arg Arg Thr Gly Arg Gln Val Ser Pro Pro Asp Asn 35 40 45Phe Thr Ala Ala Ala Gln Asp Leu Ala Gln Ser Leu Asp Ala Asn Thr 50 55 60Val Thr Phe Pro Ala Asn Ile Ser Ser Met Pro Glu Phe Arg Asn Trp 65 70 75 80Ala Lys Gly Lys Ile Asp Leu Asp Ser Asp Ser Ile Gly Trp Tyr Phe 85 90 95Lys Tyr Leu Asp Pro Ala Gly Ala Thr Glu Ser Ala Arg Ala Val Gly 100 105 110Glu Tyr Ser Lys Ile Pro Asp Gly Leu Val Lys Phe Ser Val Asp Ala 115 120 125Glu Ile Arg Glu Ile Tyr Asn Glu Glu Cys Pro Val Val Thr Asp Val 130 135 140Ser Val Pro Leu Asp Gly Arg Gln Trp Ser Leu Ser Ile Phe Ser Phe145 150 155 160Pro Met Phe Arg Thr Ala Tyr Val Ala Val Ala Asn Val Glu Asn Lys 165 170 175Glu Met Ser Leu Asp Val Val Asn Asp Leu Ile Glu Trp Leu Asn Asn 180 185 190Leu Ala Asp Trp Arg Tyr Val Val Asp Ser Glu Gln Trp Ile Asn Phe 195 200 205Thr Asn Asp Thr Thr Tyr Tyr Val Arg Ile Arg Val Leu Arg Pro Thr 210 215 220Tyr Asp Val Pro Asp Pro Thr Glu Gly Leu Val Arg Thr Val Ser Asp225 230 235 240Tyr Arg Leu Thr Tyr Lys Ala Ile Thr Cys Glu Ala Asn Met Pro Thr 245 250 255Leu Val Asp Gln Gly Phe Trp Ile Gly Gly Gln Tyr Ala Leu Thr Pro 260 265 270Thr Ser Leu Pro Gln Tyr Asp Val Ser Glu Ala Tyr Ala Leu His Thr 275 280 285Leu Thr Phe Ala Arg Pro Ser Ser Ala Ala Ala Leu Ala Phe Val Trp 290 295 300Ala Gly Leu Pro Gln Gly Gly Thr Ala Pro Ala Gly Thr Pro Ala Trp305 310 315 320Glu Gln Ala Ser Ser Gly Gly Tyr Leu Thr Trp Arg His Asn Gly Thr 325 330 335Thr Phe Pro Ala Gly Ser Val Ser Tyr Val Leu Pro Glu Gly Phe Ala 340 345 350Leu Glu Arg Tyr Asp Pro Asn Asp Gly Ser Trp Thr Asp Phe Ala Ser 355 360 365Ala Gly Asp Thr Val Thr Phe Arg Gln Val Ala Val Asp Glu Val Val 370 375 380Val Thr Asn Asn Pro Ala Gly Gly Gly Ser Ala Pro Thr Phe Thr Val385 390 395 400Arg Val Pro Pro Ser Asn Ala Tyr Thr Asn Thr Val Phe Arg Asn Thr 405 410 415Leu Leu Glu Thr Arg Pro Ser Ser Arg Arg Leu Glu Leu Pro Met Pro 420 425 430Pro Ala Asp Phe Gly Gln Thr Val Ala Asn Asn Pro Lys Ile Glu Gln 435 440 445Ser Leu Leu Lys Glu Thr Leu Gly Cys Tyr Leu Val His Ser Lys Met 450 455 460Arg Asn Pro Val Phe Gln Leu Thr Pro Ala Ser Ser Phe Gly Ala Val465 470 475 480Ser Phe Asn Asn Pro Gly Tyr Glu Arg Thr Arg Asp Leu Pro Asp Tyr 485 490 495Thr Gly Ile Arg Asp Ser Phe Asp Gln Asn Met Ser Thr Ala Val Ala 500 505 510His Phe Arg Ser Leu Ser His Ser Cys Ser Ile Val Thr Lys Thr Tyr 515 520 525Gln Gly Trp Glu Gly Val Thr Asn Val Asn Thr Pro Phe Gly Gln Phe 530 535 540Ala His Ala Gly Leu Leu Lys Asn Glu Glu Ile Leu Cys Leu Ala Asp545 550 555 560Asp Leu Ala Thr Arg Leu Thr Gly Val Tyr Pro Ala Thr Asp Asn Phe 565 570 575Ala Ala Ala Val Ser Ala Phe Ala Ala Asn Met Leu Ser Ser Val Leu 580 585 590Lys Ser Glu Ala