Patent Application
20170304350
The invention concerns a pharmaceutical composition for treating a viral infection caused by a member of the Reoviridae family; a method of treatment involving the use of same; and use of the anti-viral to treat said viral infection. The invention has use in both humans and animals.
How virus genomes, particularly those in the Reoviridae, are packaged into their protective coats, or capsids, during infection is unknown. This is especially so for viruses with multipartite genomes, as a copy of each segment of the genome must be incorporated into the capsid for the virus to be viable.
Members of the Reoviridae family can affect the gastrointestinal system (such as Rotavirus) and respiratory tract. Viruses in the family Reoviridae have genomes consisting of segmented, double-stranded RNA (dsRNA).
Reoviruses are non-enveloped and have an icosahedral capsid (T-13) composed of an outer and inner protein shell. The genomes of viruses in Reoviridae contain 10-12 segments which are grouped into three categories corresponding to their size: L (large), M (medium) and S (small). Typically segments range from about 3.9 to 1 kbp and each segment encodes 1-3 proteins.
Rotavirus is a genus of double-stranded RNA virus in the family Reoviridae. There are eight species of this virus, referred to as A, B, C, D, E, F, G and H. Rotavirus A, the most common species, causes more than 90% of rotavirus infections in humans. The genome of rotavirus is segmented and consists of 11 unique double helix molecules of RNA which are 18,555 nucleotides in total. The RNA is surrounded by a three-layered icosahedral non-enveloped protein capsid. There are six viral proteins (VPs) that form the virus particle (virion). These structural proteins are called VP1, VP2, VP3, VP4, VP6 and VP7. In addition to the VPs, there are six non-structural proteins (NSPs), that are only produced in cells infected by rotavirus. These are called NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6. Each helix, or segment, is a gene numbered 1 to 11 by decreasing size.
Bluetongue virus (BTV) is a complex, multi-layered, segmented double-stranded RNA virus and is the type member of the Orbiviruses, a genus in the family Reoviridae. As such it shares a virus family relationship with several other scientifically and medically important viruses (e.g. Rotaviruses). BTV is transmitted by insect vectors, replicating in both insect and mammalian cells, and can cause high morbidity and mortality in animals. BTV particles are non-enveloped, architecturally complex particles organised in two capsids. The outer capsid is composed of VP2 and VP5, which are responsible for virus entry in mammalian cells. The icosahedral inner capsid or core, with a diameter of 75 nm, is composed of two protein layers, the surface layer of 260 trimers of VP7 (38 kDa) which is built on a thin scaffold made up of 60 dimers of VP3 (100 kDa). The VP3 layer encloses a viral genome of 10 dsRNA segments of discrete sizes (S1-S10), together with the transcription complex of three proteins, VP1, VP4 and VP6 termed the subcore. The genome is ˜19 kb in size, separated into 10 individual segments, S1 to S10 (0.8 to 3.9 kb), which encode 7 structural (VP1-VP7) and 4 non-structural (NS1-4) viral proteins, each of which is involved in various stages of the virus replication cycle. The ten segments of BTV vary both in sequence and size (from 0.8 kb to 3.95 kb) but are clustered in three distinct size classes (large, S1-S3; medium, S4-S6 and small, S7-S10).
BTV enters mammalian cells via an endocytic pathway where the particle is uncoated (removal of VP2 and VP5) to release core particles into the cytosol. The genomic dsRNA segments are never released from the core; rather the intact core produces 10 capped mRNAs (ssRNAs), one from each genomic segment, which are repeatedly synthesised by core-associated enzymes and extruded into the cytoplasm via pores in the 12 vertices of the icosahedral structure. These newly synthesised ssRNAs serve as messenger RNAs (mRNAs) that express all viral proteins but they also serve as templates following packaging into newly formed viral cores where the synthesis of genomic dsRNAs subsequently takes place. In BTV-infected cells, ssRNA packaging and the assembly of cores occurs within virus-induced inclusion bodies (VIBs), a matrix structure that is enriched with NS2 protein. NS2 has been shown to have sequence-specific affinity for BTV ssRNA segments and it also recruits BTV ssRNAs into the viral assembly location in BTV-infected cells. However, we have recently shown that NS2 is not necessary for in vitro ssRNA packaging.
Some segmented RNA genome viruses appear to have a non-selective packaging mechanism, for example, the two segments of infectious bursal disease virus genome are randomly packaged into virions and produce a large proportion of non-infectious particles with incomplete genome. However, we suggest it is mathematically impossible for the members of Reoviridae, containing 9 to 12 genome segments, to adopt this mechanism as the percentage of infectious particles containing complete genome would be too small. Moreover, the particles to infectivity ratio for these viruses is high, also in our view suggesting that there must be a selective packaging mechanism allowing the multiple genomic segments to correctly and effectively package into newly formed capsids.
Here, we show for the first time that there is a packaging order for how ssRNA viral segments can be packaged into their protective coats, or capsids, during infection and that this can be exploited for the purpose of controlling viral replication and so infection.
According to a first aspect of the invention there is provided a pharmaceutical composition effective against a member of the Reoviridae family and comprising at least one oligonucleotide complementary to, and so able to bind with, an untranslated region (UTR) of nucleic acid located, either 5′ or 3′, adjacent the coding region of at least one of the viral genome segments that constitutes the viral genome together with at least one pharamaceutically acceptable carrier.
In a preferred embodiment of the invention said viral genome segment constitutes the smallest segment within the viral genome or the smaller of the total segments within the viral genome having regard to size and being typically, but not exclusively categorised as small (S) within the Reoviridae.
Additionally, or alternatively, said oligonucleotide is complementary to, and so binds with, the longest UTR of nucleic acid located, either 5′ or 3′, adjacent the coding region of at least one of the viral small (S) genome segments, typically but not exclusively the smallest viral (S) genome segment that constitutes the viral genome.
Most ideally, said oligonucleotide is complementary to, and so binds with, the UTR of nucleic acid located, either 5′ or 3′, adjacent the coding region of said viral genome segment selected from the group comprising S6, S7, S8, S9, S10, S11 and S12.
