SAMHD1 mediated dNTP regulation and HIV in myeloid cells

项目摘要 包括HIV-1在内的慢病毒感染活化/分裂的CD4T细胞，并最终- 分化/未分裂的髓系细胞(如巨噬细胞和小胶质细胞) 发病机制。慢病毒的逆转录会消耗感染者提供的dNTP底物。 宿主细胞。然而，有人预测，未分裂的细胞，如巨噬细胞，应该含有有限的dNTP 泳池。事实上，我们之前曾报道过，人类原代单核细胞来源的巨噬细胞 有限的dNTP水平(20-40 NM)，与激活的CD4T细胞(1-5微米)相比，这种有限的dNTP水平 巨噬细胞限制HIV-1的复制。我们还报告了主机SAM域和HD域 含有蛋白1(SAMHD1)，它能降解dNTPs，在巨噬细胞中含量丰富，是导致 巨噬细胞中dNTP水平低和HIV-1复制受限。最近，我们发现了两部小说 SAMHD1介导的dNTP代谢调控电路可在未分裂的髓系细胞中发挥作用 用于dNTP耗尽和HIV-1限制。在这项建议中，我们建议阐明病毒学、分子和 SAMHD1介导的未分裂髓系dNTP耗竭调控回路的结构性质 细胞。在目标1中，我们将探索我们的假设，即SAMHD1不仅能水解dNTPs，而且还能直接 通过与巨噬细胞中的RNR结合，抑制RNR介导的dNTP生物合成。我们的假设 预测VPX可以通过以下途径迅速提高SAMHD1降解后巨噬细胞中的dNTP水平 同时去除RNR介导的dNTP生物合成的抑制调节子。的确，我们最近 观察到SAMHD1与RNR R1亚基直接结合，支持这一假说。在这里，我们将 研究SAMHD1-RNR相互作用介导的这个负dNTP代谢调节电路 未分裂髓系细胞的dNTP耗竭和HIV-1限制。在目标2中，我们假设细胞内的PP2A- 在未分裂的髓系细胞中，B55磷酸酶是SAMHD1的关键正调控因子，可以保持SAMHD1 非磷酸化和酶活性的dNTP耗尽和HIV-1限制。事实上，我们观察到 在未分裂的髓系细胞中，SAMHD1与PP2A的B55调节亚基相互作用，支持这一点 假设。在这里，我们将研究SAMHD1-PP2A相互作用在dNTP负调控中的作用 巨噬细胞的代谢与HIV-1的限制。在目标3中，我们建议研究结构和 SAMHD1与RNR和PP2A相互作用的分子性质导致dNTP耗竭和HIV- 1冷冻电子显微镜和X射线结晶学对未分裂的髓系细胞的限制。总的来说，我们将 探索SAMHD1介导的未分裂髓系细胞中独特的dNTP代谢调节电路，这是 由两个不同的监管机构设计，这项提案旨在发现新的更好的抗病毒概念 特别针对长期存活的髓系贮存库中的艾滋病毒-1，这有助于艾滋病毒-1的持久性。

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 reservoirs that contribute to HIV-1 persistence.