The goal of the proposed currently not well understood. To achieve this goal, intracellular movement, behavior and nuclear trafficking of individual HIV-1 particles will be observed by incorporating the fluorescent reporter molecule, green fluorescent protein (GFP), fused to the HIV-1 accessory protein, Vpr, into virion particles. Recent advances in fluorescent microscopic imaging makes possible the viewing of individual particles in living and fixed cells for the first time. Because GFP-Vpr remains associated with the viral core and genome complex, post-entry events in HIV infection can be followed as discrete steps, from uncoating of the viral core, to nuclear migrating and docking to the nuclear pore complex (NPC). Particles in pathways leading to productive infection will be distinguished from those that are non-productive. Detailed analysis of the movement of these particles will define the distances and speeds traveled during transit to the nucleus (aim 1). Interactions between GFP-Vpr labeled particles and the cytoskeleton will be identified and characterized (aim 2). The importance of microtubules or other cytoskeletal elements for HIV infection and trafficking will be determined (aim 3). Additionally, the cellular nuclear import machinery exploited by HIV for nuclear entry of the viral genome complex will be identified by following individual particles. Using the same analysis with viral mutant particles, interactions between viral proteins involved in nuclear trafficking and cellular import pathways will also b characterized. Viral mutants will also be used to examine a potential link between viral uncoating and trafficking.
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