Title: Roles of Macropinocytosis in HIV-1 infection of CD4+ T Cells Project Summary: Host cell entry is the first step in the replication cycle of HIV-1, the causative agent of AIDS. Although receptor binding of viral Env glycoprotein and subsequent virus-cell fusion during the virus entry process have been described in detail, subcellular locations of these events remain to be determined. In particular, despite the importance of CD4+ T lymphocytes as a natural host cell type for HIV-1, the contribution of virion endocytosis to HIV-1 infection of CD4+ T cells remains poorly understood. Previous studies of HIV-1 entry into T cells have largely focused on clathrin-mediated endocytosis and cell surface fusion. However, our team recently discovered that primary CD4+ T cells engage in robust macropinocytosis, a large-scale, clathrin-independent form of endocytosis. Importantly, our preliminary data indicate that pharmacological inhibition of macropinocytosis blocks both internalization of HIV-1 particles and productive infection in primary human CD4+ T cells. In contrast, the same treatment does not affect HIV infection of a commonly used CD4+ T cell line. These results suggest that the macropinocytic entry is a hitherto overlooked mode of HIV-1 entry that contributes to infection of primary human CD4+ T cells. Interestingly, our preliminary study also showed that inhibition of macropinocytosis after completion of virus internalization also blocks expression of virus-encoded genes, suggesting that macropinocytosis has another later role in productive HIV infection, which is separable from virus internalization. In the current application, we propose to determine the roles of macropinocytosis in productive infection of primary CD4+ T cells. Our long-term goal is to fully describe cellular mechanisms that promote HIV-1 infection and to use the obtained knowledge for developing antiviral strategies. Our central hypothesis in this application is that macropinocytosis in primary human CD4+ T cells contributes to productive infection as a major HIV-1 entry pathway and as a cellular function necessary for maintaining a permissive environment for the virus. To test this hypothesis, we plan to determine: 1) the extent to which macropinocytic internalization contributes to productive HIV entry into primary human CD4+ T cells; 2) the roles played by Env-receptor interactions in promoting HIV-1 macropinocytosis; 3) the influence of macropinosome environment on HIV-1 entry; and 4) the nature of post-internalization macropinocytosis function that promotes productive infection. The outcomes of the experiments outlined in this proposal will likely fill the knowledge gap in our understanding of HIV-1 entry, a fundamental aspect of the HIV-1 replication cycle, and help us develop or improve antiviral strategies that target virus entry and establishment of productive infection in CD4+ T cells.