The overall goal of this renewal application is to understand how myristoylated retroviral proteins are trafficked through the cell, targeted to the plasma membrane and assembled into virions. This will be accomplished by studying the HIV-1 Gag polyprotein, Pr55gag. Use of HIV-1 as a model system is dually advantageous. First, these studies allow us to address key basic questions regarding membrane targeting of myristoylated proteins. Second, elucidation of the molecular mechanisms involved in Gag membrane association and multi-merization will provide new insights into the process of HIV infection and will serve to define novel targets for drug development that could interfere with formation of HIV-1 virions and be efficacious for the treatment of AIDS. Specifically, the proposed research will address the following issues: 1. Identification of assembly site domains in the plasma membrane Pulse/chase labeling combined with density gradient centrifugation will be used to molecularly define discrete steps of viral particle assembly at the plasma membrane. Gradient fractions containing Gag will be isolated and characterized in order to establish a temporal order to the assembly process. 2. When and where does oligomerization of Pr55gag occur? Gag multi-merization constitutes a critical step in virus assembly, yet it is not known when and where Gag-Gag interactions occur in the cell. We will determine the multi-merization state of Pr55gag at different stages during its biosynthetic trafficking and correlate the degree of multi-merization with specific assembly intermediates. 3. Visualization of Gag trafficking and assembly in live cells. Pr55gag will be fused to different color variants of GFP in order to monitor membrane localization and Gag-Gag interactions by real time confocal microscopy. These experiments will complement the biochemical studies in Aims 1 and 2 and are likely to provide a fascinating view of Gag movement and dynamics in live cells.
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