Positive strand RNA viruses are ubiquitous pathogens that cause significant disease in humans, animals and plants. They replicate their genomes in association with cellular membranes, which physically anchor the viral RNA-dependent RNA polymerase (RdRp) and its associated replication complexes. The RdRp of the nodavirus Flock House virus (FHV) is located in the outer mitochondrial membrane, where it forms invaginations also called “spherules”. A low resolution model for these spherules has previously been generated but it is descriptive and lacks mechanistic details of RNA replication. I will present new data for the structure of mitochondria in FHV-infected Drosophila cells obtained by combining electron tomography with improved protocols for EM specimen preparation. In addition, preliminary data for the structure of the RdRp will be shown. Our long term goal is to provide an in situ view of the viral RNA replication complex at the nanoscale. This will broadly extend our knowledge of the functional modules required for viral RNA synthesis and deepen our understanding of how spatial organization in the cell leads to compartmentalization of RNA replication, translation and packaging. It should also provide a strong foundation for cross-fertilization between basic and medically relevant systems to benefit human and animal health.
Previous analyses of FHV particle assembly and structure at the nanoscale have already allowed us to develop potential applications in the medical field, particularly in the area of viral diagnostics and vaccines. Virus particles are known for their strong immunogenicity, a property that is based on the ordered array of their capsid proteins, particulate nature, and ability to stimulate the innate immune response. Using a structure-based approach, we have engineered FHV particles as a platform for multivalent display of heterologous peptides and proteins. This strategy was used to induce antibodies against a highly conserved helical epitope located in the membrane-proximal region of influenza virus hemagglutinin with the goal of developing the particles into an effective human vaccine. Similar projects focusing on HCV, HIV and malaria are currently underway in our laboratory and will be discussed.