Patent Application
20060247196
Upon infection, the genomes of positive-strand RNA viruses are translated to yield a variety of proteins. Some of these direct the assembly of an RNA replication complex, which first synthesizes a negative-strand RNA replication intermediate and then uses this negative strand as a template for producing more positive-strand genomic RNAs. Several lines of evidence suggest that multiple steps in positive-strand RNA virus RNA replication depend on specific host factors. Different host cells show differing levels of permissiveness for various intracellular replication steps (W. De Jong and P. Ahlquist, J. Virol. 69:1485-1492, 1995; A. V. Gamarnik and R. Andino, EMBO J. 15:5988-5998, 1996). The replication complex of each virus assembles on specific membrane sites in the infected cell (S. Froshauer, et al., J. Cell Biol. 107:2075-2086, 1988; K. Bienz, et al., J. Virol. 66:2740-2747, 1992; M. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), and such association with cell membranes appears particularly important for positive-strand RNA synthesis (S. Wu, et al., Proc. Natl. Acad. Sci. USA 89:11136-11140, 1992). Partial purification of some positive-strand RNA replication complexes has shown them to involve complexes of viral and cellular proteins, and some of the cell proteins in such complexes have been implicated as potentially functional contributors to replication (R. Quadt, et al., Proc. Natl. Acad. Sci. USA 90:1498-1502, 1993; T. A. M. Osman and K. W. Buck, J. Virol. 71:6075-6082, 1997; Yamanaka, et al., Proc. Natl. Acad. Sci. USA 97:10107-10112, 2000).
To facilitate studying the mechanisms of positive-strand RNA virus replication and the nature and function of host proteins involved, we have shown that brome mosaic virus (BMV) RNAs and their derivatives can replicate and direct gene expression in the yeast Saccharomyces cerevisiae, the rapid growth, facile genetics, and completely sequenced genome of which offer potentially useful features for virus replication studies. BMV replication in yeast reproduces all known features of BMV RNA replication in naturally plant hosts, including localization fo replication complexes to the endoplasmic reticulum, dependence on the same viral replication factors and on the same cis-acting RNA replication signals, similar ratios of positive to negative strand RNA, and other features (M. Janda and P. Ahlquist, Cell 72:961-970, 1993; M. Sullivan and P. Ahlquist, J. Virol. 73:2622-2632; M. Ishikawa, et al., J. Virol. 71:7781-7790, 1997; M. Restrepo-Hartwig and P. Ahlquist, J. Virol. 73:10303-10309, 1999; R. Quadt, et al., Proc. Natl. Acad. Sci. USA 92:4892-4896, 1995).
BMV encodes two RNA replication factors, 1a and 2a, containing three domains conserved throughout the large alphavirus-like superfamily of animal and plant viruses (P. Ahlquist, Curr. Opin. Genet. Dev. 2:71-76, 1992). BMV1a (109 kDa) contains an N-proximal helicase-like domain, whereas 2a (94 kDa) contains a central polymerase-like domain. BMV1a and 2a interact (C. C. Kao, et al., J. Virol. 66:6322-6329, 1992; C. C. Kao and P. Ahlquist, J. Virol. 66:7293-7302, 1992; E. Smirnyagina, et al., J. Virol. 70:4729-4736, 1996) and in vivo colocalize on the endoplasmic reticulum at the sites of BMV RNA synthesis (M. Restrepo-Hartwig and P. Ahlquist, supra, 1996). BMV 1a and 2a are encoded by BMV RNA1 and RNA2, respectively. A third genomic RNA, RNA3, encodes the 3a cell-to-cell movement protein and the coat protein, which are required for BMV infection spread in its natural plant hosts but are dispensable for RNA replication (R. Allison, et al., Proc. Natl. Acad. Sci. USA 87:1820-1824, 1990; K. Mise and P. Ahlquist, Virology 206:276-286, 1995). The 3′-proximal coat gene is not translatable from RNA3 but only from a subgenomic mRNA, RNA4, synthesized from negative-strand RNA3 (
Yeast expressing 1a and 2a from DNA plasmids replicate RNA3 or RNA3 derivatives and synthesize subgenomic mRNAs to express the coat gene or other genes substituted for it. Replicatable RNA3 derivatives can be introduced into yeast by transfection of in vitro transcripts (M. Janda and P. Ahlquist, Cell 72:961-970, 1993) or by in vivo transcription of an RNA3 cDNA flanked 5′ by a DNA-dependent RNA polymerase promoter and 3′ by a self-cleaving ribozyme (M. Ishikawa, et al., J. Virol. 71:7781-7790, 1997). Such cDNA-based RNA3 launching cassettes can be carried on yeast plasmids (M. Ishikawa, et al., supra, 1997) or, as shown here, integrated into a yeast chromosome. Expression of reporter genes substituted for the coat gene in RNA3 launching cassettes provides colony-selectable or -screenable markers for all forms of BMV RNA-dependent RNA synthesis, because such expression requires 1a-, 2a-directed negative-strand RNA synthesis, and subgenomic mRNA synthesis, and is strongly reduced if RNA-dependent positive-strand RNA amplification is blocked (M. Janda and P. Ahlquist, supra, 1993; M. Ishikawa, et al., supra, 1997).
The invention described below depends on the inventors' new understanding of yeast host genes required for viral replication. This information was obtained using the above-described BMV expression system and is described in detail below. Needed in the art of antiviral techniques is a method of preventing viral replication involving knowledge of essential host genes.
In one embodiment, the present invention is an antiviral agent comprising an altered MAB1 gene, MAB2 gene, MAB3 gene, OLE1 gene, gene homolog or related gene. The agent is capable of inhibiting viral replication in a host cell.
In a preferred embodiment of the present invention, the host cell is a microbe or a eukaryotic cell. In a most preferred embodiment of the present invention, the host cell is a plant, animal, or yeast cell.
In another form, the present invention is a method of creating a virus-resistant organism comprising creating a transgenic organism containing an antiviral agent selected from the group of an altered MAB1 gene, MAB2 gene, MAB3 gene, OLE1 gene, homologs of these genes, related genes, and combinations of these genes and homologs.
The present invention also includes a method of creating a virus-resistant organism comprising creating a transgenic organism comprising an antiviral agent selected from the group of antisense sequences or sense sequences designed to alter the expression of MAB1, MAB2, MAB3 gene or OLE1 gene expression or MAB1, MAB2, MAB3 or OLE1 gene homologs or genes related to MAB1, MAB2, MAB3 or OLE1.
The present invention is also a method of increasing or optimizing replication of a virus or virus derivative by expression of MAB1, MAB2, MAB3 or OLE1 or a related or homolog gene from the same or different cell type, or combinations of such genes, or expression of modified versions of such genes, or alteration of the natural expression levels of such genes, to optimize the replication of the virus or derivative.
In a preferred form of the above-described method, the viral derivative is an expression vector derivative, the viral replication is within a plant or animal cell, or the viral replication is within a microbial cell.
It is an object of the present invention to provide antiviral agents and create transgenic virus-resistant organisms comprising these agents.
Other objects, advantages and features of the present invention will become apparent after one of skill in the art reviews the specification, claims and drawings.
A. In General
In one embodiment, the present invention is directed towards antiviral agents developed from the observation that four particular yeast genes (MAB1, MAB2, MAB3 and OLE1), have been found by the inventors to affect viral replication. By “antiviral” we mean inhibitory to RNA viruses, preferably positive strand. In another embodiment, the invention is inhibitory to double-stranded RNA viruses.
In the Examples below, we describe specific yeast genes whose mutation was found to inhibit brome mosaic virus (BMV) replication, identification of the MAB1, MAB2, MAB3 and OLE1 genes by their ability to restore BMV-directed RNA replication and expression in the mutants, and characterization of these yeast genes and yeast genes products. We inventors also describe a proposed use of the MAB1, MAB2, MAB3 and OLE1 genes (or homologs or related genes) to develop antiviral agents and vector systems.
