Like other retroviruses, HIV-1 encodes a sequence within the Gag protein termed a late or 'L'-domain that is required for the scission of a nascent virion from the virus producing cell. L-domains are functionally interchangeable among different retroviruses and act by recruiting host-cell factors. In HIV-1, this activity is provided by a short peptide sequence (PTAP) that binds directly the host-cell protein, TsglOl, and recruits it to sites of particle assembly. TsglOl is one of a group of sixteen or more proteins, termed the class E vacuolar protein sorting (VPS) factors, that are normally required for the budding of vesicles into the endosomal lumen. As part of our long term objective to understand the mechanism by which viral L-domains mediate particle budding, this proposal aims to test the hypothesis HIV-1 L-domain activity requires the same cellular activities that mediate the topologically equivalent process of endosomal vesicle budding. TsglOl itself exhibits a homo-multimerization activity and is known to bind to a second class E VPS factor, hVPS28. The experiments described herein will first determine whether these interactions are required for the HIV-1 particle budding function of TsglOl . Subsequently, the identification other cellular proteins that are associated with Tsgl 01 will be undertaken. Using biochemical yeast 2-hybrid and microscopic analyses, it will be determined whether and how they and other, already identified, class E VPS factors are recruited to sites of HIV-1 budding. Moreover, RNA duplex mediated silencing will be used to test whether these additional factors are required for L-domain mediated HIV-1 particle release. Preliminary studies indicate that a second class of retroviral L-domains, that contain a PPXY rather than a PTAP sequence motif, act by a similar mechanism to the HIV-1 L-domain. While these L-domains do not bind directly to TsglOl, they are clearly able to recruit TsglOl and hVPS28. Therefore, in some experiments, a PPXY L-domain will be included to test the hypothesis that PPXY L-domains mediate viral budding by the same mechanism as does HIV-1 , but by binding to an alternative VPS factor. Finally, a determination of whether the HIV-1 L-domain recruits a deubiqutinating activity during viral particle assembly will be undertaken. Based on findings in yeast systems, this is anticipated to be a secondary consequence of VPS factor recruitment, and is likely to explain the apparent requirement for the cellular ubiqutination machinery in retroviral particle release. Overall this project will aim to advance our understanding to the molecular mechanisms used by HIV-1 to facilitate viral budding. These studies have the potential to reveal completely novel, host-cell targets for therapeutic blockade of the replication of HIV-1, and possibly other viruses. ? ?
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