More preferably, said untranslated region of nucleic acid is located 3′ of said coding region. Alternatively, said untranslated region of nucleic acid is located 5′ of said coding region.
We demonstrate herein that among the Reoviridae family, for example in the Blue Tongue Virus (BTV), of all the genomic segments the smallest segment (such as S10 in BTV) with the longest UTRs (5′ UTR and 3′ UTR together=155 bases) initiates RNA packaging. We show herein that S10 plays a crucial role in initiating genome packaging within the viral capsid by representing the fundamental packaging member that recruits the other smaller RNA segments to form a complex or complexes that can then interact with the larger genome segments.
In other words, we show that ordered RNA-RNA interactions are required for packaging the RNA segments in segmented dsRNA viruses.
As used herein, the term “oligonucleotide” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to viral RNA comprising an UTR located adjacent a particular viral gene segment and thereby, inhibits the function of the bound RNA and so prevents viral packaging in the viral capsid.
The term “modified” also encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleotides may also include 2′ substituted sugars such as 2′-O-methyl-; 2-0-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
Modified nucleotides are known in the art and include alkylated purines and/or pyrimidines;
acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5 carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; □-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2 methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psuedouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; 1-methylcytosine. Modified double stranded nucleic acids also can include base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996). The use of modified nucleotides confers, amongst other properties, resistance to nuclease digestion and improved stability.
Those skilled in the art will recognize that the exact length of the oligonucleotide and its degree of complementarity with its UTR target will depend upon the specific UTR target selected, including the sequence of the target and the particular bases which comprise that sequence.
It is preferred that the oligonucleotide be constructed and arranged so as to bind selectively with the UTR target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
In order to be sufficiently selective and potent for inhibition, such oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 12, 13, 14, or 15 consecutive bases the majority of which are complementary to the target. Most preferably, the oligonucleotides comprise a complementary sequence of 20-30 bases. Most preferably still, the oligonucleotides comprise a complementary sequence of bases to the entire 5′ or 3′ UTR.
Most preferably the oligonucleotide of the invention is, in ascending order of preference, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical with the target UTR with which it is complementary.
The term oligonucleotide is to be construed as a material manufactured either in vitro using conventional oligonucleotide synthesising methods which are well known in the art or oligonucleotides synthesised recombinantly using expression vector constructs.
Ideally said selected viral genome segment(s) is/are the smallest or smaller in the viral genome. Ideally, any one or more of S7-10 in BTV; any one or more of S7-10 in AHSV; any one or more of S6-11 in Rotavirus; and any one or more of S6-12 in Colorado Tick Virus.
In yet a more preferred embodiment of the invention said oligonucleotide is complementary to, and so able to bind with, at least a part of a UTR selected from Table 1.
In a further preferred embodiment of the invention said oligonucleotide is complementary to the whole ora part of SEQ ID Nos: 9, 10 11, 12, 13, 14, 15, 16, 17, 18 and 19.
In a preferred embodiment of the invention, said Reoviridae virus is selected from the group comprising: Cardoreovirus, Mimoreovirus, Orbivirus, Phytoreovirus, Rotavirus, Seadornavirus, Aquareovirus, Coltivirus, Dinovernavirus, Idnoreovirus and Mycoreovirus.
More preferably still, said Reoviridae virus is selected from the group comprising: Rotavirus such as the Colorado tick virus (neurologic disease), Reovirus such as Aquareviruses (infects fish and mollusks), fusogenic orthoreviruses (causes pulmonary disease in human), orbiviruses (African horse sickness, kills horses), Bluetongue (infects sheep and cattle), Seadornavirus (infects human), Avian reovirus and Rice dwarf virus (Phytoreovirus).
In yet a further preferred embodiment of the said oligonucleotide is selected form the group comprising:
The pharmaceutical composition of the invention may comprises a plurality of said oligonucleotides each one of which is complementary to, and so able to bind with, an untranslated region of nucleic acid located, either 5′ or 3′, but ideally 3′, adjacent the coding region of at least one of the viral genome segments that constitutes the viral genome. In preferred embodiments of the invention said oligonucleotides are designed to target both the 5′ and 3′ UTR of at least one selected viral genome segment and in yet a further preferred embodiment said oligonucleotides are designed to target a UTR of a plurality of selected viral genome segments, more preferably yet, said oligonucleotides are designed to target both the 5′ and 3′ UTR of said plurality of selected viral genome segments.
The present invention, thus, includes pharmaceutical compositions containing natural and/or modified oligonucleotide molecules that are complementary to and hybridize with, under physiological conditions, an untranslated region of nucleic acid located, either 5′ or 3′, adjacent the coding region of at least one of viral genome segments that constitutes the viral genome, together with at least one pharmaceutically acceptable carrier (e.g. polymers, liposomes/cationic lipids).
Preferably, the pharmaceutical composition may include the oligonucleotide(s) in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art (e.g. liposomes). The compositions should be sterile and contain a therapeutically effective amount of the oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
Oligonucleotides may be administered as part of a pharmaceutical composition.
Formulations for administration include those suitable for oral, rectal, nasal, bronchial (inhaled), topical (including eye drops, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous and intradermal) administration and may be prepared by any methods well known in the art of pharmacy.
The route of administration will depend upon the condition to be treated but preferred compositions are formulated for intravenous, parenteral, oral, nasal, bronchial or topical administration.
The composition may be prepared by bringing into association the oligonucleotide of the invention and the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a oligonucleotide of the invention in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.
For compositions for oral administration (e.g. tablets and capsules), the term “acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
For topical application to the skin, formulations may be made into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.
Oligonucleotides of the invention may be used for the treatment of the respiratory tract by nasal, bronchial or buccal administration of, for example, aerosols or sprays which can disperse the pharmacological active ingredient in the form of a powder or in the form of drops of a solution or suspension. Pharmaceutical compositions with powder-dispersing properties usually contain, in addition to the active ingredient, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non-ionic or anionic surfactants and/or diluents. Pharmaceutical compositions in which the pharmacological active ingredient is in solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant, compressed air can also be used, it being possible for this to be produced as required by means of a suitable compression and expansion device.