The antiviral uses of the present invention include constructing a transgenic organism containing an altered MAB1, MAB2, MAB3 or OLE1 gene or containing antisense nucleic acids designed to alter the native function of the MAB1, MAB2, MAB3 or OLE1.
B. Evaluation of Altered Genes
By “altered gene” is meant mutated, enhanced, synthetic, or duplicated MAB1, MAB2, MAB3 or OLE1 genes (including some or all of the coding, non-coding, regulatory, and promoter regions that inhibit viral replication). The inventors envision that the particular gene mutations that they studied are not the only mutations of the MAB1, MAB2, MAB3 and OLE1 that would result in analogous antiviral activity. Thus, the present invention encompasses any mutation or alteration of MAB1, MAB2, MAB3 or OLE1 that results in significant alteration of viral replication. This mutation may result in the absence of gene expression or in the expression of an altered product.
One would obtain an altered gene by mutagenizing or altering a wild-type MAB1, MAB2, MAB3 or OLE1 gene. To obtain a wild-type MAB1, MAB2, MAB3 or OLE1 gene, one would most typically examine the nucleotide sequences of the gene (see Table 1, below, for reference to the yeast protein database and GenBank Accession Number and obtain primers sufficient to amplify the gene from a yeast genomic library. (GenBank Accession Nos. Z49399, U32517, Z71340 and Z72577 are incorporated by reference as if set forth entirely herein.) Of course, other methods would be known to one of skill in the art.
To mutagenize or alter the gene, one would look below to the Examples for one method of creating altered genes. Of course, other methods are known to those of skill in the art, including those described in Umen and Guthrie, Genetics 143:723-739, 1996, incorporated by reference herein.
One would test the candidate altered cene by methods described in the sections below. For example, one might test a candidate altered gene by use of a virus-directed reporter gene expression system, as exemplified below with BMV.
One would also be able to construct RNA-mediated interference agents, such as an antisense, sense or double-stranded transcript designed to inhibit or alter gene function (See Fire, et al., Nature 391:806-811, 1998, Nature 391:744-745, 1998; Bingham, et al., Cell 90:385-387, 1997, all incorporated by reference as if fully set forth herein).
Additionally, the inventors envision that virus replication can be inhibited by decreasing or increasing the expression of the gene (MAB1, MAB2, MAB3 or OLE1 or combinations of such genes), expressing a related gene or gene homologs from the same or a different cell type, or altering the natural copy or copy number of the gene or combinations of such genes. One might also express altered versions of the gene, or of related gene or genes from the same or a different cell type, or combinations of such altered genes in addition to the natural copy of the gene.
The inventors envision expressing derivatives of the gene or of a related gene or genes from the same or a different cell type, such as partial segments of the gene or fusions of the entire gene or segments thereof to other protein domains.
A preferred altered gene of the present invention is a dominant negative mutation of MAB1, MAB2, MAB3, OLE1 or related genes or homologs. One would characterize such a mutation by the ability of the mutation to shut down viral replication once the mutated gene is present in the host organism, even when the wild-type gene is also present in the organism. One of skill in the art would develop these mutations by generally known procedures. For example, one would review Herskowitz, I., et al., Nature 329:219-222, 1987 as a reference for mutations of cellular genes, Baltimore, D., et al., Nature 335:395-397, 1998 for viral genes, and Holzmayer, et al., Nucl. Acid. Res. 20:711-717, 1992 for lambda bacteriophage, and Brachmann, et al., Proc. Natl. Acad. Sci. USA 93:4091-4095, 1996 for dominant negative mutations of p53 selected in yeast.
C. Homologs and Related Genes
The inventors also envision that there are homologs to MAB1, MAB2, MAB3 and OLE1 in other organisms. For example, the inventors are aware that LSM/MAB1 and a related set of interacting LSM proteins are conserved from yeast to humans (Schweinfest, et al., Canc. Res. 57:2961-2965, 1997; Salgado-Garrido, et al., EMBO J. 18:3451-3462, 1999; Achsel, et al., EMBO J. 18:5789-5802, 1999). A “homolog” is defined herein as a gene, preferably from another species, with a sufficient sequence or functional similarity to MAB1, MAB2, MAB3 or OLE1 such that the homolog functions in a similar manner in the non-yeast species. The inventors also envision that there may be homologs of MAB1, MAB2, MAB3 and OLE1 present in yeast species. Such genes can be identified by sequence similarity or by functional screens.
With regard to OLE1, the inventors note that Δ9 fatty acid desaturase (the function encoded by OLE1 in yeast) is also the rate-limiting, initial enzyme for UFA synthesis in animals (J. M. Ntambi, J. Lipid Res. 40:1549-1558, 1999).
The inventors also envision that the manipulation of genes related to MAB1, MAB2, MAB3 and OLE1 could alter viral expression. By “related gene” is meant an associated gene. For example, MAB1, MAB2, MAB3 or OLE1 may be part of a functional complex and the alteration of a complex member may prove effective to alter viral replication. In one example, MAB3 is identified below as a member of a chaperone complex, involving many cofactors. These other cofactors may also be candidates for antiviral agents. (See Hu, et al., EMBO J. 16:59-68, 1997 for a discussion of Hepadnavirus involvement in the chaperone complex.)
D. Host Organisms
The inventors envision that the present invention will be useful in a variety of organisms, most particularly plants, human, microbe and animal cells.
E. Use of Antiviral Agents of the Present Invention
The present invention is therefore an antiviral agent comprising an altered MAB1, MAB2, MAB3 or OLE1 gene or homologs of these genes, or related genes, or combinations of these genes, wherein the agent is capable of altering viral replication in a host cell. In one embodiment, the altered gene or gene homolog is a mutated gene. In another embodiment, the altered gene is a naturally occurring gene and the host cell contains an antisense or sense transcript capable of altering gene expression.
In a preferred embodiment, the invention is directed to increasing or optimizing replication of a virus or virus derivatives by expression of the gene (MAB1, MAB2, MAB3, OLE1), a related or homolog gene from the same or a different cell type, or combinations of such genes, or expression of modified versions of such genes, or alteration of the natural expression levels of such genes, to optimize the replication of a virus or its derivatives, including expression vector derivatives, in any of the various cell types described above. To use the antiviral agents of the present invention, one might use a variety of molecular biological methods. For example, one might construct a transgenic plant cell with a altered gene corresponding to a homolog of MAB1, MAB2, MAB3 or OLE1. One would expect that this plant cell would be not capable of supporting viral replication and, when regenerated into a mature plant, would provide a virus-resistant plant.
Similarly, other transgenic viral-resistant organisms may be created through standard techniques known to those of skill in the art of molecular biology.
One could most easily determine whether an organism is virus-resistant by challenging the organism with a test virus and evaluating viral replication relative to a control challenge.
F. Use of Genes as Argets for Antiviral Screens
We envision that the MAB1, MAB2, MAB3 or OLE1 gene, regulatory sequences and regulatory factors and encoded proteins are appropriate targets for antiviral drug discovery. Such discovery can be pursued by any of a number of in vivo or in vitro methods well-known to those in the art, such as screening libraries of chemical compounds for effects on the expression, stability and activity of the OLE1 protein. Procedures to assay for gene expression, protein levels and enzyme activity are well-known in the field. Among these are, e.g., nucleic acid hybridization- and reverse transcription/PCR-based methods for assaying specific mRNA levels, and ELISA and other assays for specific protein levels. Assays for enzyme activities such as the Δ9 fatty acid desaturase activity of OLE1 could be based on a variety of detection approaches, such as the incorporation or release of a chemical group assayable by fluorescence, light absorbance, radioactivity, etc.; a conformational change in a reaction product relative to a reaction substrate, leading to changes in fluorescence, light absorption or other property, etc.; and many other approaches known to those in the art. We specifically envision Δ9 fatty acid desaturase genes and gene products in other organisms as targets for antiviral drug discovery, as described above.
