RNA packaging and virus assembly in the Flock House virus life cycle
Flock House virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs (RNA1 and RNA2) in a single virion. The virus provides a convenient model system for studying the principles underlying the packaging of multi-partite RNA genomes. Our research projects address the unresolved question of how multipartite RNA genomes are recognized and packaged into a single virion. Some of our current research projects are:
Interactions between viral RNA and coat protein that govern specific genome packaging and virion assembly
To elucidate the mechanism by which FHV is able to package two distinct RNA molecules, we are examining the FHV coat protein as well as both genomic RNAs in more detail. By mutating specific regions in FHV RNAs we generated mutant virus particles that showed different patterns in genome packaging compared to wildtype virus. We developed a real time PCR assay to quantify the packaged RNA within these mutant particles.
We are also trying to find encapsidation signals for RNAs 1 and 2 by in vitro selection using genomic SELEX (systematic evolution of ligands by exponential enrichment). Other studies in our laboratory are aimed at determining the oligomeric state of FHV coat protein at the onset of assembly. These studies generally involve techniques such as in vitro binding assays, EMSA (gel mobility shift assay) analysis and FPLC.
Coordination between synthesis of viral RNA and coat protein during assembly
Coupling between RNA replication and virus assembly is critical for efficient assembly of infectious FHV virions. A feasible explanation for this coupling event is that coat protein synthesis occurs in proximity to mitochondria, which are the sites of viral RNA synthesis, and that this facilitates the retrieval of viral RNA by the coat protein for specific genome packaging. To test this hypothesis, a variety of molecular biological techniques are used in conjunction with fluorescence and electron microscopy.