Thr Ser Ser Ile Ile Lys Ser Val Gly Glu Thr Ala 595 600 605Val Gly Ala Ala Gln Ser Gly Leu Ala Lys Leu Pro Gly Leu Leu Met 610 615 620Ser Val Pro Gly Lys Ile Ala Ala Arg Val Arg Ala Arg Arg Ala Arg625 630 635 640Arg Arg Ala Ala Arg Ala Asn 645 2479 base pairs nucleic acid unknown unknown DNA unknown CDS 283..2307 51GTTTTTCTTT CTTTACCAAG TGTGGTAAAA TTTAAACAAA GAAGAAAACC AGGACCGTAA 60CCCGGCCCTT ACACACCTCG AGTCCGTGAC CACCGGATTA TACGTCGCCC ACCACACGG 120GCCTTTTCCG ACCACTCTCG AGAGTCGTTG GGAGTTTCGT CCGTGACCAC CCGGTTGGC 180GTCGACAGAC GCTTCCGGAC CACTAGAACC TCCTCGAGCG ACGCACACAC AGCACACAC 240CCGCCTTAGC TGCACCTACG GCAGCGTTGA TAGCGCGGAT TT ATG AGC GAG CAC 294 Met Ser Glu His 1ACC ATC GCC CAC TCC ATC ACA TTA CCA CCC GGT TAC ACC CTT GCC CTA 342Thr Ile Ala His Ser Ile Thr Leu Pro Pro Gly Tyr Thr Leu Ala Leu 5 10 15 20ATA CCC CCT GAA CCT GAA GCA GGA TGG GAG ATG CTG GAG TGG CGT CAC 390Ile Pro Pro Glu Pro Glu Ala Gly Trp Glu Met Leu Glu Trp Arg His 25 30 35AGC GAC CTC ACA ACC GTC GCG GAA CCC GTA ACG TTC GGG TCA GCG CCA 438Ser Asp Leu Thr Thr Val Ala Glu Pro Val Thr Phe Gly Ser Ala Pro 40 45 50ACA CCG TCA CCG TCA ATG GTA GAA GAA ACC AAC GGC GTC GGA CCG GAA 486Thr Pro Ser Pro Ser Met Val Glu Glu Thr Asn Gly Val Gly Pro Glu 55 60 65GGC AAG TTT CTC CCC CTG ACA ATT TCA CCG CTG CTG CAC AAG ACC TCG 534Gly Lys Phe Leu Pro Leu Thr Ile Ser Pro Leu Leu His Lys Thr Ser 70 75 80CGC AAA GCC TTG ACG CCA ACA CCG TCA CTT TCC CCC GCT AAC ATC TCT 582Arg Lys Ala Leu Thr Pro Thr Pro Ser Leu Ser Pro Ala Asn Ile Ser 85 90 95 100AGC ATG CCC GAA TTC CGG AAT TGG GCC AAG GGA AAG ATC GAC CTC GAC 630Ser Met Pro Glu Phe Arg Asn Trp Ala Lys Gly Lys Ile Asp Leu Asp 105 110 115TCC GAT TCC ATC GGC TGG TAC TTC AAG TAC CTT GAC CCA GCG GGT GCT 678Ser Asp Ser Ile Gly Trp Tyr Phe Lys Tyr Leu Asp Pro Ala Gly Ala 120 125 130ACA GAG TCT GCG CGC GCC GTC GGC GAG TAC TCG AAG ATC CCT GAC GGC 726Thr Glu Ser Ala Arg Ala Val Gly Glu Tyr Ser Lys Ile Pro Asp Gly 135 140 145CTC GTC AAG TTC TCC GTC GAC GCA GAG ATA AGA GAG ATC TAT AAC GAG 774Leu Val Lys Phe Ser Val Asp Ala Glu Ile Arg Glu Ile Tyr Asn Glu 150 155 160GAG TGC CCC GTC GTC ACT GAC GTG TCC GTC CCC CTC GAC GGC CGC CAG 822Glu Cys Pro Val Val Thr Asp Val Ser Val Pro Leu Asp Gly Arg Gln165 170 175 180TGG AGC CTC TCG ATT TTC TCC TTT CCG ATG TTC AGA ACC GCC TAC GTC 870Trp Ser Leu Ser Ile Phe Ser Phe Pro Met Phe Arg Thr Ala Tyr Val 185 190 195GCC GTA GCG AAC GTC GAG AAC AAG GAG ATG TCG CTC GAC GTT GTC AAC 918Ala Val Ala Asn Val Glu Asn Lys Glu Met Ser Leu Asp Val Val Asn 200 205 210GAC CTC ATC GAG TGG CTC AAC AAT CTC GCC GAC TGG CGT TAT GTC GTT 966Asp Leu Ile Glu Trp Leu Asn Asn Leu Ala Asp Trp Arg Tyr