Parenteral formulations will generally be sterile.
The precise amount of a oligonucleotide of the present invention which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the tissue level of the agent to the concentration required to have a therapeutic effect.
According to a further aspect of the invention there is provided a combined pharmaceutical composition comprising a pharmaceutical composition according to the invention and one or more different additional anti-viral agents.
Those skilled in the art will appreciate that the additional anti-viral agents are conventionally known.
According to a second aspect of the invention there is therefore provided a method for treating a viral infection comprising administering to an individual to be treated an effective amount of a pharmaceutical effective against a member of the Reoviridae family and comprising an oligonucleotide complementary to, and so able to bind with, an untranslated region of nucleic acid located, either 5′ or 3′, adjacent the coding region of at least one of the viral genome segments that constitutes the viral genome.
According to a further aspect of the invention there is therefore provided a method for treating a viral infection comprising administering to an individual to be treated a pharmaceutical composition according to the invention.
According to a yet further aspect of the invention there is provided the pharmaceutical composition of the invention to treat a viral infection.
According to a yet further aspect of the invention there is provided use of the pharmaceutical composition of the invention in the manufacture of a medicament to treat a viral infection.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.
Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
An embodiment of the present invention will now be described by way of example only with reference to the following wherein:
Table 1. Nucleic acid sequences of the 5′ and 3′ Untranslated Regions either side of the coding sequence for the genome segment indicated in Bluetongue virus BTV-1 and BTV-10 (rows 1-2); AHSV-4 row 3; Rotavirus C (row 4); Colorado tick virus (rows 5-6).
Table 2. The 2′O-methyl modified antisense oligoribonucleotides (ORNs) used for in vivo and in vitro studies. The ORN name, sequence (5′-3′), length, target BTV RNA segments regions are listed.
Table 3. Summary of RNA-RNA interactions studies. Left panel: Interactions between two RNA segments of purified or co-transcribed RNAs. Right panel: Interactions among three or four segments by co-transcription. Values (%) are from mean and standard deviation of >3 independent experiments (n=3-5).
Table 4, Interactions of multiple BTV segments in the presence or absence of ORNs (left panel) and S7/S8/S9 with S10 VVT or S10 with deletion mutants (right panel). Values (%) are the mean and standard deviation of >3 independent experiments made relative to control (no ORN or S10 WT) set at 100% (n=3-5).
Table 5. Summary of the inhibitory effects of ORNs. Plus (+) sign indicates inhibitory effects and negative (−) non-inhibitory effects deduced from different assays performed.
Plasmids and DNA templates. To generate T7 transcripts, template plasmids containing a T7 promoter and a specific restriction enzyme site flanking cDNA of exact copies of each BTV-1 genome segment (South African reference strain, Genbank accession numbers FJ969719-FJ969728), BTV-10 S10 (U.S. isolate, NC006015), AHSV-4 S10 (FJ183368), and Rhesus Rotavirus (RRV) S9 (EU636932.1) derived from viral dsRNA using the method of full-length amplification of cDNA (FLAC) were used. Chimeric S10 constructs were generated using 5′ primers encoding T7 promoter and 3′ primers (available upon request). A sequencing marker, replacing the sequence of 384-399 nt from 5′-GTTGAAAAGTGACCTA-3′ (SEQ ID No: 1) to 5′-ACTAAAGAGCGATTTG-3′ (SEQ ID No: 2) was also introduced in each chimeric S10 construct.
For the generation of S10 RNA deletion mutants, two S10 deletion constructs corresponding to the target sequences of S10.2 (39 nts) and S10.5 (34 nts) ORNs were generated by polymerase chain reaction (PCR) through site-directed mutagenesis (37). Amplicons were then treated with Dpnl to digest the parental plasmid prior to transformation into competent cells. For the generation of four S10 RNA substitution mutants S10.2713-718, S10.2725-730, S10728-732 and S10.5743-748 site directed mutagenesis was performed by overlapping PCR using S10 specific primers. Deletion and interchanging 3′UTRs of S8 and S10 were also generated by overlapping PCR followed by Dpn 1 treatment. Capped BTV RNA transcripts for in vitro translation assay were generated using mMESSAGE mMACHINE® Kit (Ambion) as described previously. For generation of uncapped ssRNA for cell-free assembly, linearized DNA were incubated at 37° C. for 2 h with 40 U of T7 RNA polymerase (Thermo Scientific), 50 mM DTT, 0.5 mM each rNTP and 10 U RNase inhibitor (Thermo Scientific).
Bluetongue virus serotype 1 (BTV-1) South African reference strain was plaque purified and amplified in BSR cells, a BHK 21 clone derivative of baby hamster kidney cells (American Type Culture Collection) grown in Dulbecco modified Eagle medium containing 5% fetal calf serum (FCS) penicillin, streptomycin and amphotericin B at 35° C. with 5% CO2. Virus stocks were maintained by infecting BSR cells at multiplicity of infection (MOI) of 0.1 and harvested at 48-72 hpi.
Generation of T7 transcripts. Capped and uncapped ssRNAs were generated as previously described (17) using mMessage RNA (Ambion) and T7 High (Thermos) respectively.
Cell-free in vitro assembly assay. Packaging of viral ssRNAs was investigated using a recently established cell-free assembly (CFA) assay (8). Packaging efficiency was estimated using either 32P-labelled ssRNAs or non-radioactive qRT-PCR. For 32P-labelled ssRNAs, T7 transcripts were 3′ end-labelled with 10 μCi 5′-32P-cytidine (Perkin-Elmer) using T4 RNA ligase (Fermentas). The CFA assay was carried out as described previously (8). Briefly, VP1, VP4, VP6, VP3 and VP7 were sequentially in vitro translated from capped ssRNA of coding regions, followed by incubation with full-length 10 BTV uncapped ssRNAs to allow viral core assembly. The whole mixture was loaded onto a continuous sucrose gradient and fractions were collected after ultracentrifugation. In the relevant fraction (fraction 6), unpackaged RNAs were eliminated by RNase One (Promega) digestion. Packaged RNA was extracted and analysed by denaturing agarose gel electrophoresis. To detect radiolabelled RNA, the gel was dried and exposed to a Storage Phosphor screen and analysed with Phosphor-imager and ImageQuantTL software (GE Healthcare).
