Monocytes are precursors of dendritic cells and macrophages, and these cell types play an important and multifaceted role in HIV-1 infection and pathogenesis. Undifferentiated primary monocytes are resistant to HIV-1 postentry infection, which is likely due to the expression of a potential restriction factor. A challenge in he field is to identify the host factor and understand its mechanisms underlying HIV-1 restriction of primary monocytes. In our preliminary study, we identified a cellular protein named UBE2V1 (ubiquitin-conjugating enzyme E2 variant 1) that can inhibit HIV-1 infection in monocytic cells. The goal of this R21 proposal is to explore the function and mechanisms of UBE2V1 in blocking HIV-1 infection in primary monocytes. We propose to test the hypothesis that post-translationally modified UBE2V1 inhibits HIV-1 replication in primary monocytes. The expected outcome of our proposed studies is to define a novel mechanism by which post-translational modifications of UBE2V1 regulate HIV-1 infection in monocyte-lineage cells. UBE2V1 (also called UEV1, UEV1A or CROC-1) belongs to the subfamily of ubiquitin-conjugating enzyme variant proteins and acts as a regulatory protein in DNA damage and cell differentiation. A recent study indicated that the retroviral restriction factor TRIM5 functions as an innate immune sensor for the retrovirus capsid lattice by interacting with the ubiquitin-conjugating enzyme complex UBC13-UBE2V1. This finding suggests that UBE2V1 is a novel component of the innate immune response. Interestingly, our preliminary studies indicate that post-translational modification of UBE2V1 is associated with HIV-1 restriction in primary monocytes. We propose to explore the role and mechanisms of UBE2V1 in HIV-1 restriction in primary monocytes in two specific aims. Aim 1. Characterize UBE2V1-mediated restriction of the HIV-1 early lifecycle in primary monocytes. Aim 2. Define the critical domains and residues of the modified UBE2V1 responsible for HIV-1 restriction in primary monocytes. The significant impact of our proposed studies will be to open a new area in the study of post- translational modifications of host factor involved in HIV-1 replication. We will define the molecular mechanism of HIV-1 restriction in monocyte-lineage cells by characterizing the function of UBE2V1 in HIV-1 infection. Accomplishing the proposed aims has the potential to advance basic knowledge of HIV-host interactions. Ultimately, the knowledge learned from the proposed studies will enable design of novel approaches to block HIV-1 replication in target cells.