Val Val 215 220 225GAC TCT GAA CAG TGG ATT AAC TTC ACC AAT GAC ACC ACG TAC TAC GTC 1014Asp Ser Glu Gln Trp Ile Asn Phe Thr Asn Asp Thr Thr Tyr Tyr Val 230 235 240CGC ATC CGC GTT CTA CGT CCA ACC TAC GAC GTT CCA GAC CCC ACA GAG 1062Arg Ile Arg Val Leu Arg Pro Thr Tyr Asp Val Pro Asp Pro Thr Glu245 250 255 260GGC CTT GTT CGC ACA GTC TCA GAC TAC CGC CTC ACT TAT AAG GCG ATA 1110Gly Leu Val Arg Thr Val Ser Asp Tyr Arg Leu Thr Tyr Lys Ala Ile 265 270 275ACA TGT GAA GCC AAC ATG CCA ACA CTC GTC GAC CAA GGC TTT TGG ATC 1158Thr Cys Glu Ala Asn Met Pro Thr Leu Val Asp Gln Gly Phe Trp Ile 280 285 290GGC GGC CAG TAC GCT CTC ACC CCG ACT AGC CTA CCG CAG TAC GAC GTC 1206Gly Gly Gln Tyr Ala Leu Thr Pro Thr Ser Leu Pro Gln Tyr Asp Val 295 300 305AGC GAG GCC TAC GCT CTG CAC ACT TTG ACC TTC GCC AGA CCA TCC AGC 1254Ser Glu Ala Tyr Ala Leu His Thr Leu Thr Phe Ala Arg Pro Ser Ser 310 315 320GCC GCT GCA CTC GCG TTT GTG TGG GCA GGT TTG CCA CAG GGT GGC ACT 1302Ala Ala Ala Leu Ala Phe Val Trp Ala Gly Leu Pro Gln Gly Gly Thr325 330 335 340GCG CCT GCA GGC ACT CCA GCC TGG GAG CAG GCA TCC TCG GGT GGC TAC 1350Ala Pro Ala Gly Thr Pro Ala Trp Glu Gln Ala Ser Ser Gly Gly Tyr 345 350 355CTC ACC TGG CGC CAC AAC GGT ACT ACT TTC CCA GCT GGC TCC GTT AGC 1398Leu Thr Trp Arg His Asn Gly Thr Thr Phe Pro Ala Gly Ser Val Ser 360 365 370TAC GTT CTC CCT GAG GGT TTC GCC CTT GAG CGC TAC GAC CCG AAC GAC 1446Tyr Val Leu Pro Glu Gly Phe Ala Leu Glu Arg Tyr Asp Pro Asn Asp 375 380 385GGC TCT TGG ACC GAC TTC GCT TCC GCA GGA GAC ACC GTC ACT TTC CGG 1494Gly Ser Trp Thr Asp Phe Ala Ser Ala Gly Asp Thr Val Thr Phe Arg 390 395 400CAG GTC GCC GTC GAC GAG GTC GTT GTG ACC AAC AAC CCC GCC GGC GGC 1542Gln Val Ala Val Asp Glu Val Val Val Thr Asn Asn Pro Ala Gly Gly405 410 415 420GGC AGC GCC CCC ACC TTC ACC GTG AGA GTG CCC CCT TCA AAC GCT TAC 1590Gly Ser Ala Pro Thr Phe Thr Val Arg Val Pro Pro Ser Asn Ala Tyr 425 430 435ACC AAC ACC GTG TTT AGG AAC ACG CTC TTA GAG ACT CGA CCC TCC TCT 1638Thr Asn Thr Val Phe Arg Asn Thr Leu Leu Glu Thr Arg Pro Ser Ser 440 445 450CGT AGG CTC GAA CTC CCT ATG CCA CCT GCT GAC TTT GGA CAG ACG GTC 1686Arg Arg Leu Glu Leu Pro Met Pro Pro Ala Asp Phe Gly Gln Thr Val 455 460 465GCC AAC AAC CCG AAG ATC GAG CAG TCG CTT CTT AAA GAA ACA CTT GGC 1734Ala Asn Asn Pro Lys Ile Glu Gln Ser Leu Leu Lys Glu Thr Leu Gly 470 475 480TGC TAT TTG GTC CAC TCC AAA ATG CGA AAC CCC GTT TTC CAG CTC ACG 1782Cys Tyr Leu Val His Ser Lys Met Arg Asn Pro Val Phe Gln Leu Thr485 490 495 500CCA GCC AGC TCC TTT GGC GCC GTT TCC TTC AAC AAT CCG GGT TAT GAG 1830Pro Ala Ser Ser Phe Gly Ala Val Ser Phe Asn Asn Pro Gly Tyr Glu 505 510 515CGC ACA CGC GAC CTC CCG GAC