Studies of the cell cycle, nuclear transport, RNA processing, chromatin structure, the cytoskeleton, and vesicular trafficking in the model organism Saccharomyces cerevisiae have provided paradigms for characterization of analogous systems in metazoans. We and others have characterized Ty retrotransposons in S. cerevisiae as models of retroviruses. We have recently used high throughput screening to identify roughly 300 genes, expression of which affects retrotransposition of Ty3, a yeast retrovirus-like element. These genes include ones related to DNA repair, RNA processing, protein folding, lipid metabolism, and vesicular trafficking. In the yeast system, we are studying the mechanism of action of a small number of these genes in considerable detail. Here we propose to survey a subset of nineteen genes for effects on replication of a simple mammalian retrovirus, Moloney murine leukemia virus. The goals of this application are: 1) Target gene function will be disrupted by shRNA. Candidate genes will be chosen based on several criteria, including existence of a nonessential mouse homolog, severity of defect, and possible defects for transposition of Ty1 a retrotransposon more distantly related to Ty3 than retroviruses. Activity of these genes will be disrupted by shRNA. 2) Effects on retrovirus infectivity and production will be determined using transfection and infection assays. 3) The effect of overexpression of negatively acting genes will be determined. Some genes identified in the screen act negatively on Ty3 transposition. Whether overexpression of these genes decreases retrovirus replication will be determined. 4) The biochemical point at which retrovirus replication is affected by effective candidate genes will be determined. For genes where an effect on retrovirus replication of shRNA or overexpression is determined, protein, RNA and DNA intermediates in replication will be assayed in order to identify the stage in the lifecycle where replication is impacted by the candidate gene. Although effects could be direct or indirect, knowledge of the function of the gene together with the point of effect in the lifecycle, should offer significant insight as to the mechanism of the effect. This approach has high potential for opening unexpected avenues in retrovirus research and identifying novel targets for therapeutics.