In one embodiment, the present invention is a method of evaluating a substance as an antiviral therapy, comprising the steps of (a) exposing a substance to a protein selected from the group consisting of the MAB1, MAB2, MAB3 or OLE1 expression products, and (b) evaluating the effect of the substance on the stability or activity of the protein expression product, wherein the inhibition of the activity or stability expression product indicates that the substance is a possible antiviral therapy. Screens for compounds that inhibit enzyme activity would likely be conducted with extracts of cells rather than purified protein and might also be conducted with whole cells. For example, using OLE1 as an example, screens for compounds selectively affecting OLE1 protein stability would preferably be conduced in cells or animals. Selective effects on OLE1 protein (Ole1p) stability would likely result from the effects of a compound on the complex in vivo systems affecting Ole1p intracellular localization and on systems like ubiquitin ligase, the proteasome, etc. that affect protein degradation.
In another embodiment, the present invention is a method of evaluating a substance as antiviral therapy, comprising the step of (a) exposing the substance to a protein expression system, such as whole cells in culture or a purified transcription or protein expression system, wherein the system expresses a protein selected from the group consisting of MAB1, MAB2, MAB3 or OLE1 expression products, and (b) evaluating the effect of the substance on the expression level of the expression product, wherein the inhibition of the expression level indicates that the substance is a possible antiviral therapy.
Screens for compounds that induce changes in gene expression and protein expression most likely would be conducted using cells in culture, not purified transcription or protein expression systems. Such tests would eventually also be conducted in whole animals. Using OLE1 as an example, selective changes in OLE1 transcription, e.g., might be inducible by a drug because OLE1 expression is regulated by extracellular levels of some lipids and related products. Such lipids likely bind to one or more cell surface receptors that in turn send specific signals to the nucleus to alter OLE1 transcription. The candidate drug might interact with this cell surface receptor (or a downstream factor in the signal transduction pathway) to induce selective effects on OLE1 transcriptional regulation. In a preferred form of the invention, we would employ a cell culture system because in a simplified in vitro transcription system, it is less likely that we could find selective inhibitory effects on OLE1 transcription without inducing general transcription inhibition.
In another embodiment, the invention is a method of evaluating a substance as an antiviral therapy, comprising the step of (a) exposing a substance to a transcription system, wherein the system transcribes an mRNA product selected from the group consisting of MAB1, MAB2, MAB3 or OLE1 mRNAs, and (b) evaluating the effect of the substance on the expression level or stability of the mRNA product, wherein the inhibition of the expression level or decrease in stability indicates that the substance is a possible antiviral therapy. As discussed above, we envision that a preferable system will be a whole cell or cell extract system rather than a purified transcription system.
In another embodiment, the present invention is the above-identified screens for protein activity, expression and stability, and mRNA expression and stability for the enzyme encoded by OLE1, Δ9 fatty acid desaturase. Applicants note that this enzyme name is generally applied to the class of homologous enzymes from yeast and other cells. There is much homology and functional equivalents among such genes. It is known that the human protein can functionally replace the yeast protein. By the “A9 fatty acid desaturase” we mean to include all homologs of this enzyme and specifically note that mammalian organisms are known to contain several homologs. By “homolog” we mean to include enzymes that are functionally equivalent to the OLE1-encoded enzyme.
A. Mutations in Multiple Yeast Complementation Groups Inhibit Brome Mosaic Virus RNA Replication and Transcription and Perturb Regulated Expression of the Viral Polymerase-Like Gene.
1. In General
Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1-1, mab2-1 and mab3-1 (for maintenance of BMV functions), were selected for initial study. Each of these mab mutants has a single, recessive, chromosomal mutation that inhibits accumulation of BMV positive-strand and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic replication steps are defective.
mab1-1, mab2-1 and mab3-1 slowed yeast division to varying degrees and showed temperature sensitive restriction of growth, implying that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the levels of 2a mRNA and the polymerase-like 2a protein. In association with their other effects, mab1-1 and mab2-1 block the ability of 1a to stimulate 2a mRNA and protein accumulation. mab3-1, however, shows elevated 2a protein accumulation. Since mab3-1 is recessive and does not elevate 2a mRNA levels, this suggests that MAB3 both supports BMV RNA replication and contributes to degradation of at least some pools of 2a. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression, accumulation and turnover of 2a, possibly in connection with replication complex assembly.
2. Isolation of S. cerevisiae Mutants in which BMV RNA Replication is Reduced.
We have previously constructed plasmids from which BMV RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3′ end. (See M. Ishikawa, et al., J. Virol. 71(10):7781-7790, 1997, herein incorporated by reference.) Upon induction with galactose, yeast harboring such plasmids transcribed and accumulated BMV RNA3 derivatives but failed to express the gene placed in the coat protein (CP) position because the CP gene is located downstream of the 3a gene. In contrast, in yeast expressing the 1a and 2a proteins, GAL1 promoter-driven BMV RNA3 derivative RNAs are subjected to RNA-dependent RNA replication and subgenomic RNA4 transcription to express the gene placed in the CP gene position. In the system, the expression of the gene was shown to be dependent on both BMV RNA3 replication steps and subgenomic RNA4 synthesis. By using this system, we designed a screening strategy to select cells which express less efficiently the gene placed in the CP gene position to isolate mutant yeast with reduced BMV RNA replication, stability, or expression.
B3URA3 and B3GUS are BMV RNA3 derivatives with the CP gene replaced with the yeast uracil biosynthesis gene URA3 and E. coli β-glucuronidase (GUS) gene, respectively. The yeast strain YMI04 is a YPH500 derivative harboring two gene cassettes, [GAL1 promoter-B3URA3-ribozyme] and [GAL1 promoter-B3GUS-ribozyme], integrated in the chromosomal can1 and lys2 loci, respectively. In addition, YMI04 has pB1CT19 and pB2CT15, yeast 2μ plasmids carrying constitutive ADH1 promoter-driven BMV replication proteins 1a and 2a gene cassettes, respectively. YMI04 was [Ura+ Gus+] if cells were grown in galactose medium, and [Ura− Gus−] if cells were grown in glucose medium or either 1a or 2a plasmids was lost. In keeping with the Ura+ phenotype, YMI04 showed 1%-5% of plating efficiency on a 5-fluoroorotic acid (5-FOA)-containing galactose plate lacking His and Leu (omission of His and Leu was necessary to maintain 1a and 2a expression plasmids) compared to that on a corresponding plate without 5-FOA. By filter-lift assay using X-gluc, YMI04 cells grown in galactose medium developed detectable blue color within 20-30 minute incubation at 37° C. In contrast, a lys2::B3Gus strain lacking 1a or 2a plasmids developed no detectable blue color even after 24 hour incubation. Total protein extracts from YMI04 cells grown in galactose medium showed. GUS activity of approximately 20-50 nmol 4 MU/mg protein/m in.
We mutagenized YMI04 by ultraviolet irradiation, and after overnight growth on glucose plates lacking His and Leu, cells were harvested and plated on 5-FOA galactose plates lacking His and Leu to select mutants with reduced BMV-directed URA3 expression. From 1.6×105 viable cells plated, approximately 2,000 colonies that appeared between 5-7 day incubation were picked and streaked on galactose plates lacking His and Leu. Gus activity expressed in these cells was estimated by filter-lift/X-gluc assay, and 34 cells which showed slow or no blue color development were selected.
To distinguish between mutations in BMV components (1a, 2a, B3URA3 or B3GUS) and host genes, these 34 mutant candidates were mated with YMI06, a YPH499 derivative with LYS2+genotype, and diploids were generated. The resulting diploids were grown on galactose plates, and assayed for Gus activity by filter-lift assay. Among the mutant 34 candidates, 7 strains recovered Gus activity, suggesting that each of these 7 strains carried a recessive mutation that can be complemented by the yeast genome supplied by YMI06, i.e., recessive mutation on the yeast chromosome. The other 27 strains recovered Gus activity by mating with YMI06 with either active BMV 1a or 2a expression plasmids, suggesting that these 27 strains carried recessive defects in 1a or 2a genes. We selected four strains, designated #1-33, #16-10, #4-29 and #1-20 (note: mab1-4) from the 7 mutant candidates and analyzed further.
