Lentivirus Replication Strategy and Pathogenesis

项目概要- Kim 慢病毒感染活化/分裂的CD 4 + T细胞和终末分化/非分裂的骨髓细胞 在其发病过程中。正如我们以前报道的，宿主SAMHD 1 dNTR限制病毒 逆转录步骤，特别是在非分裂的骨髓细胞通过消耗细胞dNTPs，而HIV-2 一些SIV通过其附属蛋白（例如Vpx）通过蛋白体降解来抵消SAMHD 1。 在这次更新中，我们的目标是揭示慢病毒对其髓样细胞采用的高贵的机制策略， 感染和快速进化/逃逸。首先，慢病毒编码额外的多嘌呤轨道（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的合成。我们以前报道过，（+）链DNA的伴随启动 PPT和cPPT的合成补偿了HIV-1逆转录的动力学延迟， 通过将PPT合成的（+）链DNA的大小减半， 在目标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 SAMHD 1介导的dNTP耗竭和完成（+）链DNA合成，即使在非分裂髓系中 没有抵抗SAMHD 1的辅助蛋白的细胞。第二，我们以前报道过， HIV-1 RT紧密的dNTP结合亲和力在机制上有助于其催化执行DNA的能力 即使在低dNTP浓度下也能合成，这使得HIV-1能够在骨髓细胞中复制， 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-1 RT能够有效地 与其他逆转录病毒RT相比，在错误插入后延长错配引物，这是导致高度 HIV-1 RT的DNA合成容易出错。基于这些观察，我们提出解决X射线结构 HIV-1 RT三元复合物与错配引物，这将阐明高度错误的结构性质， 易于HIV-1复制的机制，这对病毒进化和逃逸很重要。第三，虽然致命， 尽管在其他RNA病毒中观察到了突变，但仍不清楚HIV-1的致死性突变 和慢病毒可以通过药理学手段获得。莫努匹韦的三磷酸盐（TP），b-d-N4 羟基胞苷（NHC）前药，是一种核糖核苷酸底物和RNA依赖性RNA的RNA诱变剂 包括SARS-CoV-2在内的多种RNA病毒的聚合酶，其诱导病毒致死突变。令人兴奋的是， 我们的生物化学数据表明，宿主细胞RNA聚合酶II在RNA聚合过程中也掺入NHC-TP。 NHC-TP的合成，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依赖性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死突变。总的来说，这一更新应用程序的重点是 阐明慢病毒复制机制的独特机制和结构元件， 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.