Specific Lipid Requirements and Localized Lipid Composition Changes Associated with Flock House Virus RNA Replication in Drosophila Cells
Dr. Benjamin Kopek , Biology
Positive-strand RNA [(+)RNA] viruses are significant human pathogens. A universal feature of (+)RNA viruses is that they replicate their genomes in association with host intracellular membranes. This association may be a target for broad spectrum antivirals against (+)RNA viruses. The (+)RNA virus used in our studies is the alphanodavirus Flock House virus (FHV). FHV is a simple (+)RNA virus with a 4.5 kb bipartite genome that replicates in insect cells. FHV replicates its RNA genome at the outer mitochondrial membrane of infected cells where it forms 50-70 nm in diameter invaginations that are membrane-bound RNA replication complexes. Previous work by others has shown an increase in the amount of phosphatidylcholine in FHV infected cells. Additionally, decreasing the amount of phosphatidylcholine in Drosophila cells thru targeted down regulation of biosynthetic genes decreased FHV replication. We hypothesized that increased levels of phosphatidylcholine were required for the formation and maintenance of the membrane-bound replication complexes located at mitochondria. To test this hypothesis, we synthesized the choline analog, propargylcholine, which is incorporated into phosphatidylcholine and can be “tagged” using copper(I) catalyzed cycloaddition chemistry. FHV infected cells were incubated with propargylcholine, tagged with a fluorescent dye, and visualized with confocal fluorescence microscopy. Our results suggest that there is an enrichment of phosphatidylcholine at the sites of FHV RNA genome replication. Additionally, we determined that two other major classes of cellular lipids, phosphatidylethanolamine and phosphatidylserine, were not enriched at FHV genome replication sites. Ongoing work includes a quantitative analysis of the mitochondrial lipid composition of FHV infected cells. Our work provides support for the importance of specific lipids in (+)RNA virus genome replication and the possibility that lipid biosynthetic pathways may be good antiviral targets.
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