We first examined how severely the BMV-directed Gus expression was reduced in these strains quantitatively. The strains were cultured in galactose liquid medium for two days, harvested, disrupted, and the Gus activity in the resulting cell extract was measured. Gus activity for these strains ranged from 1% to 5% levels of the parental wild-type strain, YMI04. In contrast, diploids generated by mating between the strains and YMI06 showed Gus activity ranging from 50% to 90% levels of wild-type diploids generated by mating between YMI04 and YM106. These results confirmed filter-lift assay results. In wild-type diploid cells (YMI04×YMI06), the levels of Gus activity were approximately one-fifth of those in haploid YMI04. At present, we do not know why less Gus activity is expressed in diploid cells.
The growth of #16-10, #4-29 and #1-20 at 28° C. was slower than that of the parental strain YMI04. At 37° C., #1-33, #16-10 and #4-29 showed growth defect, and #1-20 showed growth defect at 15° C. Diploid strains generated by the crosses between these four strains and wild-type yeast did not show slow growth at 28° C. or growth defect at extreme conditions, suggesting that the growth phenotypes are controlled by recessive traits.
3. Each Mutant Had a Single Recessive Chromosomal Mutation Belonging to a Distinct Complementation Group.
To characterize the genetic traits controlling the phenotype of reduced Gus activity, we sporulated diploids generated by mating #1-33, #16-10, #4-29 or #1-20 and YMI08 derivatives with lys2::[GAL1 promoter-B3GUS ribozyme] integration, dissected more than 20 tetrads for each strain, supplemented 1a and/or 2a expression plasmids if necessary, grew in galactose medium, and measured Gus activity. The tetrad analysis was consistent with each mutant strain carrying a single mutation that interfered with BMV-directed GUS expression. In addition, evidence was found for some modifier locus differences between the starting parents YMI04 and YMI08. Crosses between the mutants showed that each was in a different complementation group.
B. Characterization of MAB1, MAB2 and MAB3 Genes
1. In General
Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of positive-strand RNA viruses of animals and plants. Yeast expressing BMV RNA replication proteins 1a and 2a support the replication of BMV RNA3 derivatives. We describe above the isolation of yeast strains with mutations designated mab1-1, mab2-1 and mab3-1, which inhibit BMV RNA replication and subgenomic mRNA synthesis. In this section, we describe the identification of the MAB1, MAB2 and MAB3 genes by their ability to restore BMV-directed RNA replication and gene expression in the mutants.
Three reports from the Yeast Protein Database are summarized below in Table 1 and describe the yeast genes corresponding to the mab1-1, mab2-1, and mab3-1 mutations.
In brief, we found that MAB1 encodes a 20 kDa protein containing both Sm motifs that are conserved in the core Sm proteins of snRNPs and have been suggested to be involved in protein-protein interaction. MAB2 encodes an 88 kDa protein containing similarity to the WD-40 repeat, which is found in proteins involved in signal transduction, RNA processing, and membrane vesicle traffic, and has also been suggested to participate in protein-protein interaction. MAB2 is essential for yeast growth at 30° C., while MAB1 is dispensable at 30° C. but essential at 37° C. MAB3 encodes a 409 amino acid protein with a molecular weight of approximately 44.6 kDa. The protein is present in the cytoplasm and on the endoplasmic reticulum and nuclear envelope.
Overall, the results suggest that these host genes are required for the assembly and/or function of the BMV RNA replication complex.
2. Cloning of Yeast Genomic DNA Fragments Complementing MAB1, 2 and 3 Mutations.
Isolation of the MAB1 gene. Starting from yeast strain YMI04, we previously isolated mutant yeast strains defective in supporting brome mosaic virus (BMV) RNA replication and subgenomic mRNA synthesis (M. Ishikawa, et al., supra, 1997). YMI04 is a YPH500 derivative containing 2μ DNA plasmids expressing BMV RNA replication proteins 1a and 2a and chromosomally-integrated cDNA cassettes from which replicatable BMV RNA3 derivatives B3URA3 and B3GUS are transcribed from the galactose-inducible, glucose-repressible GAL1 promoter. In these cells, the B3URA3 and B3GUS RNAs are replicated and direct synthesis of subgenomic mRNAs that express the URA3 and GUS genes, respectively. These yeast were mutagenized with ultraviolet light and subjected to a multistep selection and screening process to isolate mutant yeast strains in which BMV-directed gene expression was inhibited. One of these yeast mutants, mab1-1, was found to have a single, recessive, chromosomal mutation that inhibits BMV-directed RNA3 replication and subgenomic mRNA synthesis, and produces temperature-sensitive inhibition of yeast growth at 36° C. (M. Ishikawa, et al., supra, 1997).
To identify the responsible mutant gene, we transformed mab1-1 yeast with a yeast genomic DNA library constructed in Ycp50, a centromeric vector carrying URA3 as a selectable marker (Rose, Gene, 60(2-3):237-243, 1987). The transformed cells were plated on minimal glucose plates lacking uracil, incubated 12 hours at 24° C., and then transferred to 36° C. to screen for clones in which the growth defect at 36° C. was rescued. From each of four transformants that grew at 36° C., the library plasmid was recovered and amplified in E. coli. Restriction mapping showed that all four plasmids contained distinct but overlapping yeast DNA fragments.
One of these plasmids, designated p1012, was introduced into mab1-1 yeast and tested again for complementation of the defects in growth at 36° C. and in BMV-directed GUS expression via B3GUS RNA replication and subgenomic mRNA synthesis.
As shown in
DNA sequencing showed that the yeast DNA insert of p1012 corresponded to a region of chromosome X containing multiple open reading frames (ORFs) (
To determine whether YJL124c corresponded to the wild-type locus of mab1-1 rather than an extragenic suppressor, URA3 was integrated next to wild-type YJL124c and the wild-type MAB1 phenotype was shown to co-segregate with URA3 after crossing this strain and with mab1-1 yeast. The 0.76-Kb EcoRV-ClaI fragment of the wild-type MAB1 gene was subcloned between the SmaI and ClaI sites of pRS306, a yeast integrating plasmid bearing URA3. The resultant plasmid was digested with EcoNI to direct its integration to YJL124c, transformed into the wild-type MATa strain YMIO8, and Ura+ transformants were selected. Integration of the plasmid at the intended site was verified by Southern analysis. The Ura+ transformants were crossed with the original mab1-1 mutant, the diploids were sporulated, and the meiotic products were tested for BMV-directed GUS activity, growth at 36° C., and growth on medium lacking uracil. All 24 tetrads analyzed were parental ditypes with 2 GUS+ Ts+ Ura+ spores and 2 GUS− Ts− Ura− spores, where GUS− reflected BMV-directed GUS expression averaging 10-fold lower than GUS+ spores. Based on this close linkage of mab1-1 complementation to YJL124c and the isogenic mutant results described below, YJL124c is referred to hereafter as MAB1.
Mab1p contains similarities to core snRNP proteins. MAB1 encodes a putative protein of 172 amino acids. To test for expression of this predicted protein, a triple HA epitope tag was inserted at the N terminus of Mab1p. When introduced on a centromeric plasmid into mab1-1 yeast, the epitope-tagged MAB1 gene restored growth at 36° C. and restored BMV-directed GUS expression to wild-type levels.
Western blot analysis demonstrated that an epitope tagged protein near the anticipated size was expressed (
Mab1p contains regions similar to the two Sm motifs, which are conserved among the eight common or core proteins of the small nuclear ribonucleoprotein particles (snRNPs) that assemble to form the spliceosome directing pre-mRNA splicing (
Mab1p has been suggested to be a possible U6 snRNP-associated protein from the Sm B family (Fromont-Racine, Nat. Genet. 16:277-282, 1997). However, to our knowledge, there is no experimental evidence to support this suggestion. Immunoprecipitation analysis of yeast Mak31p showed no association with snRNP RNAs, suggesting that some proteins bearing Sm motifs may not be snRNP-associated.
