Elucidating the role of IP6 in HIV-1 assembly, maturation, and infection. - PROJECT SUMMARY The HIV/AIDS pandemic has claimed more than 36 million lives worldwide. Although effective antiretroviral drugs are widely available, virologic failure due to drug toxicity and poor adherence leading to the development of resistance is an increasingly serious problem. This highlights the need for continued development of novel HIV inhibitors and a strong commitment to basic research aimed at elucidating the mechanisms driving HIV replication, transmission, pathogenesis, and drug resistance. The cellular metabolite inositol hexakisphosphate (IP6) is an essential co-factor for HIV-1 replication. IP6 promotes particle production in HIV-1 producer cells by binding to and stabilizing the immature Gag lattice (IGL) and subsequently promotes particle infectivity by binding to and stabilizing the capsid, which is assembled during particle maturation. Importantly, these functions of IP6 suggest interplay between IP6 and two classes of HIV inhibitors: maturation inhibitors (MIs) and capsid inhibitors (CIs). During the mentored phase of this award, the applicant will investigate the separate roles of IP6 in each of these key steps in the HIV-1 replication cycle, informing the development of novel MIs and CIs. A forced evolution approach will be used in which replication-deficient mutant viruses will be propagated in T cell lines to determine how HIV-1 adapts to the loss of IP6 binding. In Aim 1, the role of IP6 during IGL assembly and particle production will be investigated. The interaction between IP6 and the IGL is hypothesized to ensure the packaging of IP6 into viral particles at levels required to promote capsid assembly during maturation. HIV-1 Gag mutant viruses that assemble independently of IP6 will be used to test this hypothesis. This work builds on the applicant’s recently published research showing that the replication of Gag mutants deficient for IP6 binding can be restored by IGL stabilization. In Aim 2, the role of IP6 binding to the HIV-1 capsid and the implications of this interaction for capsid assembly and virion infectivity will be investigated. The capsid, which assembles in the virion, must remain stable and intact during the post-entry steps of the virus replication cycle. Mutant viruses unable to interact with IP6 in the context of the assembled capsid are highly deficient in replication and particle infectivity due to an inability to assemble stable capsids. The mechanisms by which mutations within capsid that restore capsid assembly and particle infectivity to capsids unable to bind IP6 will be studied using cell-based assays and structural analyses via collaborations with expert investigators. In addition to research, the applicant will engage in a wide variety of career development activities including technical training and management, leadership, and diversity training. The applicant will also meet regularly with his advisory committee to discuss research progress and to receive advice on the academic job search. During the independent phase of this award, additional factors, including antiviral compounds, that affect capsid stability will be investigated. In Aim 3, the mechanisms of action of novel CIs and pathways of resistance to these compounds will be investigated. Additionally, host factors that affect capsid stability post-entry will be investigated in the context of CI activity and resistance.
