The goal of this proposal is to elucidate the role of host proteins in the function of HIV-1 Gag and Gag- Pol during the late stages of the viral replication cycle. We have recently identified ALP1, the human homologue of the yeast class E vacuolar protein sorting (Vps) protein Brol, as a novel binding partner for the Gag proteins of HIV-1 and other lentiviruses. AlP1 also interacts with Tsgl01 and with homologues of a subunit of the yeast class E Vps protein complex ESCRT-III. Further, our data show that mutant versions of AlP1 and of putative mammalian ESCRT-III components potently inhibit HIV-1 particle formation. Unexpectedly, disrupting the binding site for AlP1 in the p6 domain of Gag did not prevent the incorporation of AlP1 into HIV-1 virions, and we thus propose to investigate whether Gag harbors additional contact sites for ALP1. We also propose to examine why the primary AlP1 binding site becomes significantly more important for HIV-1 particle production in a minimal Gag context. Further, the roles of individual AlP1 domains and of known AlP1 interaction partners in HIV-1 Gag function remain to be determined. In particular, we propose experiments to test the model that ALP1, together with Tsg101, links HIV-1 Gag to the llate-acting endosomal sorting complex ESCRT-III. We find that ESCRT-III remains important for HIV-1 tparticle production even if the known interaction sites for class E Vps proteins are absent from Gag. We thus Iplan to examine the roles of other Gag regions in ESCRT-III recruitment, and in the sensitivity of Gag to Imutant ESCRT-III components. Further, we propose to investigate the mechanism of inhibition of HIV-1 particle formation by a dominant-negative ESCRT-III subunit, in particular the role of phosphoinositide binding and of autoinhibitory interactions. We have also identified endosome-associated host proteins whose specific incorporation into HIV-1 virions is dependent on Gag-Pol, and is prevented by mutations in integrase that affect HIV-1 particle production. We thus propose to determine the mechanism of incorporation of these )roteins, and their relevance for HIV-1 particle formation and infectivity.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Sharma, Opendra K
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University of Massachusetts Medical School Worcester
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