Despite clear evidence that host factors play crucial roles in the replication of many important (+) strand RNA viruses, little is known about these host factors. The long term objectives of this research are to identify all host factors required for the intracellular replication of a representative (+) strand RNA virus; to determine the roles of each host factor in viral replication, cell function, and infection pathology; to determine how widespread the use of-particular cell factors, pathways or subassemblies are among closely or distantly related (+) strand RNA viruses; and to use the resulting knowledge to develop more effective antiviral strategies. The results will enhance understanding of a broad range of important viruses and provide a basis for new virus control measures and improved use of RNA viruses in genetic engineering. Previously, identification and study of the host factors exploited by viruses of higher eukaryotes have been severely hindered by the complexity of higher eukaryotic genomes and the limited genetic tools available. The Principal Investigator's laboratory has now overcome these barriers by showing that the genetically tractable yeast S. cerevisiae will support RNA replication and gene expression by a higher eukaryotic virus, brome mosaic virus (BMV). BMV is a representative member of the alphavirus-like superfamily of viruses and an advanced model system for studying RNA virus replication. This unique, new yeast/BMV system and powerful techniques of yeast genetics and molecular biology will now be used to identify the potentially wide range of host factors required for BMV replication and gene expression, and to ascertain the contribution of each host gene to infection. Proven genetic approaches will be used to identify the relevant host factor genes through the effects of host mutations on BMV-directed replication and expression of selectable and counterselectable marker genes, through effects induced by yeast gene overexpression libraries, and by other criteria. Each yeast gene found to be required for BMV replication will be cloned and sequenced. For each host factor, existing in vivo and in vitro tests will determine which step(s) of viral RNA synthesis, accumulation, or expression require the factor; whether or not the factor is a component of the BMV RNA replication complex or is required for its assembly or function; whether or not the gene product interacts with the BMV RNA replication proteins or BMV RNA; and what function(s) the factor contributes to infection. Recently cloned yeast genes will be used to explore known interactions of BMV RNA with cellular tRNA nucleotidyltransferase and tyrosyl-tRNA synthetase; the observed role of cellular RNA polymerase Ill signals in the BMV genome as cis-acting elements in BMV RNA amplification; and other selected issues. The relationship between each factor's normal role, its role in viral replication, its intracellular localization, and infection-specific cytopathic effects will be explored.
