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 of 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 (FIG. 1). Host factor involvement in BMV RNA replication is suggested by host-specific replication effects, biochemical studies, and cell biology studies as noted above and by the presence of multiple tRNA-related sequences and functions in the cis-acting replication signals on BMV RNAs (W. De Jong and P. G. Ahlquist, supra, 1995; M. Restrepo-Hartwig and P. Ahlquist, supra, 1996; R. Quadt, et al., supra, 1993; P. Ahlquist, supra, 1992; M. Sullivan and P. Ahlquist, Sem. Virol. 8:221-230, 1997).
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.
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