The cell-free system for BTV was carried out as described (10) with some modifications. Briefly, VP1, VP4 and VP6 were synthesized from RRL system followed by incubation with the complete set of 10 full-length (300ng each) uncapped ssRNAs with or without 20 pmol S10.1, S10.2, S10.4, S10.5, S10 AUG and Scr ORNs. In vitro synthesized VP3 and VP7 were then added to the mixture and further incubated to allow viral core assembly. After eliminating unpackaged RNA by RNase One (Promega) digestion, the assembled particles in the reaction mixture were isolated by a 15% to 65% continuous sucrose gradient followed by fractionation as described previously (10). For positive control, S10.2 and S10.5 ORN gradients, packaged RNAs were extracted from fractions 5, 6 and 7 and analysed by denaturing 1% agarose gel electrophoresis to identify the packaged 10 ssRNAs (10). Only fraction 6 was collected for samples with S10.1, S10.4, S10.5, S10 AUG and Scr (packaged ssRNAs are previously shown to be present at this fraction) (10). For analysis of in vitro incorporated proteins, the in vitro synthesized viral proteins were radio labelled with 35S-methionine, analysed in 9% SDS-PAGE and detected by autoradiography.
Quantitative RT-PCR. For detection of non-radioactive ssRNA, BTV-1 S6 or chimeric S10 were analysed by qRT-PCR using either primers reported by Toussaint et al (18) or BTV-1 S10 335F: 5′-GTTGAAAAGTGACCTAGGAGGC -3′ (SEQ ID No: 3) and BTV-1 S10 492R: 5′-TTCACCACACCTAACATTGGG -3′ (SEQ ID No: 4), respectively. BTV RNAs from the packaging assay were reversely transcribed (RT) into cDNA using ReverseAid Premium Reverse Transcriptase (Thermo) and quantified with suitable primers using 7500 Fast Real-Time PCR system and SYBR select Master Mix (Applied Biosystems). Three independent experiments were undertaken and qPCR was performed in duplicate. Standard deviations from the three experiments were calculated.
In vivo Packaging Assay. 106 BSR cells were transfected as previously described (19) with 2 μg of uncapped T7 transcripts of wild-type or chimeric S10. The cells were subsequently infected with BTV-1 at a multiplicity of infection (MOI) of 3. After 12 h, allowing for one replication cycle to be completed, cells were lysed and aliquots were stored for transfection control. Viral cores were then purified from the major portions of lysates as previously described. Unpackaged RNAs were digested with RNase at a final concentration of 1 μg/μl. Viral genomic RNA was then extracted, precipitated and subjected to qRT-PCR with a primer specific for the marker sequence (5′-ACTAAAGAGCGATTTG-3′) (SEQ ID No; 2) located in non-UTR or BTV-1 S10: BTV-1 S10 marker R: 5′-CCCAAATCGCTCTTTAG-3′ (SEQ ID No: 5). Copy number of marked S10 was correlated to the total BTV-1 S6 representing the number of total viral cores. Transfection discrepancy was further correlated with the copy number of marked S10 detected in the stored cell lysate aliquots.
Reverse Genetics (RG) System. To generate the virus with chimeric S10, BSR cells were firstly transfected with pCAG plasmid encoding primary replication complex (VP1, VP4 and VP6), VP3 and NS2 as described previously (17), followed by a second transfection with capped chimeric S10 ssRNA together with the remaining 9 BTV-1 ssRNAs. Replication of recovered viruses was visualised by plaque assay. To confirm the recovery of mutant virus, genomic dsRNAs were purified from the infected cells, reverse transcribed and the integrity of chimeric S10 was confirmed by nucleotide sequencing (Source Bioscience).
To generate the virus with S10 mutants (S10.2713-718, S10.2725-730, S10728-732 and S10.5743-748, and chimeric S10 and S8) BSR cells were transfected with mutated S10 ssRNA together with the remaining 9 BTV-1 ssRNAs as described previously (12, 40). For combined chimeric S10 and S8, BSR cells were transfected with mutated S10 ssRNA together with the remaining 8 BTV-1 ssRNAs. Replication of recovered viruses was visualised by crystal violet staining. Virus recovery was quantified by qRT-PCR using specific BTV genomic primers as previously described (9). To confirm the recovery of mutant virus, genomic dsRNAs were purified from the infected cells, reverse transcribed and the mutated sequences of S10 was confirmed by nucleotide sequencing (Source Bioscience).
RNA interaction assay. ssRNA of BTV-1 S10 was attached to beads leaving its 5′ and 3′ ends free by the following methods: Streptavidin agarose beads (Novagen) were coated with a biotin-labelled primer which annealed to nt 401-700 in the coding region of S10 (5′- biotin-TTTTTTTTTTTGTATTAT AGCTCTTTTCTTCTTTAAGCCTC -3′) (SEQ ID No: 6). The beads were incubated with poly-A RNA to decrease non-specific binding. BTV-1 S10 was then incubated with the coated beads followed by the addition of other 32P-labelled or non-labelled RNAs in an RNA folding buffer previously described (20). After 20 min incubation at 30° C., the beads were washed three times with excess folding buffer followed by 1 min heating at 90° C. to release the RNA. For the radiolabelled RNA assay, samples were analysed by a denaturing gel and phosphor screen exposure. For non-labelled RNA, samples were analysed by qRT-PCR using primers specific for the target RNA, as described above. The S8 coated beads and S3 coated beads were similarly prepared using the biotin-labelled primers: 5′-biotin-TTTTTTTTTTGC TTCATCATCATCCAGCGTGACTCTTCCCTTGGC -3′ (SEQ ID No: 7) for S8 beads and 5′-biotin-TTTTTTTTTTCAAC ATCTATTGTAGCCCATCCATTAT ATCCTGTTCCTG -3′ (SEQ ID No: 8) for S3 beads.
