Project Summary Lentiviruses including HIV-1 infect both activated/dividing CD4+ T cells and terminally- differentiated/nondividing myeloid cells (e.g., macrophages and microglia) during the course of their pathogenesis. The reverse transcription of lentiviruses consumes dNTP substrates provided from the infected host cells. However, it was predicted that nondividing cells such as macrophages should contain limited dNTP pools. Indeed, we previously reported that human primary monocyte derived macrophages harbor extremely limited dNTP levels (20-40 nM), compared to activated CD4+ T cells (1-5 µM), and this limited dNTP level in macrophages restricts HIV-1 replication. We also reported that the host SAM domain and HD domain containing protein 1 (SAMHD1), which hydrolyzes dNTPs and is abundant in macrophages, is responsible for the low dNTP levels and the restricted HIV-1 replication in macrophages. Recently, we discovered two novel regulatory circuits of SAMHD1 mediated dNTP metabolism that can operate in nondividing myeloid cells for dNTP depletion and HIV-1 restriction. In this proposal, we propose to elucidate virological, molecular and structural natures of these regulatory circuits of SAMHD1-mediated dNTP depletion in nondividing myeloid cells. In Aim 1, we will explore our hypothesis that SAMHD1 not only hydrolyzes dNTPs but also directly suppresses the RNR-mediated dNTP biosynthesis by binding to RNR in macrophages. Our hypothesis predicts that Vpx can rapidly elevate dNTP levels in macrophages following SAMHD1 degradation by simultaneously removing a suppressive regulator of RNR mediated dNTP biosynthesis. Indeed, we recently observed the direct binding of SAMHD1 to RNR R1 subunit, supporting this hypothesis. Here, we will investigate this negative dNTP metabolic regulatory circuit mediated by the SAMHD1-RNR interaction for dNTP depletion and HIV-1 restriction in nondividing myeloid cells. In Aim 2, we hypothesize that cellular PP2A- B55 phosphatase is a key positive regulator of SAMHD1 in nondividing myeloid cells that can keep SAMHD1 un-phosphorylated and enzymatically active for dNTP depletion and HIV-1 restriction. Indeed, we observed the interaction of SAMHD1 with B55 regulatory subunit of PP2A in nondividing myeloid cells, supporting this hypothesis. Here, we will investigate the roles of SAMHD1-PP2A interaction in the negative regulation of dNTP metabolism and HIV-1 restriction in macrophages. In Aim 3, we propose to investigate the structural and molecular natures of the SAMHD1 interactions with RNR and PP2A that contribute to dNTP depletion and HIV- 1 restriction in nondividing myeloid cells by employing cryo-EM and X-ray crystallography. Overall, we will explore the unique SAMHD1-mediated dNTP metabolic regulatory circuits in nondividing myeloid cells, which are engineered by two distinct regulators, and this proposal aims to discover new and better antiviral concepts specifically targeting HIV-1 in long-living myeloid reservoir...