TAC ACT GGC ATC CGT GAC TCA TTC GAC 1878Arg Thr Arg Asp Leu Pro Asp Tyr Thr Gly Ile Arg Asp Ser Phe Asp 520 525 530CAG AAC ATG TCC ACC GCT GTG GCC CAC TTC CGC TCA CTC TCC CAC TCC 1926Gln Asn Met Ser Thr Ala Val Ala His Phe Arg Ser Leu Ser His Ser 535 540 545TGC AGT ATC GTC ACT AAG ACC TAC CAG GGT TGG GAA GGC GTC ACG AAC 1974Cys Ser Ile Val Thr Lys Thr Tyr Gln Gly Trp Glu Gly Val Thr Asn 550 555 560GTC AAC ACG CCT TTC GGC CAA TTC GCG CAC GCG GGC CTC CTC AAG AAT 2022Val Asn Thr Pro Phe Gly Gln Phe Ala His Ala Gly Leu Leu Lys Asn565 570 575 580GAG GAG ATC CTC TGC CTC GCC GAC GAC CTG GCC ACC CGT CTC ACA GGT 2070Glu Glu Ile Leu Cys Leu Ala Asp Asp Leu Ala Thr Arg Leu Thr Gly 585 590 595GTC TAC CCC GCC ACT GAC AAC TTC GCG GCC GCC GTT TCT GCC TTC GCC 2118Val Tyr Pro Ala Thr Asp Asn Phe Ala Ala Ala Val Ser Ala Phe Ala 600 605 610GCG AAC ATG CTG TCC TCC GTG CTG AAG TCG GAG GCA ACG TCC TCC ATC 2166Ala Asn Met Leu Ser Ser Val Leu Lys Ser Glu Ala Thr Ser Ser Ile 615 620 625ATC AAG TCC GTT GGC GAG ACT GCC GTC GGC GCG GCT CAG TCC GGC CTC 2214Ile Lys Ser Val Gly Glu Thr Ala Val Gly Ala Ala Gln Ser Gly Leu 630 635 640GCG AAG CTA CCC GGA CTG CTA ATG AGT GTA CCA GGG AAG ATT GCC GCG 2262Ala Lys Leu Pro Gly Leu Leu Met Ser Val Pro Gly Lys Ile Ala Ala645 650 655 660CGT GTC CGC GCG CGC CGA GCG CGC CGC CGC GCC GCT CGT GCC AAT 2307Arg Val Arg Ala Arg Arg Ala Arg Arg Arg Ala Ala Arg Ala Asn 665 670 675TAGTTTGCTC GCTCCTGTTT CGCCGTTTCG TAAAACGGCG TGGTCCCGCA CATTACGC 2367ACCCTAAAGA CTCTGGTGAG TCCCCGTCGT TACACGACGG GTCTGCCGCG GTTCGATT 2427ATTCCCAAGC GGCAAGAAGG ACGTAGTTAG CTCTGCGTCC CTCGGGATAC CA 2479 675 amino acids amino acid linear protein unknown 52Met Ser Glu His Thr Ile Ala His Ser Ile Thr Leu Pro Pro Gly Tyr 1 5 10 15Thr Leu Ala Leu Ile Pro Pro Glu Pro Glu Ala Gly Trp Glu Met Leu 20 25 30Glu Trp Arg His Ser Asp Leu Thr Thr Val Ala Glu Pro Val Thr Phe 35 40 45Gly Ser Ala Pro Thr Pro Ser Pro Ser Met Val Glu Glu Thr Asn Gly 50 55 60Val Gly Pro Glu Gly Lys Phe Leu Pro Leu Thr Ile Ser Pro Leu Leu 65 70 75 80His Lys Thr Ser Arg Lys Ala Leu Thr Pro Thr Pro Ser Leu Ser Pro 85 90 95Ala Asn Ile Ser Ser Met Pro Glu Phe Arg Asn Trp Ala Lys Gly Lys 100 105 110Ile Asp Leu Asp Ser Asp Ser Ile Gly Trp Tyr Phe Lys Tyr Leu Asp 115 120 125Pro Ala Gly Ala Thr Glu Ser Ala Arg Ala Val Gly Glu Tyr Ser Lys 130 135 140Ile Pro Asp Gly Leu Val Lys Phe Ser Val Asp Ala Glu Ile Arg Glu145 150 155 160Ile Tyr Asn Glu Glu Cys Pro Val Val Thr Asp Val Ser Val Pro Leu 165 170 175Asp Gly Arg Gln Trp Ser Leu Ser Ile Phe Ser Phe Pro Met Phe Arg 180 185 190Thr Ala Tyr Val Ala Val Ala Asn Val Glu Asn Lys Glu Met Ser Leu 195 200 205Asp Val Val Asn Asp