Design of Antisense Oligoribonucleotides with 2′O-Methyl Modifications Based on Prediction of ssRNA Structures
A series of thirteen antisense oligoribonucleotides (ORNs) were designed to hybridize either the 5′UTR including the AUG initiating codon, the internal coding region or the 3′ UTR of segments S1, S9 and S10 (Table 2). These ORNs were modified at the ribose with 2′O-methyl group (Integrated DNA Technologies) and named by their target position in each segment (
Optimization of Inhibitory Conditions of 2′OMe ORNs and Challenge with BTV-1
To determine the optimal inhibitory condition for each ORNs, a concentration range (0.5, 1.5 and 2.5 μM) of S10 AUG, S10 3′ UTR and SCR were transfected to BSR cells using Lipofectamine 2000 (Life Technologies). After 3 h incubation, the cells were infected with BTV-1 at MOI 0.1 for 1 h. The inoculum was removed by 3 washes with low pH medium (DMEM-HCl, pH 6) to inactivate free virus, twice with normal medium to restore pH and incubated with DMEM supplemented with 1% FCS and the appropriate ORNs for one virus replication cycle of 16-18 h. Cells were harvested and the virus titre was analysed by plaque assay. The virus yield was calculated as the mean of plaque forming units per ml (PFU/ml) of three independent transfection assays with each 2′OMe ORNs and expressed as the relative PFU/ml of BTV1 transfected without ORNs, consider as 100%. Cytotoxicity was determined by cell staining at the end of the treatment. The optimal concentration for the ORNs was 1.5 μM.
Different concentration range (0.5, 2 and 4 μM) of ORNs S1 AUG, S1.3′, S9 AUG, S9.1, S9.2, S10.1, S10.2, S10.3, S10.5, S10 AUG or Scr were incubated with BTV transcripts (300 ng) for 20 min at 37° C. and added to a reaction mix containing 7.5 μl of nuclease-treated rabbit reticulocyte lysate (RRL, Promega), 1 mM amino acid mix minus methionine and 6 μCi 35S-methionine. Translation reaction was incubated at 30° C. for 90 min and treated with 1 μl of 1 μg/μl RNase A for 10 min at room temperature. Labelled proteins were quantified by densitometry using PhosphorlmagerTM screen. The inhibition of BTV protein expression was calculated relative to the control lacking ORNs. The experiment was repeated at least three times.
For RNA-RNA interactions of individual RNA segments, 1 μg of linearized plasmid was transcribed in a buffer containing 40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20 mM NaCl2, 3 mM spermidine, 50 mM DTT, 5 mM each rNTPs, 10 U RNase inhibitor and 40 U of T7 RNA polymerase (Thermo Scientific) for 3 hours at 37° C. followed by RNase free DNase 1 treatment. Transcribed RNAs were extracted by standard phenol-chloroform method and re-suspended in RNase free water. RNAs were individually heated at 80° C. for 1 min, ice chilled and mixed in pairs in folding buffer (50 mM Na cacodylate pH 7.5, 300 mM KCl and 10 mM MgCl2) (38) and RNA-RNA complexes were allowed to form for 90 min at 30° C. and immediately analysed by electrophoresis in 1% agarose gel supplemented with 0.1 mM MgCl2. Electrophoresis gel was run for 180 minutes min at 150 V in TBM buffer (45 mM Tris, pH 8.3, 43 mM boric acid, 0.1 mM MgCl2) and stained with 0.01% (w/v) ethidium bromide. The integrity of transcribed RNA was checked by denaturing gel electrophoresis.
For co-transcription experiments, 150 ng linearized plasmid of each segments (S7-S10) were transcribed either in pairs or combinations of 3 to 4 plasmids (S7, S8, S9 and S10 or S10 mutants). RNA transcription was carried out in the same condition as individual RNA segments. Immediately after transcription and DNase 1 treatment, the reaction was analysed on a 1% agarose gel as described above. The percentage of the retarded RNA in each lane was determined against the total mass of input RNA (%) by densitometry (Gene Tools, Syngene). For RNA complex inhibition assay with ORNs, the simultaneous transcription of S7-S10 (combination of 3 or 4) was performed in the presence or absence of 20 pmol of S10.1, S10.2, S10.4, S10.5 and Scr ORNs and analysed as described above. Non-specific yeast tRNA (20 and 50 pmol) was incorporated in the co-transcription reaction as a control. Quantification of intermolecular RNA complex was performed as described above.
For RNA-ORN hybridization assay, 10 pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3′ end labelled with 10 μCi [32P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4° C. overnight. Unincorporated 32P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare). Prior to hybridization, unlabelled S10 RNA was denatured at 80° C. for 1 min, immediately chilled and then mixed with folding buffer (50 mM sodium cacodylate pH 7.5, 100 mM KCl and 10 mM MgCl2). RNA-ORN hybridization was performed with 0.5 pmol of pre-folded S10 RNA annealed with 32P labelled ORNs (1, 2 and 5 pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs) in folding buffer in 10 μl final volume (39). The complex was allowed to form for 30 min at 30° C. followed by electrophoresis in 4% native acrylamide gel at 4° C. for 50 min. at 150V in TBM buffer, dried and exposed by autoradiography.