MAB1 is not essential for yeast growth at 30° C. To determine if MAB1 was required for cell growth, a mab1::URA3 disruption allele was constructed by replacing 58% of the MAB1 coding region with the URA3 gene. The resulting locus can express at most only the N-terminal 33 amino acids of the 172 amino acid Mab1p, while even a much shorter C-terminal truncation abolished Mab1p function (p1138 in
Identification of the mab1-1 mutation and construction of a mab1-1 strain isogenic to YPH500. To identify the causal mutation, the mab1-1 allele was cloned by gap repair (Orr-Weaver, Proc. Natl. Acad. Sci. USA 78:6354-6358, 1981) and sequenced. Briefly, we generated two deletion derivatives of the MAB1 locus on TRP1-containing plasmids. These derivatives had deletions of the PstI/ClaI or ClaI/EcoNI fragments, which together encompass the MAB1 gene. These plasmids were independently transformed into mab1-1 yeast, resulting in recombination with the chromosomal mab1-1 allele to repair the gap and thus circularize the plasmid for replication. Such gap-repaired plasmids were recovered from Trp+ transformants, amplified in E. coli, and retransformed into mab1-1 yeast to verify lack of complementation. Sequencing revealed a single change in the form of the deletion of one adenine from a run of 7 adenines at nucleotide position 463468 in the ORF. This frameshifted the ORF after amino acid 156, resulting in translation of an additional 68 amino acids from an alternate frame prior to termination.
To insure the absence of extraneous mutations such as might be present in the original UV-mutagenized mab1-1 strain, the mab1-1 point deletion was transferred into wild-type YPH500 yeast, the progenitor of YMI04. To obtain this strain, designated mab1i, mab1Δ yeast cells were transformed with a DNA fragment containing the mab1-1 allele, and plated on 0.1% 5-fluorootic acid, which selects against yeast expressing active URA3 and thus for cells in which the mab1::URA3 locus has been replaced by recombination with the mab1-1 DNA fragment. The resulting Ura− mab1i strain showed the expected temperature-sensitive growth phenotype.
To compare BMV RNA replication and subgenomic mRNA synthesis in mab1i and mab1Δ yeast with the original mutant strain, mab1i and mab1Δ yeast were transformed with plasmids expressing 1a, 2a, and wild-type BMV RNA3.
Northern blot analysis (
mab1-1 mutation inhibits 1a-induced stabilization of 2a mRNA. In wild-type yeast, co-expression of 1a increases 2a mRNA accumulation approximately 5-fold, with a concomitant increase in 2a protein accumulation (M. Ishikawa, et al., supra, 1997 and
Isolation of the MAB2 gene. Subcloning the implicated yeast genomic DNA region in YCplac22 as discussed above showed that the mab2-1 mutation was complemented by a DNA fragment spanning from coordinates 1114114 to 1119120 in yeast chromosome IV. This fragment contains one complete open reading frame (ORF) larger than 100 codons and the N-terminal portion of a second open reading frame. However, overlapping fragments that contained the second, partial open reading frame but truncated the first open reading failed to complement mab2-1, showing that complementation was due to the first ORF. This ORF extends from coordinates 1114470 to 1116800 and is designated YDR324C in the standard nomenclature for yeast open reading frames, denoting that it is located in the right arm of chromosome IV, relative position 324 from the centromere, and is in the Crick (3′ to 5′) strand orientation. We propose to name this gene MAB2.
MAB2 is predicted to encode a 776 a protein (MW≈88 kDa) of unknown function. Following the standard yeast ORF nomenclature, this protein is designated YDR324C in the Yeast Protein Database. The MAB2 protein or Mab2p contains amino acid sequence similarities to the WD40 repeat motif, which is found in G-protein β subunits and in proteins involved in RNA processing, membrane vesicle traffic and signal transduction. This WD-40 motif has been suggested to participate in protein-protein interactions (Neer, E. J., et al., Nature 371:297-300, 1994).
An engineered mab2 mutant in which most of the MAB2 coding region was replaced with the yeast URA3 gene did not grow at 30° C., showing that MAB2 is an essential gene.
Isolation of MAB3 gene. Subcloning the implicated yeast genomic DNA region in YCplac22 as discussed above showed that the mab3-1 mutation was complemented by a DNA fragment spanning from coordinates 505713 to 507844 in yeast chromosome XIV. This fragment contains only one complete open reading frame (ORF) larger than 100 codons. This ORF extends from coordinates 507095 to 505866 and is designated YNL064C in the standard nomenclature for yeast open reading frames, denoting that it is located in the left arm of chromosome XIV, relative position 64 from the centromere, and is in the Crick (3′ to 5′) strand orientation. In our proposed nomenclature, this gene would be designated MAB3. This ORF has already been reported to encode a yeast dnaJ homolog, YDJ1/MAS5 (Caplan and Douglas, J. Cell Biol. 114:609-621, 1991; Atencio and Yaffe, Mol. Cell. Biol. 12:283-291, 1992).
The MAB3/YDJ1/MAS5 locus encodes a 409 aa protein (MW≈44.6 kDa) that can be farnesylated at a site in the C-terminal region (Caplan, et al., J. Biol. Chem. 267:1889-18895, 1992). This protein is present in the cytoplasm and on the endoplasmic reticulum and nuclear envelope (Caplan and Douglas, J. Cell Biol. 114:609-621, 1991). Yeast with a complete deletion of this gene grow very slowly at 30° C. and do not grow at 37° C. (Atencio and Yaffe, Mol. Cell. Biol. 12:283-291, 1992; Caplan and Douglas, J. Cell Biol. 114:609-621, 1991). Thus, MAB3/YDJ1/MAS5 is essential for viability at elevated temperatures and important for optimal growth at lower temperatures.
With HSP70 or HSP90, Ydj1p functions as a molecular chaperone, and is involved in various processes such as, e.g., protein import into the endoplasmic reticulum (Caplan, et al., Cell 71:1143-1155, 1992) and mitochondria (Atencio and Yaffe, Mol. Cell. Biol. 12:283-291, 1992; Caplan, et al., Cell 71:1143-1155, 1992), activation of protein kinase p60v-src (Kimura, et al., Science 268:1362-1365, 1995; Dey, et al., Mol. Biol. of the Cell 7:91-100, 1996) and steroid hormone receptors (Caplan, et al., J. Biol. Chem. 270:5251-5257, 1995) and ubiquitin-dependent protein degradation (Lee, et al., Mol. Cell. Biol. 16:4773-4781, 1996).
C. Isolation of OLE-1, a Yeast Mutant Strongly Inhibiting BMV-Directed Gene Expression.
To isolate mutants with reduced ability to support BMV-directed gene expression, we used yeast strain YM104 (M. Ishikawa, et al., Proc. Natl. Acad. Sci. USA 94:19810-13815, 1997a). YM104 contains plasmids expressing BMV 1a and 2a from the constitutive ADH1 promoter and chromosomally-integrated cassettes expressing B3URA3 and B3GUS from the galactose (gal)-inducible GAL1 promoter. B3URA3 and B3GUS are BMV RNA3 derivatives with the coat gene replaced by the URA3 and GUS genes, respectively. URA3 or GUS expression requires both gal to induce B3URA3 and B3GUS transcription, and BMV 1a- and 2a-directed RNA replication and subgenomic mRNA synthesis (
Referring to
For mutant isolation, UV-mutagenized YM104 yeast cells were plated on gal medium containing 0.1% 5-fluororotic acid to select against cells with BMV-directed URA3 expression. After 5-7 days, about 0.1% of the plated cells developed into colonies. 6,000 such colonies were examined for BMV-expressed GUS activity by filter lift assays. 300 isolates with blue color development lacking or delayed relative to wt YM104 were selected and mated with YM106, which contained no BMV sequences and had the mating type (MATα) opposite to that of YM104 (MATα). Of the resulting 300 diploids, 100 showed restored GUS activity, implying that inhibition of BMV-directed GUS expression in the corresponding YM104-derived parental haploids was due to recessive yeast chromosomal mutations complemented by the YM106 genome. One such GUS-haploid isolate, in which BMV-directed GUS expression was reduced 20-fold, was chosen for further analysis. Complementation studies showed that this mutation was independent of previously described BMV-inhibiting yeast mutations mab1, 2 and 3 (M. Ishikawa, et al., supra, 1997a).
