HIV-1 infection is initiated by fusion of virus and target cell membranes, resulting in delivery of the ribonucleoprotein core into the host cell cytoplasm. The ensuing events that immediately follow fusion are poorly understood for all retroviruses. It is generally assumed that the viral core undergoes an uncoating process involving dissolution of the viral capsid. Studies in our laboratory have shown that relatively subtle perturbations in HIV-1 capsid stability result in poorly infectious virions that are impaired for viral DNA synthesis in target cells, suggesting that uncoating is a finely tuned process and is crucial for efficient reverse transcription. The high sensitivity of HIV-1 infection to alterations in capsid stability, and the ability of the restriction factor TRIM51 to block HIV-1 infection by targeting CA, suggest that uncoating may be an attractive target for antiviral therapy. Besides CA, other viral and cellular molecules are likely to participate in the regulation of HIV-1 uncoating in vivo. In this application, we propose to use biochemical and cell-based assays to better understand the mechanism of HIV-1 uncoating and to identify relevant virus-host cell interactions suitable for therapeutic targeting. The work will be planned according to five Specific Aims: 1. To analyze the uncoating of poorly infectious HIV-1 CA mutants in target cells. 2. To identify novel pseudorevertants of HIV-1 uncoating mutants. 3. To identify structural intermediates in HIV-1 uncoating. 4. To identify host cell factors which regulate HIV-1 uncoating. 5. To determine the effect of TRIM51 on HIV-1 uncoating in vitro. Collectively, these studies will elucidate an obscure stage of the HIV-1 life cycle and reveal new possibilities for HIV/AIDS therapy. PUBLIC HEALTH RELEVANCE: HIV-1 infection is critically dependent on the poorly understood process of uncoating of the viral core. We will perform biochemical and molecular genetic studies to identify viral and cellular determinants of uncoating and to define stages in the process. Ultimately, these studies will help determine whether uncoating can be a useful target in the development of new antiretroviral therapies.