The importance of viral persistence in pathogenesis is just beginning to be fully appreciated. Viral persistence is especially puzzling in RNA viruses that do not produce proviruses (integrated DNA copies). There are double-stranded RNA viruses of animals, plants, insects, and fungi that exhibit persistence. The fungal viruses are uniformly and permanently persistent: they have no natural infectious cycle. Their replication must therefore be regulated by the cell, since they neither kill the cell nor fail to infect all progeny of an infected cell. Among the fungal viruses, the Saccharomyces cerevisiae , or yeast virus, ScV, is the simplest and best understood. Yeast is an ideal system in which to address the nature of persistence: the ease of its sophisticated molecular and classical genetics is unmatched in other eucaryotes. In ScV, there is only one essential viral dsRNA, L1, with a known sequence, and only one major capsid polypeptide (the cap gene product). L1 has only two large open reading frames, cap and pol. The pol protein, expressed by a retrovirus-like frameshift event contains an RNA-dependent RNA polymerase (RDRP). In the present application, we seek to understand (1) the mechanism of frameshifting in ScV and its possible relationship to cellular helicases, and (2) the functional domains in the pol open reading frame. ScV frameshifting, the first system in which it has been shown that frameshifting is correlated with a ribosomal pause, will be exploited to understand in detail the relationship between RNA structure and the frameshifting event. Further mutants will be created to test more complete models for the ScV frameshift pesudoknot and these will be analyzed by frameshifting in vitro and in vivo in appropriate heterologous contexts, as well as by heelprinting experiments to determine the location of paused ribosomes in the frameshift region. The structure of the frameshift region will also be analyzed directly by sensitivity to single and double strand specific nucleases. The role of cellular helicases in frameshifting will be ascertained by overexpression of helicase genes in cells producing beta-galactosidase by virtue of a frameshift event. The cap-pol fusion protein is not processed; all of its activities are expressed in viral particles in the form of the fusion protein. These, including the RNA binding domain, the nucleotide phosphohydrolase (NPH), and the RDRP, will be further defined by site-directed mutagenesis and expression of cDNAs. The NPH activity appears to have been localized to a 75 amino acid region, which when expressed as a beta-galactosidase fusion protein retains its activity. Mutants lacking this activity will be constructed in full-sized cDNA clones that can be used for complementation tests in vivo, in order to determine the viral function of the NPH. Similarly, the RNA binding activity appears to have been localized to a 100 amino acid region of pol. Putative loci for interaction with cellular proteins, within the pol open reading frame, have been identified. These will be altered in a systematic way to define their functions in vivo.
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