Biophysics of Protein-Mediated Membrane Fusion

HIV remains a major health threat worldwide. Whereas effective antiviral treatments are available, the emergence of drug resistant variants warrants continued efforts to identify novel viral and cellular targets to combat infection. In-depth understanding of the early steps of HIV infection will reveal critical interactions and processes that support or inhibit the virus entry and, thereby, suggest potential drug targets. Our focus has been the investigation of mechanisms of HIV-1 fusion and its restriction by host factors. Over many years, we have developed innovative functional and fluorescence microscopy approaches to delineate key determinants and cellular sites of HIV-1 entry. Our preliminary results and novel technologies implemented during the past grant cycle provide unique opportunities to gain critical mechanistic insights into the architecture and dynamics of HIV-1 membrane, define the sites of HIV-1 fusion with target cells following cell-free and cell-cell transmission routes, and elucidate the molecular mechanisms by which host restriction factors antagonize HIV-1 entry. To achieve these goals, we propose the following Specific Aims. First, we will capitalize on our novel virus labeling strategies to elucidate the determinants of HIV-1 entry pathways. We will employ a panel of functional, single virus imaging, and biochemical approaches to delineate the basis for a surprising HIV-1's preference for fusion with pH-neutral intracellular vesicles described in our recent publication. Second, we propose to define the mechanism of HIV-1 cell-cell transmission via tunneling nanotubes, the sites of fusion with target cells after transmission, and the role of restriction factors, such as SERINC5 and IFITM3, in regulating these processes. Third, we will utilize specific membrane probes and novel imaging approaches to elucidate the nanoscale organization and dynamics of HIV-1 membrane and their modulation by host restriction factors. Specifically, we will investigate the roles of viral lipid asymmetry, membrane tension and Env glycoprotein clustering on virions in regulating the efficiency of virus-cell fusion. Completion of the proposed experiments will provide unprecedented mechanistic insights into regulation of HIV-1 entry and fusion by viral membrane architecture/dynamics and by host restriction factors. RELEVANCE (See instructions):