| Positive strand (+) RNA viruses are a significant health threat today, yet the diseases that they cause are difficult to treat due to a lack of antiviral drugs, which is in part due to the tight association of their replication with host cellular functions. In order to design improved therapeutic agents for use against (+) RNA viruses, we must increase our understanding of their replication mechanisms in host cells, and what cellular factors are involved in their life cycle. The early events in (+) RNA virus replication include translation of the genome into the viral replication proteins, followed by the folding, trafficking, and assembly of the viral replication factories on intracellular membranes, which are critical for all (+) RNA viruses, and, therefore viable targets for potential antivirals. To better understand these early events and the cellular proteins that are involved, I used Flock House virus, a model (+) RNA virus, in a Saccharomyces cerevisiae model system to take advantage of the facile and well understood genetics of yeast. In these studies, I examined the impact of the major chaperone and cochaperone systems in FHV RNA replication in yeast. I identified a number of chaperones that impacted FHV RNA replication both negatively and positively, including the J-domain family of Hsp40s, the main cellular Hsp70 chaperone system, as well as a number of minor chaperones. In addition, I found that some cellular requirements were membrane-specific, and that there were differences between the requirements seen in Drosophila cells and those in yeast. Finally, I began to develop a method for high-throughput analysis of the early events in FHV RNA replication based on the accumulation of the viral polymerase. Therefore, in this body of work, I confirmed that cellular chaperones play a role in the genome replication of a positive strand RNA virus, and identified a number of proteins in the cell as targets for future study. |
