Lentivirus Replication Strategy and Pathogenesis

项目摘要-Kim 慢病毒感染活化/分裂的CD4+T细胞和终末分化/未分裂的髓系细胞 在它们的发病过程中。正如我们之前报道的，宿主SAMHD1 dNTPase限制病毒 逆转录步骤通过耗尽细胞dNTPs而在未分裂的髓系细胞中特异性地进行，而HIV-2 一些SIV通过其辅助蛋白(如VPX)通过蛋白酶体降解来抵消SAMHD1。 在这次更新中，我们的目标是揭示慢病毒对其髓系细胞所采用的高尚的机制策略。 感染和快速进化/逃逸。首先，慢病毒编码一个额外的多嘌呤跟踪(PPT)序列， 称为中央PPT(CPPT)，位于病毒基因组的中心，用于额外的启动 (+)链DNA合成。我们之前报道过伴随的(+)链DNA的启动 PPT和cPPT的合成补偿HIV-1在体内的动态延迟逆转录 通过将从PPT合成的(+)链DNA的大小减半来不分裂具有有限dNTP池的细胞。 在目标1中，我们将检验我们的假设，即HIV-1的额外cPPT允许HIV-1克服 SAMHD1介导的dNTP耗尽和完成(+)链DNA合成即使在未分裂的髓系中也是如此 不含抗SAMHD1的辅助蛋白的细胞。其次，我们之前曾报道过， HIV-1RT紧密的dNTP结合亲和力对其执行DNA的催化作用 即使在低浓度的dNTP下也能合成，这使得HIV-1能够在非常低的浓度下在髓系细胞中复制 DNTP池。重要的是，我们还报道了这种紧密的dNTP结合亲和力使HIV-1RT能够有效地 与其他逆转录病毒逆转录酶相比，延长错配引物插入后，可导致高度 容易出错的HIV-1RT的DNA合成。在这些观测的基础上，我们提出了解决X射线结构 用错配引物构建HIV-1 RT三元复合体，这将阐明高度错误的结构性质 容易感染HIV-1病毒的复制机制，这对病毒的进化和逃逸很重要。第三，虽然是致命的 在其他RNA病毒中也观察到了突变，但尚不清楚HIV-1的致命性突变 慢病毒可以通过药物手段获得。三磷酸莫那普拉韦，b-d-N4 羟基胞苷(NHC)前药，是一种核糖核苷酸底物，也是RNA依赖的RNA的诱变剂 包括SARS-CoV-2在内的多种RNA病毒的聚合酶，可诱导病毒致死突变。令人兴奋的是， 我们的生化数据表明，宿主细胞RNA聚合酶II在RNA过程中也整合了NHC-TP 合成，支持NHC-TP通过宿主RNA聚合酶掺入细胞RNA的可能性。 由于慢病毒rna基因组是由宿主dna依赖的rna聚合酶II合成的，我们假设 NHC可能能够在慢病毒中诱导致命的突变。总体而言，此续订申请侧重于 阐明慢病毒复制机制的独特机制和结构元素，最终目的是 致力于开发新的抗病毒概念和工具。

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.