CRYO-EM TOMOGRAPHY OF ISOLATED MITOCHONDRIA FROM FHV INFECTED CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. General description of the biological problem: Flock House virus (FHV) is a prototypical bipartite positive-stranded RNA virus, that has been an excellent model system for biochemically and genetically investigating virus genome replication. The viral RNA is replicated by the viral RNA dependant RNA Polymerase (RdRp), which forms small (~60 nm) invaginations on the outer mitochondrial membrane called spherules. Each spherule is open to the cytoplasm through a small neck, which is presumably where the newly synthesized RNA is released. EM tomography of samples where the membrane is not stained reveals that the viral polymerase protein and RNA form a ring shaped complex inside the spherules. Although, these studies are informative about the morphology of the mitochondria inside the cell, the structural information obtainable from these samples are limited due to the heavy metals used for staining the biological components for EM. The first goal of this study is to use cryo-EM tomography to determine the three-dimensional structure of the RdRp complex in mitochondria isolated from FHV infected Drosophila cells in a near to native state. The second goal will be to extract the density for these RdRp complexes from the tomogram and use them to construct a higher resolution averaged structure. Ultimately these higher resolution cryo-EM tomograms will be combined with data from cellular tomography studies of the mitochondria inside FHV infected cells and atomic structures of the isolated RdRp protein (currently being investigated by John Johnson) to create a comprehensive model that spans from the cellular resolution to the atomic resolution. Significance: All RNA viruses replicate their genome using viral encoded RNA polymerases, which primarily function as oligomers. Disrupting the formation of these polymerase complexes represents a new therapeutic target for many viruses. However, the structure of the oligomers are not well understood since they typically form in association with membrane organelles. Flock House virus is unique in that the viral polymerase forms replication complexes in association with the mitochondria, which is an organelle that can be isolated from the cell for EM tomography studies.