This original mutant strain will be designated ole1w yeast because, as shown below, the causal mutation that inhibits BMV RNA replication maps to the yeast OLE1 gene. w is an allele designation to distinguish this mutation from other ole1 mutations. Ole1w yeast grew normally. Its doubling time in defined gal medium, about 5 hours, paralleled that of wt YM104 yeast. Nevertheless, BMV-directed gene expression was strongly inhibited: GUS activity per mg of total protein in extracts of ole1w yeast averaged 5% of wt YM104 yeast (
Materials and Methods
Plasmids. pB1CT19 and pB2CT15 (M. Janda and P. G. Ahlquist, supra, 1993) and pB1YT3H and pB2YT5 (J. Chen and P. G. Ahlquist, supra, 2000) were used to express 1a and 2a from the ADH1 and GALL promoters, respectively. pB1YT3H was made by substituting the HIS3 marker gene for the URA3 gene in pB1YT3 (Y. Tomita and M. Ishikawa, unpublished results), a yeast centromeric plasmid with the 1a ORF linked to the GAL1 promoter. All plasmids expressing RNA3 or its derivatives were derived from pB3RQ39 (M. Ishikawa, et al., supra, 1997b) as described. Yeast genomic DNA library ATCC77164 containing yeast strain YPH1 DNA fragments in centromeric vector pRS200 (J. Halbrook and M. F. Hoekstra, supra, 1994) was used to identify the complementing gene.
Yeast strains, cell growth, and transformation. Yeast strain YPH500 and its derivatives (M. Ishikawa, et al., supra, 1997a) were used throughout, except that YMI06 (M. Ishikawa, et al., supra, 1997a) was used for mating. YMI04, the parental strain for mutant isolation, was a YPH500 derivative containing chromosomally integrated B3URA3 and B3GUS expression cassettes and plasmids pB1CT19 and pB2CT15. Ole1Δ::URA3 yeast was constructed by integrative transformation of the YM104 with the NheI-BsrG1 fragment of
Yeast cultures were grown and harvested in mid-logarithmic phase (optical density at 600 nm=0.5-0.7) as described (M. Ishikawa, et al., supra, 1997b). Cell pellets were stored at −70° C. for RNA or protein extraction. Tergitol Nonidet P-40 (1%) was added to medium to solubilize unsaturated fatty acids (UFAs) (J. Stukey, et al., supra, 1989). Plasmid transformation was performed with the FROZEN-EZ™ yeast transformation kit (Zymo Research).
RNA Transfection. Capped in vitro RNA transcripts of B3CAT were synthesized from pB3CA101, spheroplasts were prepared from yeast grown 24 hours in gal medium, and RNA transfections were performed as described (M. Janda and P. G. Ahlquist, supra, 1993).
GUS and CAT Assays. GUS filter lifts and quantitative assays were performed as described (M. Ishikawa, et al., supra, 1997b). For CAT assays, yeast lysate was prepared as for quantitative GUS assay but using a different extraction buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 0.1% N-lauroylsarcosine, 0.1% Triton X-100, and 1× protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 1 μg/ml pepstatin A and 2.5 μg/ml each aprotinin, and leupeptin). CAT protein levels were measured with a CAT ELISA kit (Boehringer Mannheim) and total protein was determined with a Bradford protein assay kit (Bio-Rad).
Western blotting. Protein was prepared as for CAT assays except that the extraction buffer was augmented with 20 mM 2-mercaptoethanol and 2× protease inhibitors and clarified cell lysate was supplemented with 1% SDS and boiled for 5 minutes to inactivate proteases. Total protein was determined with the SDS-tolerant Bio-Rad DC Protein assay (Lowry assay). Cell lysate was electrophoresed and Western blotted as described (M. Restrepo-Hartwig and P. G. Ahlquist, supra, 1996).
Northern blotting. Total yeast RNA isolation, RNA concentration determination by absorbance at 260 nm, agarose-formaldehyde gel electrophoresis and transfer to nylon membrane were performed as described (F. M. Ausubel, et al., supra, 1987; M. Janda and P. G. Ahlquist, supra, 1993). Positive-strand RNA3 and RNA4 were detected with a 32P-labeled RNA probe complementary to their 3′ 200 bases. Negative-strand RNA3 was detected with a 32P-labeled RNA probe corresponding to the CAT gene (for B3CAT) or coat gene (for B3 and B3CPfs) coding sequence (M. Janda and P. G. Ahlquist, supra, 1993). Radioactive signals were measured with a Molecular Dynamics Phosphorimager.
Yeast OLE1 complements mutant defect in BMV-directed gene expression. To identify genes able to complement this recessive defect in supporting BMV-directed gene expression, ole1w yeast cells were transformed with a yeast genomic DNA library carried by shuttle vector pRS200, which bears the yeast TRP1 gene (J. Halbrook and M. F. Hoekstra, Mol. Cell. Biol. 14:8037-8050, 1994). Of 20,000 transformants screened by filter lift assays for BMV-directed GUS activity, 5 reproducibly showed a wild-type blue color development. From each of these transformants, a pRS200-based plasmid was isolated by its ability to permit E. coli auxotrophic strain KC8 to grow on medium lacking tryptophan (F. M. Ausubel, et al., Current Protocols in Molecular Biology, 1987). Each of these plasmids complemented the ole1w mutation when re-transformed into ole1w yeast. Sequencing both ends of the yeast genomic DNA in these plasmids revealed two overlapping fragments of yeast chromosome VII: bases 397187-406757 and bases 398499-407045. The 8.25 kb region common to both fragments contained 5 open reading frames (ORFs) of 100 or more codons and 2 tRNA genes.
By deletion mapping and filter lift assays for BMV-directed GUS activity, complementing activity was assigned to a 2.9 kb NbeI-BsrG1 fragment containing only the OLE1 ORF (
To determine whether OLE1 was the originally mutated gene or an extragenic suppressor, the ole1w gene was cloned from the mutant yeast by gap repair and used to replace the OLE1 gene in wt YM104 yeast by integrative transformation. The resulting ole1w isogenic strain reproduced the original ole1w mutant phenotype, inhibiting BMV-directed GUS expression to 5% of wt, and this phenotype was suppressed by a plasmid bearing the wt OLE1 gene (
To identify the causal mutation in the ole1w allele, restriction fragments were exchanged between the mutant and wt OLE1 genes and the recombinant plasmids were tested for ability to complement ole1w yeast. The mutant phenotype was mapped to a 280 bp DNA fragment encoding Arg167-Leu262 of the OLE1-encoded protein, Ole1p. DNA sequencing of this region in the wt and mutant genes revealed a single A to G substitution, causing a Tyr262 (TAT) to Cys (TGT) substitution in Ole1p.
UFAs restore BMV-directed gene expression in ole1w and ole1Δ yeast. Ole1p encodes the Δ9 fatty acid desaturase, an integral ER membrane protein that converts saturated palmitic (16:0) and stearic (18:0) acids into unsaturated palmitoleic (16:1) and oleic (18:1) acids (
Because BMV RNA synthesis is also associated with yeast ER membranes (M. A. Restrepo-Hartwig and P. G. Ahlquist, J. Virol. 73:10303-130309, 1999), the function and localization of OLE1 suggested two possible explanations for the inhibition of BMV-directed gene expression in mutant yeast. First, the ole1w mutation might alter the level of UFAs in yeast membranes, which might inhibit BMV RNA replication, subgenomic mRNA synthesis, or both through effects on membrane fluidity or other physical properties. In keeping with this hypothesis, the ole1w mutation (Tyr212 to Cys) is located in the predicted catalytic domain of OLE1 (J. Stukey, et al., J. Biol. Chem. 265:20144-20149, 1990). Alternatively, Ole1p itself, as an integral membrane protein, could be required as an anchor for the BMV RNA replication complex on the ER.