To investigate if there is a preferential packaging of BTV genome segments, we sequentially excluded one RNA segment from mixtures of the full 10 RNA segments and used the recently developed in vitro cell-free assembly (CFA) assay to determine the RNA packaging into the assembled core (8). Each of the BTV inner core proteins was translated sequentially in an in vitro translation system together with ten full-length +ve sense single-stranded ssRNA segments. To avoid any interference between protein translation and ssRNA packaging, only the coding region of each BTV transcript was used for translation assay while uncapped full-length ssRNAs were used for packaging. In brief, VP1, VP4 and VP6, the proteins that form the polymerase complex, were first generated individually using S1, S4 and S9 segments respectively and then all 3 proteins were mixed and incubated with a set of 32P-labelled T7-driven 10 full-length BTV transcripts. For each experiment either a set of the complete 10 ssRNA segments, or a set of 9 segments excluding one large (S2), one medium (S5) or one small (S10) ssRNA segment was used. The reaction mixture was then incubated sequentially with in vitro expressed VP3 to form the subcore and VP7 to form a stable core structure. The newly assembled cores were purified by a sucrose gradient centrifugation and the fraction containing cores (fraction 6,
To further examine and quantify the effect of exclusion of each RNA segment, we undertook a further set of packaging experiments. Except for S6, all segments were excluded individually from the set of non-radioactive 10 ssRNA segments in the CFA assay. In vitro assembled cores were purified and treated with RNase as before. An aliquot of each sample was stored for protein analysis and packaging was measured using quantitative RT-PCR (qRT-PCR) for a marker BTV ssRNA, S6. The S6 packaging efficiency from each set of 9 ssRNA segments packaging versus the complete 10 ssRNA set of control experiments was assessed. The qRT-PCR comparison results demonstrated that when S1, S2 or S3 was excluded, the packaging efficiency was ˜50%, while the exclusion of S4, S5 or S7, the medium size ssRNAs, reduced packaging further (15˜30%); but most strikingly, the packaging efficiency was as little as 10% or less when any of the smaller segments (S8, S9 and S10) were excluded (
As S10 appears to play a critical role in BTV genome packaging, we investigated the relative roles of size and sequence identity. In many RNA viruses, specific packaging signals are mainly located in UTRs. Furthermore, among BTV RNA segments, although S10 is the smallest of all BTV RNA segments (822 bases), the 3′ UTR of S10 is unusually long (118 bases) when compared to the UTRs of other 9 RNA segments of BTV and is highly conserved among all serotypes. To verify if BTV S10 5′ and the long 3′ UTRs contain packaging signals, we designed chimeric ssRNA segments based on the coding region of BTV S10, with UTRs from different sources. To identify if S10 UTRs are essential for genomic RNA packaging, the UTRs of BTV-1 S10 were substituted with the UTRs of BTV-1 S3, S5, or S8, which are all different in both size and sequence. To verify the specificity of S10 UTR sequences, the UTRs of BTV-1 S10 were substituted with the UTRs of an alternate BTV serotype, BTV-10. The S10 of these two serotypes have similar but not identical sequences. In addition, S10 UTRs of a related orbivirus, African Horse Sickness Virus (AHSV), were also used to replace the UTRs of BTV-1 S10 (
To determine the effects of altered UTRs on RNA packaging, each chimeric S10 together with the remaining 9 BTV-1 ssRNA transcripts were used in the CFA system described above. In parallel, wild-type S10 transcripts were used as a positive control. The chimeric S10 transcripts that were packaged into the newly constituted cores were quantified with qRT-PCR and the packaging efficiency compared to that of the control. When the BTV-1 S10 UTRs were substituted with the UTRs of S3, S5, or S8 of the same serotype, the packaging was significantly reduced in each case, indicating that the UTRs of S10 were important for S10 incorporation into the core. Similarly, changing the S10 UTRs of BTV-1 to the UTRs of AHSV-4 also reduced packaging substantially. However, replacement with S10 UTRs of an alternate BTV serotype (BTV-10), to BTV-1 UTRs, influenced the packaging only moderately (˜70% efficiency,
To determine if this effect can also be reproduced in vivo, we adapted a recently established in vivo single cycle packaging assay (19). The principle of this assay is that when BTV replicates in the cell, the progeny assembling cores will incorporate viral ssRNA segments from the cytoplasm. Therefore, if viral ssRNAs are transfected into cells prior to infection, both newly synthesised transcripts and transfected transcripts will be encapsidated. To perform the in vivo experiment, each chimeric S10 was introduced with a modified sequence in the coding region (nt 395, sufficiently distant to the UTRs) to facilitate specific detection and quantification by RT-PCR. This modification does not alter the amino acid sequence or the length of the segment (
These effects were not restricted to BTV, but also confirmed in other members of the Reoviridae family (
The above studies demonstrate that changing the UTRs of S10 influenced packaging both in vitro and in vivo. To further determine if poor levels of packaging can be compensated in the cellular environment, we verified the effect of the chimeric S10 constructs on viral replication using a BTV reverse genetic (RG) system which allows for the introduction of an altered genome segment in a replicating virus (17). Accordingly, the five chimeric S10 constructs described above were introduced together with the remaining 9 other wild-type BTV segments using the RG system in an attempt to recover mutant viruses. Among the five chimeric mutants, only BTV carrying BTV-1/BTV-10 chimeric S10 was successfully recovered, as examined by plaque morphology and titres, in comparison to that of the wild-type virus (
Since S10 UTRs appear to be essential for BTV RNA packaging, we determined if certain specific region/regions in the UTRs are involved in RNA packaging. The 3′ terminal nucleotides of S10 was sequentially deleted from 12 to 60 nucleotides (12, 35 and 60) and each of these truncated S10 ssRNAs, together with remaining 9 full-length ssRNAs, were used for packaging in the CFA assay. When the packaging efficiency of each set was assessed, even the deletion of 12 nucleotides from the 3′ terminus suppressed packaging by more than 50%, and additional deletions further decreased packaging (
S10 Interacts with Other BTV RNA Segments.
