Lentivirus Replication Strategy and Pathogenesis

慢病毒复制策略和发病机制

• 批准号: 10319982
• 负责人: Baek Kim
• 金额: $ 38.55万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-19 至 2023-04-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319982
• 关键词: Adopted; Animals; Antiviral Therapy; Biochemical; Biology; Brain; CD4 Positive T Lymphocytes; Cell Death; Cells; Chemistry; DNA; DNA Damage; DNA biosynthesis; Environment; Enzyme Kinetics; Equine Infectious Anemia Virus; Evolution; Exhibits; Feline Immunodeficiency Virus; Funding; Genetic; Genomics; HIV; HIV-1; HIV-2; Hot Spot; Infection; Interphase Cell; Kinetics; Knowledge; Lentivirus; Mediating; Metabolic; Microglia; Molecular Conformation; Mutagenesis; Mutation; Myelogenous; Myeloid Cells; Pathogenesis; Proteins; RNA-Directed DNA Polymerase; Reporting; Research; Ribonucleosides; SAM Domain; SIV; Serum; Testing; Therapeutic; Tissues; Viral; Viral Proteins; Viral Reverse Transcription; Viral reservoir; Virus; Virus Replication; base; cell type; comparative; in vivo; macrophage; mutant; novel; tripolyphosphate

Project Summary – Kim Lentiviruses including HIV-1, HIV-2 and SIV replicate in both activated CD4+ T cells and terminally- differentiated/non-dividing myeloid cells (e.g. macrophages). While HIV-1 rapidly replicates in activated CD4+ T cells, HIV-1 replication in macrophages is kinetically suppressed. Our previous studies found that macrophages harbor an extremely low dNTP concentration (20-40 nM), which kinetically restricts viral reverse transcription, and later that the host SAMHD1 dNTPase is responsible for the limited dNTP level in macrophages, which restricts HIV-1 replication. However, SIVsm and HIV-2 efficiently replicate even in macrophages due to its viral protein X (Vpx) that proteosomally degrades SAMHD1 and then elevates cellular dNTP levels in macrophages. Our long-term premise is that the extremely limited cellular dNTP pool in nondividing myeloid cells creates unique biochemical and virological features of HIV-1 replication, which directly influence viral replication kinetics, genomic diversity, evolution, and ultimately, pathogenesis of HIV-1. Our previously funded research revealed that HIV-1 reverse transcriptase (RT) uniquely displays efficient DNA synthesis capability even at the low macrophage dNTP concentrations, which enables HIV-1 to overcome the SAMHD1-mediated limited dNTPs in macrophages. However, we found that RTs from many SIV and HIV-2 strains exhibit significantly reduced DNA synthesis capability at the low macrophage dNTP concentrations, compared to HIV-1 RTs, supporting that HIV-2/SIV RTs did not evolve to be highly efficient, possibly because Vpx of these viruses elevates dNTP concentrations for their RTs in macrophages. Based on these findings, first, we hypothesize that the RT enzyme kinetics can counteract SAMHD1-mediated limited dNTP pools in the absence of Vpx. This hypothesis predicts that RT of a SIV mutant with Vpx deletion should evolve to be more efficient in DNA synthesis during the in vivo replication in animals in order to overcome the SAMHD1- mediated low dNTP pools in macrophages (as HIV-1 does). Second, ancestral non-primate lentiviruses such as FIV, BIV and EIAV also efficiently replicate in macrophages, and these ancestral lentiviruses do not encode Vpx. Here we will test whether these non-primate lentiviruses counteract their own host SAMHD1 proteins by proteosomally degrading SAMHD1 (as HIV-2/SIV do) or 2) evolving to harbor enzymatically efficient RTs (as HIV-1 does). Third, we reported that HIV-1 frequently incorporates highly abundant non-canonical/mutagenic ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, specifically in macrophages due to the limited canonical dNTP substrates. Since the incorporation of rNTPs is the most abundant DNA damages in cells and is also sequence-specific, we hypothesize that there are rNTP incorporation hot spots throughout HIV-1 genomic sequences, which become mutational hot spots and ultimately enhance HIV-1 mutagenesis in macrophages. Overall, this application aims at gaining knowledge on unique HIV-1 replication mechanism in myeloid cells that serve as long-living viral reservoirs, ultimately developing myeloid specific anti-HIV-1 agents.

项目概要- Kim 包括HIV-1、HIV-2和SIV在内的慢病毒在活化的CD 4 + T细胞和终末- 分化的/非分裂的骨髓细胞（例如巨噬细胞）。虽然HIV-1在活化的CD 4 + T细胞中快速复制， 细胞中，巨噬细胞中的HIV-1复制受到动力学抑制。我们之前的研究发现， 巨噬细胞具有极低的dNTP浓度（20-40 nM），其在动力学上限制病毒逆转 转录，后来，宿主SAMHD 1 dNTP负责有限的dNTP水平， 巨噬细胞，限制HIV-1复制。然而，SIVsm和HIV-2即使在 巨噬细胞由于其病毒蛋白X（Vpx），蛋白体降解SAMHD 1，然后提高细胞 巨噬细胞中的dNTP水平。我们的长期假设是， 不分裂的骨髓细胞产生了HIV-1复制的独特生化和病毒学特征， 直接影响病毒复制动力学、基因组多样性、进化，并最终影响HIV-1的发病机制。 我们先前资助的研究表明，HIV-1逆转录酶（RT）独特地显示高效的DNA 即使在低巨噬细胞dNTP浓度下也具有合成能力，这使得HIV-1能够克服 巨噬细胞中SAMHD 1介导的有限dNTPs。然而，我们发现许多SIV和HIV-2的RT 菌株在低巨噬细胞dNTP浓度下表现出显著降低的DNA合成能力， 与HIV-1 RTs相比，支持HIV-2/SIV RTs没有进化成高效的，可能是因为 这些病毒的Vpx提高了巨噬细胞中RT的dNTP浓度。根据这些发现， 首先，我们假设RT酶动力学可以抵消SAMHD 1介导的有限dNTP池， 没有Vpx。这一假说预测，Vpx缺失的SIV突变体的RT应该进化为 在动物体内复制过程中更有效地进行DNA合成，以克服SAMHD 1- 介导巨噬细胞中的低dNTP池（如HIV-1所做的那样）。第二，祖先非灵长类慢病毒， 因为FIV、BIV和EIAV也在巨噬细胞中有效复制，并且这些祖先慢病毒不编码 Vpx。在这里，我们将测试这些非灵长类慢病毒是否通过以下方式抵消它们自己的宿主SAMHD 1蛋白： 蛋白酶体降解SAMHD 1（如HIV-2/SIV）或2）进化为具有酶促有效RT（如 HIV-1确实如此）。第三，我们报道了HIV-1经常结合高度丰富的非典型/致突变的 核糖核苷三磷酸（rNTPs）在前病毒DNA合成过程中，特别是在巨噬细胞中，由于 有限的典型dNTP底物。由于rNTPs的掺入是最丰富的DNA损伤， 细胞，也是序列特异性的，我们假设有rNTP掺入热点整个 HIV-1基因组序列，成为突变热点，并最终增强HIV-1的诱变， 巨噬细胞总的来说，这个应用程序的目的是获得知识的独特艾滋病毒-1复制机制， 骨髓细胞作为长期存活的病毒库，最终开发骨髓特异性抗HIV-1药物。