To determine whether BMV-directed gene expression required Ole1p itself or only URAs, we used integrative transformation to delete the OLE1 ORF of wt YMI04 yeast and replace it with the URA3 gene, creating yeast strain ole1Δ::URA3. As expected, ole1Δ::URA3 yeast was unable to grow in medium lacking UFAs (
Supplementing the original ole1w yeast with UFAs also restored BMV-directed GUS expression (
1a and 2a protein accumulation and membrane association in mutant yeast. To facilitate viral RNA accumulation studies below, we made an additional isogenic yeast strain, ole1wi′, bearing the ole1w allele but lacking the chromosomally integrated B3URA3 and B3GUS expression cassettes of YMI04 and ole1wi. This ole1wi′ strain allowed studying wt RNA3 and RNA3 derivatives introduced on plasmids, while avoiding interference from B3URA3 and B3GUS RNAs in Northern blot analysis of BMV RNA replication products. The initial BMV RNA template used was B3CAT, which combines an easily assayed reporter gene with higher accumulation of BMV RNA replication products than B3GUS.
Wt and ole1wi′ yeast were transformed with plasmids expressing B3CAT, 1a, and 2a. With ADH1-expressed 1a and 2a, ole1w1′ yeast showed wt 1a protein accumulation and slightly reduced 2a protein accumulation (
BMV-directed CAT expression in ole1wi′ yeast with ADH1-expressed 1a and 2a was 5% of wt (
Confocal microscopy and cell fractionation show that 1a and 2a are associated with ER membrane in wt yeast replicating BMV RNA (J. Chen and P. Ahlquist, J. Virol. 74:4310-4318, 2000; M. Restrepo-Hartwig and P. G. Ahlquist, supra, 1999). To determine if the ole1w mutation inhibited membrane association of 1a and 2a, ole1wi′ yeast with GAL1-expressed 1a, 2a and RNA3 were lysed, membranes were pelleted at 10,000×g, and Western blotting was used to examine the distribution of 1a and 2a between the membrane and soluble cytoplasmic fractions (J. Chen and P. G. Ahlquist, supra, 2000). As shown in
Inhibited accumulation of BMV RNA replication products in ole1wi′ yeast. To determine whether inhibition of BMV-directed gene expression by ole1w mutation was due to a defect in subgenomic mRNA (RNA4) synthesis or translation, we measured B3CAT RNA4 accumulation in wt and ole1wi′ yeast. Positive-strand RNA4 accumulation in ole1wi′ yeast was only 2% of wt (
Since B3CAT is not a natural BMV RNA replication template, we also tested wt RNA3 replication in ole1wi′ yeast. As shown in
Normal 1a-induced RNA3 stabilization in ole1wi′ yeast. In wt yeast lacking 2a, 1a acts through the cis-acting intergenic replication enhancer (RE) of positive-strand RNA3 (
In the absence of 1a and 2a, plasmid-derived, positive-strand RNA3 transcripts accumulated to equal levels in ole1wi′ yeast with or without the UFA supplementation that suppresses the ole1w phenotype (
Unexpectedly, the ole1w-dependent inhibition of RNA3 replication in lane 5 revealed that less positive-strand RNA3 accumulated in the presence of 1a+2a than with 1a alone (lanes 3-4). Further results below (
Inhibition of negative-strand RNA3 synthesis in ole1wi′ yeast. The negative-strand RNA3 synthesis pathway in yeast is not saturated by DNA-transcribed positive-strand RNA3 templates, so that negative-strand RNA3 accumulation is stimulated by RNA-dependent amplification of positive-strand RNA3 templates (M. Ishikawa, et al., supra, 1997b). Consequently, due to the cyclical nature of wt RNA3 replication (
To block RNA-dependent positive-strand RNA synthesis and test negative-strand RNA synthesis directly, the wt BMV 5′ non-coding region (NCR) of B3CPfs was replaced with the 5′ NCR of the yeast GAL1 mRNA in an expression plasmid designated B3(5′GAL,CPfs) (
Thus, for B3(5′GAL,CPfs), the only templates for negative-strand RNA3 synthesis were provided by GAL1-promoted DNA transcription, which was unaffected by the ole1wi′ mutation (
Discussion
The studies presented here show that BMV RNA replication in yeast is severely inhibited by mutation of OLE1, an essential yeast gene encoding the Δ9 fatty acid desaturase required for UFA synthesis. UFA supplementation of an engineered ole1 deletion strain showed that BMV RNA replication did not require the Ole1 protein but rather required UFA levels well above those required for cell growth. These results demonstrate in vivo the functional importance of lipids for BMV RNA replication and, as discussed below, imply an intimate and potentially dynamic relationship between RNA replication factors and the lipid bilayer.
The RNA replication defect in ole1w mutant yeast was traced to a narrow interval in early replication. In ole1w yeast, RNA replication factor 1a carried out several normal functions. 1a still became membrane associated and directed the membrane association of 2a (
Recently BMV RNA replication was also found to be inhibited by mutation of yeast gene LSM1 (J. Diez, et al., supra, 2000). LSM1 and OLE1 show many disparate characteristics and appear to be involved in distinct aspects of BMV RNA replication. Unlike OLE1, LSM1 is dispensable for yeast growth in minimal medium at 30° C., though it is required at 37° C. The LSM1-encoded protein, Lsm1p, is not membrane associated, but distributed throughout the cytoplasm. Lsm1p is not a biosynthetic enzyme but rather is related to RNA splicing factors and implicated in the metabolism of viral and cellular mRNAs, including the transition of mRNAs from translation to other fates such as degradation and replication (R. Boeck, et al., Mol. Cell. Biol. 18:5062-5072, 1998; J. Diez, et al., supra, 2000). Accordingly, LSM1 mutation inhibits 1a-induced stabilization of RNA3, which is unimpaired in ole1wi′ mutants (
UFA dependence of RNA replication. Cerulenin, an inhibitor of lipid synthesis, inhibits RNA replication by poliovirus and the alphavirus SFV (R. Guinea and L. Carrasco, Virology 185473476, 1990; L. Perez, et al., Virology 183:74-82, 1991). While alternate interpretations cannot be ruled out due to cerulenin's ability to inhibit processes other than lipid synthesis (T. Oda and H. C. Wu, J. Biol. Chem. 268:12596-12602, 1993), this suggests a possible requirement for continued lipid and/or membrane synthesis. The inhibition of BMV RNA replication in ole1w yeast, however, is not due to a general block to lipid or membrane synthesis. Ole1p is the desaturase that converts newly synthesized SFAs to UFAs. When UFA levels in yeast are limited by ole1 mutations, membrane synthesis proceeds at normal rates but the UFA:SFA ratio in membrane phospholipids drops (J. Stukey, et al., supra, 1989). Moreover, our studies showed that ole1w yeast cells had normal growth rate and size, and this did not change when the cells expressed 1a+2a+RNA3.
