SAMHD1 mediated dNTP regulation and HIV in myeloid cells

SAMHD1 介导的 dNTP 调节和骨髓细胞中的 HIV

• 批准号: 10616679
• 负责人: Baek Kim
• 金额: $ 68.89万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10616679
• 关键词: Anabolism; Antiviral Therapy; Binding; Biological Assay; Brain; CD4 Positive T Lymphocytes; CDC2 gene; CDK2 gene; Caenorhabditis elegans; Cells; Consumption; Cryoelectron Microscopy; DNA biosynthesis; Drosophila genus; Engineering; Exposure to; HIV; HIV-1; HIV-2; Health; Homeostasis; Host Defense Mechanism; Human; Infection; Interphase Cell; Investigation; Kinetics; Lentivirus; Life Cycle Stages; Macrophage; Mediating; Metabolic; Metabolism; Microglia; Mitotic; Modeling; Molecular; Myelogenous; Myeloid Cells; Nature; Orthologous Gene; Pathogenesis; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Regulation; Reporting; Reverse Transcription; Ribonucleotide Reductase; Role; SAM Domain; SIV; Tail; Viral; Viral Proteins; Viral reservoir; X-Ray Crystallography; cell type; cyclin A2; enzyme biosynthesis; inhibitor; meter; monocyte; multicatalytic endopeptidase complex; neuroAIDS; novel; prevent

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.

项目概要 包括 HIV-1 在内的慢病毒感染激活/分裂的 CD4+ T 细胞并最终感染 分化/非分裂骨髓细胞（例如巨噬细胞和小胶质细胞） 发病。慢病毒的逆转录消耗受感染者提供的 dNTP 底物 宿主细胞。然而，据预测，非分裂细胞（例如巨噬细胞）应含有有限的 dNTP 水池。事实上，我们之前报道过人类原代单核细胞衍生的巨噬细胞极其具有 与激活的 CD4+ T 细胞 (1-5 µM) 相比，dNTP 水平有限 (20-40 nM)，并且这种有限的 dNTP 水平 巨噬细胞限制 HIV-1 复制。我们还报告了主机 SAM 域和 HD 域 含有蛋白质 1 (SAMHD1)，可水解 dNTP，并且在巨噬细胞中含量丰富，负责 低 dNTP 水平和巨噬细胞中 HIV-1 复制受限。最近，我们发现了两本小说 SAMHD1 介导的 dNTP 代谢调节回路可在非分裂骨髓细胞中发挥作用 用于 dNTP 耗尽和 HIV-1 限制。在本提案中，我们建议阐明病毒学、分子学和 非分裂骨髓中 SAMHD1 介导的 dNTP 消耗的这些调节回路的结构性质 细胞。在目标 1 中，我们将探索我们的假设，即 SAMHD1 不仅水解 dNTP，而且还直接水解 通过与巨噬细胞中的 RNR 结合来抑制 RNR 介导的 dNTP 生物合成。我们的假设 预测 Vpx 可以在 SAMHD1 降解后迅速提高巨噬细胞中的 dNTP 水平 同时去除 RNR 介导的 dNTP 生物合成的抑制性调节因子。确实，我们最近 观察到 SAMHD1 与 RNR R1 亚基的直接结合，支持了这一假设。在这里，我们将 研究由 SAMHD1-RNR 相互作用介导的负 dNTP 代谢调节回路 不分裂骨髓细胞中 dNTP 耗尽和 HIV-1 限制。在目标 2 中，我们假设细胞 PP2A- B55磷酸酶是非分裂骨髓细胞中 SAMHD1 的关键正调节因子，可以保持 SAMHD1 非磷酸化且具有酶活性，可消除 dNTP 并限制 HIV-1。事实上，我们观察到 SAMHD1 与非分裂骨髓细胞中 PP2A 的 B55 调节亚基的相互作用，支持了这一点 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 巨噬细胞中的代谢和 HIV-1 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 耗尽和 HIV- 通过采用冷冻电镜和 X 射线晶体学对非分裂骨髓细胞进行 1 限制。总体而言，我们将 探索非分裂骨髓细胞中独特的 SAMHD1 介导的 dNTP 代谢调节回路， 由两个不同的监管机构设计，该提案旨在发现新的、更好的抗病毒概念 专门针对导致 HIV-1 持久存在的长寿命骨髓库中的 HIV-1。