As the smallest BTV RNA segment, S10, appears to initiate the packaging of the remaining RNA segments, we investigated if S10 RNA recruits other segments by direct interaction. To detect interactions between different RNA segments, we designed a primer binding assay based on streptavidin beads as shown in a schematic (
Isolated S10 exhibited an affinity in vitro for the smaller BTV segments but not the medium or large segments. However, for BTV genome packaging, all 10 segments have to be included to form a complete genome set. To enable this, the smaller segments plausibly form a complex which is then linked to other segments. To verify this hypothesis, we added S6, S7, S8 and S9 onto the S10 beads followed by incubation of the mixture with S1 or S5, as representatives of large and medium size segments respectively, each of which previously failed to bind to S10 directly. Clearly, in the presence of other small segments, both S1 and S5 were successfully pulled-down but not by S10 alone, while there was no change for the RRV RNA control (
Further, we investigated whether the increased affinity was mediated by one or more specific segments of the four RNA segments, S6-S9, or if all RNA segments formed a complex to recruit larger segments. When each of the small segments, S6 to S9 was added separately into the interaction assay of S10 with S5, only S9 slightly enhanced the interaction (
This was substantiated further by using an alternate smaller segment, S8, instead of S10, where the data obtained was similar (
The aforementioned data demonstrates that smaller segments are more important for BTV RNA packaging and that BTV RNA segments may form networks of size-related groups. Based on these, we hypothesised that such networking is important for BTV genome packaging. To demonstrate this, only certain genome segments were used in CFA system (
The data above showed that smaller size class RNA segments (S7-S10), in particular, S10, triggers a series of RNA-RNA interactions that initiate the recruitment and packaging of positive sense ssRNAs during BTV assembly. Based on these data, we sought to investigate whether small specific antisense oligoribonucleotides (ORNs) targeting the 3′ terminal sequences of these smaller segments would interfere with BTV growth. A set of oligonucleotides (ORNs) complementary to the UTRs of positive sense ssRNA segments, S9 and S10 were designed (
Six ORNs complementary to different regions including the 3′ conserved terminus of the S10 (FFIG. 1C) were designed to interfere with the RNA structures, and three of which encompass the entire length of the S10 3′ UTR. S10.1 was complementary to the extreme 41 nt (nt822-782) including the conserved sequence, S10.2 should base pair 39 nt from the stop codon towards the 3′ terminus (nt737-699) and the 34 nt of S10.5 complimentary to nt781-748, the gap between S10.1 and S10.2. The other ORNs targeted the structure outside of the 3′UTR; S10.3 to the terminal 35 nucleotides of the coding region (ORF), S10.4 in the ORF (nt595-561) and S10AUG, the initiation codon. For segment 9 (S9), the 3′ UTR consists of 44nts (nt1049-1006), and thus, three ORNs encompassed part of the UTR and part of the 3′ ORF (
After optimizing the concentration of ORNs for in vivo assay, BSR cells were transfected with each ORNs and Scr ORNs at an optimal concentration of 1.5 μM. At 3 hours post transfection (hpt), cells were infected with BTV-1 of MOI of 0.1 and virus titres were monitored 16 hpi. Analysis of each ORN-transfected BSR cells followed by infection with BTV-1 showed ORNs had a negative effect on virus yield albeit to a varying degree with all S10 ORNs. Specifically, ORN S10.2 was the most inhibitory where virus yield was reduced by ˜90% while S10.3 had also a significant effect on virus replication with ˜70% reduction in comparison to that of the control (
Based on the inhibitory results of the ORN targeting the 3′UTR, we also investigated the effect of an ORN that encompasses an entire 3′UTR. We selected S1 as it possesses the shortest 3′UTR (24 nt) of all BTV RNA segments. To this end, we designed an ORN complementary to the entire length of the 3′UTR and, as positive control, another to the 5′UTR including the AUG codon (
Since antisense oligonucleotides could trigger stearic blocking of viral mRNA and thereby perturb the translation of viral mRNAs, we examined if the inhibition of virus growth was due to the interfering effect of ORNs on the efficiency of virus protein expression. To validate this, we performed a cell-free translation in the presence or absence of ORNs complementary to the initiation codons of S1 (VP1), S9 (VP6) and S10 (NS3/NS3A) or the 3′ UTR region. Analysis of translated products showed that VP1, VP6, NS3/NS3a viral proteins were efficiently translated in the presence of ORNs complementary to the 3′UTR regions (
Complex Networks of ssRNA Segments and Disruption by ORNs
Previous RNA-RNA interaction data have shown that small size class RNA segments (S7-S10) interact with each other and package prior to the recruitment of medium and large RNA segments. To obtain direct evidence for interactions between smaller segments, we used electrophoretic mobility shift assay (EMSA) to measure RNA interaction following two different experimental approaches: (1) Co-incubation of two purified ssRNA segments for hybridisation assay and (2) Co-transcription of T7 cDNA copies of segments in pairs or in combinations of 3 or 4. The EMSA analysis of co-incubation products exhibited shifted weak bands for combinations S7+S8, S7+S9 and S7+S10 (
S7 interacts with each of the other three small segments. Other RNA segment combinations did not show any distinct retarded bands (
In three or four co-transcribed RNA segments, stronger intermolecular interactions were detected with additional shifted bands each case and the amount of free, unbound RNA was also less than when only two segments were co-transcribed (
The specificity of RNA-RNA interactions was tested in the presence of non-specific competitor yeast tRNA at 20-50-fold molar mass excess and the level of complex formation was not significantly reduced (
To determine if the RNA complexes following co-transcription of multiple segments could be disrupted by ORNs targeting the S10 3′UTR, all four small RNA segments or different combinations of three (S7+S8+S9, S7+S8+S10, S7+S9+S10, S8+S9+S10) were co-transcribed in the presence or absence of 20 pmol of either S10.2 and S10.5 ORNs (most inhibitory ORNs in virus replication) or S10.4 ORN (non-inhibitory ORNs targeting the ORF) (see
The specificity of the ORN to inhibit RNA-RNA interactions was also shown by the non-inhibitory capacity of the Scr to disrupt the RNA complexes (
Hybridization assay also showed that ORN S9 AUG and ORN S9.2 hybridized with S9 mRNA. Similarly, ORN S10 AUG and ORNs S10.2, S10.3, S10.5 annealed to S10 mRNA. No hybridization with Scr control was detected when incubated with S10 and S9 mRNAs.
Identification of Regions in S10 Responsible for Interactions with Other Segments
The decreased RNA complex formation in the presence of S10 3′UTR ORNs prompted us to explore the key regions in S10 RNA responsible for recruiting other segments to form a complex. Deletion mutants in S10 which spanned the sequence of inhibitory ORN binding region were constructed and used in the RNA-RNA interactions with other segments (
Table 4 summarizes the results obtained from RNA-RNA interaction studies in the presence or absence of ORNs and S10 deletion mutants.