Leu Ile Glu Trp Leu Asn Asn Leu Ala Asp Trp 210 215 220Arg Tyr Val Val Asp Ser Glu Gln Trp Ile Asn Phe Thr Asn Asp Thr225 230 235 240Thr Tyr Tyr Val Arg Ile Arg Val Leu Arg Pro Thr Tyr Asp Val Pro 245 250 255Asp Pro Thr Glu Gly Leu Val Arg Thr Val Ser Asp Tyr Arg Leu Thr 260 265 270Tyr Lys Ala Ile Thr Cys Glu Ala Asn Met Pro Thr Leu Val Asp Gln 275 280 285Gly Phe Trp Ile Gly Gly Gln Tyr Ala Leu Thr Pro Thr Ser Leu Pro 290 295 300Gln Tyr Asp Val Ser Glu Ala Tyr Ala Leu His Thr Leu Thr Phe Ala305 310 315 320Arg Pro Ser Ser Ala Ala Ala Leu Ala Phe Val Trp Ala Gly Leu Pro 325 330 335Gln Gly Gly Thr Ala Pro Ala Gly Thr Pro Ala Trp Glu Gln Ala Ser 340 345 350Ser Gly Gly Tyr Leu Thr Trp Arg His Asn Gly Thr Thr Phe Pro Ala 355 360 365Gly Ser Val Ser Tyr Val Leu Pro Glu Gly Phe Ala Leu Glu Arg Tyr 370 375 380Asp Pro Asn Asp Gly Ser Trp Thr Asp Phe Ala Ser Ala Gly Asp Thr385 390 395 400Val Thr Phe Arg Gln Val Ala Val Asp Glu Val Val Val Thr Asn Asn 405 410 415Pro Ala Gly Gly Gly Ser Ala Pro Thr Phe Thr Val Arg Val Pro Pro 420 425 430Ser Asn Ala Tyr Thr Asn Thr Val Phe Arg Asn Thr Leu Leu Glu Thr 435 440 445Arg Pro Ser Ser Arg Arg Leu Glu Leu Pro Met Pro Pro Ala Asp Phe 450 455 460Gly Gln Thr Val Ala Asn Asn Pro Lys Ile Glu Gln Ser Leu Leu Lys465 470 475 480Glu Thr Leu Gly Cys Tyr Leu Val His Ser Lys Met Arg Asn Pro Val 485 490 495Phe Gln Leu Thr Pro Ala Ser Ser Phe Gly Ala Val Ser Phe Asn Asn 500 505 510Pro Gly Tyr Glu Arg Thr Arg Asp Leu Pro Asp Tyr Thr Gly Ile Arg 515 520 525Asp Ser Phe Asp Gln Asn Met Ser Thr Ala Val Ala His Phe Arg Ser 530 535 540Leu Ser His Ser Cys Ser Ile Val Thr Lys Thr Tyr Gln Gly Trp Glu545 550 555 560Gly Val Thr Asn Val Asn Thr Pro Phe Gly Gln Phe Ala His Ala Gly 565 570 575Leu Leu Lys Asn Glu Glu Ile Leu Cys Leu Ala Asp Asp Leu Ala Thr 580 585 590Arg Leu Thr Gly Val Tyr Pro Ala Thr Asp Asn Phe Ala Ala Ala Val 595 600 605Ser Ala Phe Ala Ala Asn Met Leu Ser Ser Val Leu Lys Ser Glu Ala 610 615 620Thr Ser Ser Ile Ile Lys Ser Val Gly Glu Thr Ala Val Gly Ala Ala625 630 635 640Gln Ser Gly Leu Ala Lys Leu Pro Gly Leu Leu Met Ser Val Pro Gly 645 650 655Lys Ile Ala Ala Arg Val Arg Ala Arg Arg Ala Arg Arg Arg Ala Ala 660 665 670Arg Ala Asn 675 59 base pairs nucleic acid unknown unknown DNA unknown 53GGGGATCCAC AGTTCTGCCT CCCCCGGACG GTAAATATAG GGGAACCATG GTCTAGAGG 59 29 base pairs nucleic acid unknown unknown DNA unknown 54GGCCGCTTAA TTAAGGATCC GGCGCGCCA 29 29 base pairs nucleic acid unknown unknown DNA unknown 55CGAATTAATT CCTAGGCCGC GCGGTGATC 29 8 base pairs nucleic acid unknown unknown DNA unknown 56TTAATTAA 8 8 base pairs nucleic acid unknown unknown DNA unknown 57GGCGCGCC 8

