Lentivirus Replication Strategy and Pathogenesis

慢病毒复制策略和发病机制

• 批准号: 10700321
• 负责人: Baek Kim
• 金额: $ 46.17万
• 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 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700321
• 关键词: 2019-nCoV; Acceleration; Affinity; Binding; Biochemical; CD4 Positive T Lymphocytes; Cells; Compensation; Complex; DNA; DNA Sequence Alteration; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Development; Elements; Enzyme Kinetics; Evolution; Genome; Genomics; HIV-1; HIV-2; Health; Infection; Interphase Cell; Investigation; Kinetics; Lentivirus; Macrophage; Mediating; Microglia; Mutagenesis; Mutagens; Mutation; Myelogenous; Myeloid Cells; Nature; Pathogenesis; Phenotype; Prodrugs; Proteins; RNA; RNA Polymerase II; RNA Viruses; RNA chemical synthesis; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Reporting; Retroviridae; Reverse Transcription; Ribonucleotides; Roentgen Rays; SIV; Series; Structure; Testing; Tropism; Viral; Viral Genome; Viral Reverse Transcription; Virus Replication; X-Ray Crystallography; molnupiravir; multicatalytic endopeptidase complex; novel; pharmacologic; tool; tripolyphosphate; viral genomics

Project Summary – Kim Lentiviruses infect both activated/dividing CD4+ T cells and terminally differentiated/nondividing myeloid cells during the course of their pathogenesis. As we previously reported, host SAMHD1 dNTPase restricts viral reverse transcription step specifically in nondividing myeloid cells by depleting cellular dNTPs whereas HIV-2 and some SIVs counteract SAMHD1 by proteosomal degradation through their accessary proteins (e.g. Vpx). In this renewal, we aim to reveal noble mechanistic strategies that lentiviruses employ for their myeloid cell infection and rapid evolution/escape. First, lentiviruses encode an additional polypurine track (PPT) sequence, called central PPT (cPPT), that locates at the center of the viral genome and is used for the additional initiation of the (+) strand DNA synthesis. We previously reported that the concomitant initiation of the (+) strand DNA synthesis from both PPT and cPPT compensates the kinetically delayed HIV-1 reverse transcription in nondividing cells with limited dNTP pools by cutting the size of the (+) strand DNA synthesis from PPT by half. In Aim 1, we will test our hypothesize that the additional cPPT of HIV-1 allows HIV-1 to overcome the SAMHD1-mediated dNTP depletion and complete the (+) strand DNA synthesis even in nondividing myeloid cells without accessary proteins counteracting SAMHD1. Second, we previously reported that the uniquely tight dNTP binding affinity of HIV-1 RT mechanistically contributes to its catalytic capability to execute DNA synthesis even at low dNTP concentrations, which enables HIV-1 to replicate in myeloid cells with very low dNTP pools. Importantly, we also reported that this tight dNTP binding affinity enables HIV-1 RT to efficiently extend mismatch primer post misinsertion, compared to other retroviral RTs, which is responsible for the highly error prone DNA synthesis of HIV-1 RT. Based on these observations, we propose to solve the X-ray structure of HIV-1 RT ternary complex with mismatch primer, which will elucidate the structural nature of the highly error prone HIV-1 replication machinery which is important for viral evolution and escape. Third, while lethal mutagenesis has been observed in other RNA viruses, it remains unclear that the lethal mutagenesis of HIV-1 and lentiviruses can be achieved by pharmacological means. Triphosphate (TP) of Molnupiravir, b-d-N4 hydroxycytidine (NHC) prodrug, is a ribonucleotide substrate and an RNA mutagen for RNA-dependent RNA polymerases of multiple RNA viruses including SARS-CoV-2, which induces viral lethal mutagenesis. Excitingly, our biochemical data demonstrate that host cellular RNA polymerase II also incorporates NHC-TP during RNA synthesis, supporting the likelihood of the NHC-TP incorporation into cellular RNAs by host RNA polymerases. Since lentivirus RNA genomes are synthesized by host DNA-dependent RNA polymerase II, we hypothesize that NHC may be able to induce lethal mutagenesis in lentiviruses. Overall, this renewal application focuses on elucidating the unique mechanistic and structural elements of lentivirus replication machinery, ultimately aiming at developing novel antiviral concepts and tools.

项目摘要 - 金 慢病毒感染了激活/分裂的CD4+ T细胞和终末分化/非分裂的髓样细胞 在其发病机理过程中。正如我们先前报道的那样，宿主SAMHD1 DNTPase限制了病毒 逆转录步骤是通过耗尽细胞DNTP而专门针对非分裂细胞的，而HIV-2 一些SIV通过蛋白质体降解通过其辅助蛋白（例如VPX）来抵消SAMHD1。 在这种续约中，我们旨在揭示慢烟病毒雇主为髓样细胞的贵族机械策略 感染和快速演变/逃生。首先，慢病毒编码一个附加的息肉轨道（PPT）序列， 称为中央PPT（CPPT），位于病毒基因组中心，用于附加启动 （+）链DNA合成。我们先前报道了（+）链DNA的伴随倡议 来自PPT和CPPT的合成可以补偿动力学延迟的HIV-1逆转录 通过将PPT的（+）链DNA合成的大小切成一半，使DNTP池有限的细胞具有有限的DNTP池。 在AIM 1中，我们将测试我们的假设，即HIV-1的额外CPPT允许HIV-1克服 SAMHD1介导的DNTP部署并完成（+）Strand DNA合成，即使在非各个髓样中 没有辅助蛋白抵消SAMHD1的细胞。其次，我们以前报道了独特的 HIV-1 RT的紧密DNTP结合亲和力机械地有助于其催化能力执行DNA 即使在低DNTP浓度下合成，这使HIV-1在非常低的髓样细胞中复制 DNTP池。重要的是，我们还报告说，这种紧密的DNTP结合亲和力使HIV-1 RT有效 与其他逆转录病毒RT相比，扩展了不匹配的引物后插入后的插入后，这是负责高度造成的 HIV-1 RT的误差下降DNA合成。基于这些观察结果，我们建议解决X射线结构 与不匹配底漆的HIV-1 RT三元复合物的结构性 俯卧的HIV-1复制机制，这对于病毒进化和逃脱很重要。第三，而致命 在其他RNA病毒中已经观察到诱变，尚不清楚HIV-1的致命诱变 可以通过药物手段来实现慢病毒。 Molnupiravir的三磷酸（TP），B-D-N4 羟基丁胺（NHC）前药是核糖核苷酸底物和RNA诱变剂RNA依赖性RNA 多种RNA病毒的聚合酶，包括SARS-COV-2，可诱导病毒致死诱变。令人兴奋的是 我们的生化数据表明，宿主细胞RNA聚合酶II还合并了RNA期间的NHC-TP 合成，支持宿主RNA聚合酶将NHC-TP工业的可能性投入细胞RNA。 由于慢病毒RNA基因组由宿主DNA依赖性RNA聚合酶II合成，因此我们假设 NHC可能能够在慢病毒中诱导致命诱变。总体而言，此续约申请的重点是 阐明慢病毒复制机制的独特机械和结构元素，最终针对 在开发新颖的抗病毒概念和工具方面。