To further understand the mechanism of action of S10.2 and S10.5 ORNs and to determine if the inhibitory effects of ORNs on virus growth and RNA-RNA interactions were directly related to BTV RNA packaging during capsid assembly, we utilised a unique cell-free core assembly system for packaging BTV RNAs that has been successfully used to understand the order of capsid assembly and the genomic segment packaging. For this study, S10.1, S10.2, S10.5, S10.4, S10 AUG and Scr ORNs were pre-annealed to S10 transcripts prior to mixing with the remaining 9 BTV ssRNA segments and subsequently incubated with pre-translated transcription complex (VP1, VP4 and VP6) before adding two major core proteins, VP3 and VP7. After removing the unpackaged ssRNAs by RNase treatment, the putative cores in in vitro assembled complexes were centrifuged on a sucrose gradient, fractionated, ssRNAs isolated and analysed as described in Methods and Materials. Only S10.2 or S10.5 ORNs, (in fraction 6) inhibited the packaging of 10 BTV ssRNA with ˜80% reduction for S10.2 ORN and ˜60% reduction for S10.5 ORN (
To confirm that core proteins were synthesised efficiently in the cell-free assembly assay, each protein was 35S-labeled and the fractionated complex was analyzed by SDS-PAGE. The 35S-labelled reconstituted protein products showed the complete set of core proteins, the three proteins of transcription complex (VP1, VP4 and VP6) and the two major core proteins (VP3 and VP7) from fraction no. 6 in the presence or absence of S10.2 ORN (data not shown) which demonstrated that the transcription complex (TC) and the subcore proteins were efficiently synthesized and assembled and were not hindered in the presence of S10.2 ORN. The effects of different ORNs in RNA packaging by in vitro assembly, in vivo virus replication, in vitro protein synthesis and RNA-RNA interactions are summarized in Table 5.
To confirm if the sequences within the identified 3′UTR regions in S10 RNA are important for RNA packaging in vivo, four substitution mutants were introduced by targeting five or six nucleotides in the putative binding sites of S10.2 and S10.5 regions at the S10 3′UTR (
To investigate further if the identified packaging signals in S10 3′UTR are interchangeable with other segments, 3′ UTRs of S8 and S10 were exchanged (S8-UTR10 and S10-UTR8) and chimeric ssRNAs were synthesized. When BSR cells were transfected with each of the chimeric RNA segments together with 9 WT ssRNA segments or all 10 WT ssRNAs as control, only control VVT virus was recovered while both chimeric segments failed in virus recovery. Further, virus recovery with combined S8-UTR10 and S10-UTR8 was unsuccessful. These data suggest that the packaging signals in the UTRs were not functional when interchanged between different segments.
For the Reoviridae members with a multiple dsRNA segmented genome and complex capsid assembly process, understanding viral packaging, and so replication or viral yield, is highly challenging process. The data described in this report shows that members of the Reoviridae family assemble ssRNA segments of the viral genome through a selective and sequential process prior to packaging same as an RNA complex, moreover, this assembly is orchestrated by the smallest segment (e.g. S10 in BTV).
The core of BTV and other members of the family is a highly rigid icosahedral structure with 12 pores, one in each fivefold vertex, through which the newly synthesised viral positive strand ssRNAs extrude. Our recent data has shown that transcripts of each segment extrude through a specific pore and that it is not a random process. For the reverse process, i.e. the packaging of ssRNA through these narrow channels, RNA segment entry would have to occur one at a time, making it difficult to explain how excluding a single segment influences the packaging of other segments.
Our data showed that ORNs targeting the 3′ UTRs of the small segments (e.g. S9 and S10) had strong inhibitory effects on virus growth but not on protein synthesis suggesting that the inhibition occurred after viral protein synthesis and prior to genome encapsidation, at the stage of genome packaging.
Our data strongly suggests that the smallest segment, especially its UTRs, play a crucial function in RNA packaging and it is likely that interaction of the 5′ and 3′ UTRs drive the formation of secondary structure of ssRNAs, necessary for recruiting other ssRNAs.
However, other small segments (S7, S8 and S9) also interact with the smallest segment (S10) to trigger the assembly of all (10) ssRNAs. The data rationalises this finding by showing that the smaller segments form a complex, which then recruits the other segments. Interestingly, although the largest segment, S1, was captured by the small segment complex the interaction was enhanced significantly when large segments S2 and S3 were added. This data suggests that the 10 BTV segments may form several complexes which combine to result in a form compatible for packaging. Moreover, although S10 is crucial to BTV RNA packaging, neither S10 alone nor S10 plus other smaller segments were packaged efficiently. Only when larger segments were included were all segments equally packaged. This is consistent with an “all-in” genome incorporation model despite the fact that RNA-RNA interactions adopt a “follow-the-leader” model to assemble the packaging complex.
Our data indicates that the UTRs of S10 are critical for BTV assembly through a sequence-specific or secondary structure-specific mechanism. Even a short deletion (12 NT) from the 3′ terminus of S10 perturbed the packaging of the ssRNAs during assembly. Alignment of the different BTV serotypes shows a high level (over 80%) of conservation in the unusually long S10 UTR (shown in Table 1). It is possible that S10 interacts with other segments in some of these regions, which is consistent with the model that RNA secondary structure serves as the genome packaging signal for segmented viruses.
The complex formation through specific RNA-RNA interactions and the critical role of Small Segment 3′ UTRs were also substantiated by the reduction of RNA complex formation in the presence of ORNs (S10.2 and S10.5), which blocked critical motifs in the 3′ UTR of BTV S10, in particular, the predicted structure that consisted of GC rich hairpin loops and bulges. Blocking these motifs with ORNs affected the binding capacity of S10 for the other interacting RNA segments which was further confirmed by deleting regions corresponding to S10.2 and S10.5 ORN binding regions. Reduction in RNA complexes detected with deletion mutants AS10.2 and AS10.5 suggested that either the deleted sequences may form a part of the binding site necessary for RNA-RNA interactions or the deletions might have disturbed the secondary structure in these regions.
Our work suggests a critical role for the untranslated region of viral small segments in the virus life cycle, likely at the packaging and assembly level. This also suggests that the key interacting sequences in small segments are located at the 3′UTR of RNA segments.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1418135.8 | Oct 2014 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2015/052925 | 10/7/2015 | WO | 00 |