Claims

1. A protein capsovector for use in controlling insect pests, the capsovector consisting of modified capsid protein or modified capsid protein and capsid protein of a small RNA insect virus, said modified capsid protein comprising said capsid protein of a small RNA insect virus fused to an exogenous insecticidal protein toxin, wherein said modified capsid protein or said modified capsid protein and said capsid protein are assembled into a viral capsid encapsidating said protein toxin within the assembled capsid, wherein, following ingestion of the capsovector by an insect, the assembled capsid protects the protein toxin from inactivation in the insect gut, and wherein said small RNA insect virus belongs to the family Tetraviridae or Nodaviridae and possesses a gene encoding said capsid protein, which provides all of the sequence information required for viral capsid assembly, encapsidation of viral RNA, binding of viral capsid to host cells, and release of viral RNA for translation in said host insect cells.

2. A capsovector as claimed in claim 1 in which the insect small RNA insect virus is HaSV.

3. A capsovector as claimed in claim 1 in which the capsid protein is P71 (SEQ ID No. 50).

4. A capsovector as claimed in claim 1 in which the insecticidal toxin is of plant origin.

5. A capsovector as claimed in claim 1 in which the insecticidal toxin is Ricin A or diphtheria toxin.

6. A capsovector as claimed in claim 1 in which the small RNA insect virus belongs to the family Tetraviridae.

7. An RNA capsovector for use in controlling insect pests, the capsovector consisting of capsid protein of a small RNA insect virus which is assembled into a viral capsid encapsidating an exogenous nucleic acid which encodes an insecticidal protein toxin, wherein following ingestion of the capsovector by an insect, the assembled capsid protects the nucleic acid molecule from inactivation in the insect gut, and wherein said small RNA insect virus belongs to the family of Tetraviridae or Nodaviridae and possesses a gene, encoding said capsid protein, which provides all of the sequence information required for viral capsid assembly, encapsidation of viral RNA, binding of viral capsid to host insect cells, and release of viral RNA for translation in said host insect cells.

8. A capsovector as claimed in claim 7 in which the nucleic acid is RNA.

9. A capsovector as claimed in claim 7 in which the small RNA insect virus is Heliothis armigera stunt virus (HaSV).

10. A capsovector as claimed in claim 7 in which the capsid protein is P71 (SEQ ID No. 50).

11. A capsovector as claimed in claim 7 in which the insecticidal toxin is of plant origin.

12. A capsovector as claimed in claim 6 in which the insecticidal toxin Ricin A or diphtheria toxin.

13. A capsovector as claimed in claim 7 in which the capsovector includes a further nucleic acid sequence, the further nucleic acid sequence encoding the capsid protein.

14. A capsovector as claimed in claim 7 in which the small RNA insect virus belongs to the family Tetraviridae.