The work in my laboratory is focused on understanding a variety of aspects of HIV-1 assembly, release, and maturation. The major areas of investigation include the subcellular targeting of HIV-1 assembly, the role of lipid rafts in HIV-1 replication, viral and host factors in HIV-1 budding, and mechanism of action of the HIV-1 maturation inhibitor PA-457 (Bevirimat). The host factors Tsg101 and Alix have been shown to interact with the p6 region of HIV-1 Gag, and we previously demonstrated that overexpression of the p6-interacting region of Tsg101 impairs HIV-1 budding in a dominant-negative manner. We now find that overexpression of the Gag-binding domain from Alix also potently inhibits HIV-1 Gag processing and virus release. These studies indicate that the budding step of the retroviral replication cycle can be targeted in a late-domain-specific manner. We have also investigated the role of plasma membrane cholesterol in HIV-1 assembly and release, following up on our finding that virus assembly and budding take place in cholesterol-enriched lipid raft microdomains. We observe that cholesterol depletion disrupts both binding of Gag to the plasma membrane and Gag multimerization, suggesting that plasma membrane cholesterol, and probably lipid rafts, help create a platform for stable Gag membrane binding, Gag multimerization, and virus release. We have also defined the target and mechanism of action of the cholesterol-binding compound AME.In collaboration with Panacos Pharmaceuticals, we have continued to characterize the mechanism of action of the anti-HIV-1 compound PA-457. Our data indicate that PA-457 blocks virus replication by interfering with a specific step in the Gag processing cascade: the conversion of the capsid-spacer peptide 1 (CA-SP1) processing intermediate to mature CA. This defect in Gag processing abolishes virus infectivity by preventing maturation of HIV-1 virions following release from drug-treated cells. We sought to identify the full spectrum of replication-competent HIV-1 variants capable of conferring PA-457 resistance. Six single amino acid substitutions, which independently confer PA-457-resistance, were isolated. All PA-457-resistance-conferring mutations mapped to the CA-SP1 junction of Gag. The PA-457-resistant isolates were characterized with respect to their effects on virus replication, the kinetics of Gag processing, virus particle assembly and release, and virion morphology. The results demonstrate that acquisition of PA-457-resistance is associated with a variety of viral phenotypes, suggesting multiple mechanisms of resistance. This study provides significant and novel insights into the emergence of resistance to PA-457. The results presented here will inform both the analysis of PA-457 resistance in patients and the development of additional compounds that, like PA-457, block HIV-1 maturation by disrupting specific steps in the Gag processing pathway. During the past year, PA-457 has demonstrated the ability to reduce viral loads in infected patients and expanded clinical trials of this drug have been initiated.