The UFA:SFA ratio affects many membrane-associated functions because of its strong effect on membrane fluidity and other physical properties (P. J. Emmerson, et al., J. Neurochem. 73:289-300, 1999; M. Shinitzky, Physiology of Membrane Fluidity, 1984). Wt BMV RNA replication required approximately 5 times more UFA supplementation than normal growth of mutant yeast (
In addition to kinetic effects, reduced UFA levels could also impede BMV RNA synthesis by perturbing the form or stability of replication factor interactions. Under reduced UFA levels, increased lipid packing density and membrane microviscosity tend to displace membrane-associated proteins farther into the aqueous phase, altering their potential for interacting with other factors and the position of such interactions relative to the membrane (R. J. Cherry, et al., Biochimica et Biophysica Acta 596:145-151, 1980; M. Shinitzky, supra, 1984). Since introducing a cis double bond shifts lipids from a cylindrical to a more cone-shaped profile, UFAs also influence membrane curvature and flexibility (R. Schneiter and S. D. Kohiwein, Cell 88:431-434, 1997). Modulating any of these parameters may impede functional interaction of 1a, 2a, viral RNA or host components with each other. Since ole1w mutation did not inhibit 1a association with membrane or 1a-directed membrane association of 2a (
While negative-strand RNA synthesis was strongly dependent on UFAs in vivo, a preformed, template-dependent negative-strand RNA synthesis activity can be solubilized from membranes of BMV-infected plant cells or yeast expressing 1a, 2a and RNA3 (R. Quadt, et al., supra, 1995). Thus, the UFA requirement may lie in assembly of a functional RNA synthesis complex. Alternatively, in vivo UFA dependence and membrane association of negative-strand RNA synthesis may relate to functions missing from the solubilized, in vitro negative-strand synthesis activity. Anomalous characteristics of the in vitro system include low efficiency of template usage (<0.1% of added template) and a lack of response to the intercistronic replication enhancer, which in vivo directs 1a-dependent RNA3 stabilization and stimulates negative-strand RNA3 synthesis and RNA3 replication approximately 100-fold (R. Quadt, et al., supra, 1995; M. L. Sullivan and P. G. Ahlquist, supra, 1999).
While oleic and/or palmitoleic UFAs were required for BMV RNA replication, oleic acid disrupts poliovirus RNA replication in HeLa cells (R. Guinea and L. Carrasco, supra, 1991) or HeLa cell extracts (A. Molla, et al., J. Virol. 67:5932-5938, 1993). These results may be related to more complex effects of oleic acid on HeLa cells. Supplementing ole1 mutant yeast with oleic acid, palmitoleic acid, or other UFAs yields a direct increase in membrane glycerophospholipids containing these UFAs (J. Stukey, et al., 1989). However, treating of HeLa cells with oleic acid resulted in major changes in the synthesis of many lipids, including dramatic increases in synthesis of cholesterol and other neutral lipids, a reduced phosphatidylserine:phosphatidylcholine ratio, and other changes (R. Guinea and L. Carrasco, supra, 1991). Similarly, in HeLa cell extracts, oleic acid inhibited in vitro translation as well as poliovirus RNA replication (A. Molla, et al., supra, 1993).
In conclusion, we find that BMV RNA replication is strongly dependent on UFA levels in vivo. When UFA was limited, ER-associated RNA replication was blocked after 1a and 2a membrane association and RNA3 template recognition and stabilization, but before negative-strand RNA synthesis. The ability to use ole1w mutation to block RNA replication at this stage should help to elucidate the early events in initiating RNA synthesis. Dependence of BMV RNA replication on UFA levels in particular implies a requirement for host membrane fluidity, suggesting that the membrane is not just a static anchoring site for RNA replication complexes. Accordingly, further study of ole1w yeast should help to illuminate the nature and function of membrane association in positive-strand RNA viruses RNA replication.
Since membrane-associated RNA replication appears to be a universal feature of positive-strand RNA viruses of eukaryotes, the replication of other viruses in this class may also be dependent on the fatty acid composition of membrane lipids. The finding that BMV RNA replication is much more sensitive than normal cell growth to reduced levels of UFAs thus suggests that genetic or pharmacological approaches to modulate the lipid composition of host membranes may provide useful antiviral strategies.
D. Proposed Use of MAB1, MAB2. MAB3 and OLE1 Genes to Develop Antiviral Agents and Vector Systems
Increasing evidence shows that virus replication involves a complex interplay between viral and host factors at multiple steps of replication. Before the present invention, most cellular factors on which viral replication depends, or that are able to influence viral replication, remained unknown. Identification of such factors herein enables a number of applications to interfere with, to permit, or to optimize virus replication in various cell types. Illustrative, but not exhaustive, examples of the kinds of applications that we envision are given below. The term “host factor” used herein is exemplified by any of the proteins encoded by MAB1, MAB2, MAB3 and OLE1.
As described above, one may obtain the altered genes of the present invention by various means known to one of skill in the art of microbiology. Most simply, one may obtain the yeast gene by probing a yeast gene library with probes obtained by studying the sequence of the gene. These sequences may be obtained from the yeast protein database at YJL124C for MAB1, YDR324C for MAB2 and YDJ1 for MAB3. The nucleic acid sequences are also disclosed below at SEQ ID NO:1, 2 and 3.
Moreover, conservation of many replication principles, sequences and functions across a wide range of different viruses of humans, animals, plants and microbes and conservation of many structures, sequences and functions across a wide range of human, animal, plant and microbe cells means that host factors and host factor genes identified for one virus and cell type will frequently have important practical implications for similar applications regarding other viruses and cell types. Thus, host factors and host functions identified as influencing BMV RNA replication suggest the involvement of related host factors, assemblies and processes in the replication of other viruses whose replication strategy and/or replication genes are related to those of BMV. Using the sequence and other characteristics of host factors involved in BMV replication, directed searches and tests for related factors involved in the replication of other viruses can be conducted, leading to similar applications.
Host factors involved in virus replication will include factors that interact directly with viral proteins, viral nucleic acids, or both. By virtue of their interaction, such factors offer multiple ways to inhibit virus replication. By point mutation, truncation, or similar approaches, derivatives of such host factors could be created that still bind to their respective viral component but lack other functions necessary to support virus replication. Expression of such derivatives can therefore sequester viral components in a nonproductive complex, interfering with viral replication.
Consistent with the above and with other mechanisms of host factor involvement in virus replication, libraries of mutagenized derivatives of one or more host factors involved in virus replication may be created in expression vectors and screened en masse in cells for antiviral activity. Thus, effective antiviral activity may be derived practically from such a host factor gene by empirical means, without requiring detailed understanding of the normal function of the host factor in virus replication or of the mechanism by which resistance is achieved. Moreover, such empirical mutagenesis and screening approaches can be used to optimize or enhance the virus resistance activity of any existing host factor gene or derivative, and/or to lower its cytotoxicity or other side effects.
Alternatively, understanding of host factor function in infected and/or uninfected cells may exist or can be obtained and used to deliberately devise an engineered resistance strategy. For example, host factors or host factor domains able to bind viral proteins or nucleic acids can be identified and linked to other protein domains able to direct the degradation of proteins or nucleic acids, respectively, thus targeting these viral factors for destruction.
In many cases the proper assembly and function of biological complexes is inhibited by altering the normal balance of expression of the components involved, including the overexpression of one or more components relative to other components. Thus, antiviral effects may be achievable not only by decreasing but also by increasing the expression of host factors involved in viral replication.
Homologs of a host factor from the same or other cells may have natural antiviral activity by virtue of being compatible for normal cellular functions but incompatible for interaction with viral components. Overexpression of such homologs could competitively interfere with virus replication by blocking virus access to necessary cellular assemblies or pathways, or by binding non-productively to viral components as envisioned above for host factor mutants.
In some other cases a virus or viral derivative may be unable to replicate or replicate poorly in a particular cell type due to limiting amounts of a host factor or due to imperfect compatibility between that host factor and a viral component. In such cases, increased expression of the relevant host factor, or expression of a more virus-compatible homolog of the host factor from another cell type, may allow or enhance replication of the virus or its derivatives. Such expression might be engineered into the virus itself, or into the cell independently from the virus, and could be useful for enhanced use of viral gene expression vectors, among other uses.
This application claims the benefit of priority from U.S. Ser. No. 60/049,439, filed Jun. 12, 1997 and U.S. Ser. No. 09/094,069, filed Jun. 9, 1998. Both of these applications are incorporated by reference herein.
This invention was made with United States Government support awarded by AID, Grant No. DHR-5542-G-SS-9034-00; NSF Grant No. DMB-8451884; MCB-9004385; IBN-9018503; and NIH, Grant No. GM35072; GM51301; A123742. The United States Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60049439 | Jun 1997 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09760040 | Jan 2001 | US |
| Child | 10618896 | Jul 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09094069 | Jun 1998 | US |
| Child | 09760040 | Jan 2001 | US |
