This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The three-dimensional nature of electron microscope tomography (EMT) overcomes many serious limitations of two-dimensional EM analysis to reveal many fundamental features not accessible from conventional transmission EM analyses. We employ EMT and the advanced model viruses, flock house virus (FHV) and brome mosaic virus (BMV), to understand the organization of the membrane-bounded, vesicle-like RNA replication complexes (spherules) induced by many important positive-strand RNA viruses. Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. RNA replication by positive-strand RNA viruses is universally associated with membranes and membrane rearrangements such as single or double membrane vesicles. However, in most cases the site(s) of RNA synthesis relative to these rearranged membranes and the topological relationships among membranes, vesicle interiors, lumenal compartments, and the cytoplasm are poorly defined. Recent results suggest that, in at least some cases, viral RNA replication occurs inside such vesicles. However, these findings leave many unresolved questions, in many ways due to limitations inherent to the two-dimensional analyses used. The best-studied member of the nodavirus family, FHV, replicates at the outer mitochondrial membranes of infected Drosophila cells where membrane-bound spherules form in the intermembrane space, similar to multi-vesicular structures observed for many positive-strand RNA viruses.
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