Lentivirus Replication Strategy and Pathogenesis

项目摘要-Kim 包括HIV-1、HIV-2和SIV在内的慢病毒可以在激活的CD4+T细胞中复制，并最终- 分化/未分裂的髓系细胞(如巨噬细胞)。而HIV-1在激活的CD4+T细胞中快速复制 细胞内，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， 巨噬细胞含有极低的dNTP浓度(20-40 nM)，这在动力学上限制了病毒的逆转。 转录，后来认为宿主SAMHD1 dNTPase负责在 巨噬细胞，它限制艾滋病毒-1的复制。然而，SIVsm和HIV-2即使在 巨噬细胞由其病毒蛋白X(VPX)引起，该蛋白可降解SAMHD1，然后升高细胞 巨噬细胞的dNTP水平。我们的长期前提是极其有限的移动dNTP池 未分裂的髓系细胞创造了HIV-1复制的独特生化和病毒学特征，这 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的致病机理。 我们之前资助的研究表明，HIV-1逆转录酶(RT)独特地显示了有效的DNA 即使在低巨噬细胞dNTP浓度下也具有合成能力，这使HIV-1能够克服 SAMHD1介导的巨噬细胞内限制性dNTPs。然而，我们发现许多SIV和HIV-2 RT-S 在低巨噬细胞dNTP浓度下，菌株的DNA合成能力显著降低， 与HIV-1 RTS相比，支持HIV-2/SIV RTS没有进化到高效率，可能是因为 这些病毒的VPX可提高巨噬细胞中其RTS的dNTP浓度。基于这些发现， 首先，我们假设RT酶动力学可以抵消SAMHD1介导的有限的dNTP池 VPX的缺失。这一假说预测带有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合成DNA，以克服SAMHD1- 巨噬细胞中介导的低dNTP池(如HIV-1)。第二，祖先的非灵长类慢病毒 由于FIV、BIV和EIAV也在巨噬细胞中有效复制，而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中和自己的宿主SAMHD1蛋白 蛋白质组降解SAMHD1(如HIV-2/SIV)或2)进化为含有酶有效的RTS(AS HIV-1就是这样)。第三，我们报告了HIV-1经常合并高度丰富的非正规性/诱变性 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 有限的标准dNTP底物。由于rNTPs的加入是最丰富的DNA损伤 细胞，也是序列特异性的，我们假设在整个过程中都有rNTP掺入热点 HIV-1基因组序列，成为突变热点，最终增强HIV-1在 巨噬细胞。总体而言，这项申请的目的是获得关于艾滋病毒-1独特复制机制的知识 髓系细胞作为长寿的病毒库，最终开发出髓系特异性的抗HIV-1药物。

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.