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: 10398255
• 负责人: Baek Kim
• 金额: $ 41.53万
• 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/10398255
• 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; Mediating; Metabolic; Metabolism; Microglia; Mitotic; Modeling; Molecular; Myelogenous; Myeloid Cells; Nature; Orthologous Gene; Pathogenesis; Phosphoric Monoester Hydrolases; Phosphorylation; 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; macrophage; monocyte; 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.

项目摘要 慢病毒在内 分化/非分明的髓样细胞（例如巨噬细胞和小胶质细胞）在其过程中 发病。慢病毒的逆转录消耗受感染的DNTP底物 宿主细胞。但是，据预测，诸如巨噬细胞之类的非各个细胞应包含有限的DNTP 游泳池。确实，我们以前报道说，人类原发性单核细胞衍生的巨噬细胞极为有 与活化的CD4+ T细胞（1-5 µM）相比，DNTP水平有限（20-40 nm），并且该有限的DNTP水平 巨噬细胞限制了HIV-1复制。我们还报告了主机SAM域和高清域 含有蛋白质1（SAMHD1），水解DNTP并在巨噬细胞中丰富，是负责 巨噬细胞中的低DNTP水平和受限的HIV-1复制。最近，我们发现了两本小说 SAMHD1介导的DNTP代谢的调节回路可以在非分散的髓样细胞中起作用 用于DNTP部署和HIV-1限制。在此提案中，我们建议阐明病毒学，分子和 SAMHD1介导的dNTP耗竭的这些调节回路的结构性本质 细胞。在AIM 1中，我们将探讨SAMHD1不仅水解DNTP，还直接的假设 通过在巨噬细胞中与RNR结合来抑制RNR介导的DNTP生物合成。我们的假设 预测，通过SAMHD1降解后，VPX可以迅速升高巨噬细胞中的DNTP水平 同时删除RNR介导的DNTP生物合成的抑制性调节剂。确实，我们最近 观察到SAMHD1与RNR R1亚基的直接结合，支持这一假设。在这里，我们会的 研究由SAMHD1-RNR相互作用介导的负DNTP代谢调节电路 非分裂髓样细胞中的DNTP耗竭和HIV-1限制。在AIM 2中，我们假设细胞PP2A-- B55磷酸酶是非分裂髓样细胞中SAMHD1的关键阳性调节剂，可以保持SAMHD1 用于DNTP耗竭和HIV-1限制的非磷酸化和酶活性。确实，我们观察到 SAMHD1与pp2a的B55调节亚基的相互作用在非分裂的髓样细胞中，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 巨噬细胞中的代谢和HIV-1限制。在AIM 3中，我们建议研究结构和 SAMHD1与RNR和PP2A相互作用的分子本质有助于DNTP耗竭和HIV- 1通过采用冷冻EM和X射线晶体学来限制非各个髓样细胞。总体而言，我们会的 探索非各种髓样细胞中独特的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，更好的抗病毒毒概念 专门针对长寿的髓样储层中的HIV-1，这有助于HIV-1持久性。