SAMHD1 controls dNTP pool and HIV sensitivity to NRTIs

描述(申请人提供)：我们先前报道，人原代巨噬细胞的dNTP浓度(20-40 nm)比活化的CD4+T细胞(2-5？M)低约100倍。有限的dNTP池对巨噬细胞中的HIV逆转录施加了强烈的限制。最近发表在《自然》杂志上的几篇论文报道，VPX是HIV-2和SIV的共同包装辅助蛋白，通过降解髓系细胞特异性宿主病毒限制因子SAMHD1增强巨噬细胞的病毒感染性，SAMHD1抑制巨噬细胞中HIV-1的复制。另外两项后续研究发现，SAMHD1是一种依赖dGTP的dNTP水解酶，可以降解和灭活细胞内的dNTP，这表明SAMHD1通过降低细胞内的dNTP池来限制HIV-1在未分裂的巨噬细胞中的感染，这在逻辑上与我们观察到的巨噬细胞中dNTP的浓度极低是一致的。事实上，我们最近在《自然免疫学》上发表的报告显示，虽然SAMHD1的表达减少了细胞dNTP池，但VPX通过降解巨噬细胞中的SAMHD1有效地提高了细胞dNTP浓度，支持VPX通过促进髓系细胞类型的前病毒DNA合成动力学来增强HIV的传染性。我们预测，细胞内dNTP的可获得性将机械地影响发生在HIV生命周期早期的三个重要事件：(1)病毒对NRTI的敏感性：在急性感染的巨噬细胞中，NRTI介导的抑制病毒复制的EC50大约比T细胞低10倍。我们假设VPX通过增加细胞dNTP池来降低病毒对NRTIs的敏感性，而SAMHD1的表达通过在巨噬细胞中降解细胞dNTP来增强病毒对NRTIs的敏感性，但在激活的CD4+T细胞中不能。(2)病毒重组频率：HIV-1逆转录酶暂停是由DNA合成过程中dNTP浓度降低引起的，启动RNA模板切换和重组。因此，我们假设VPX和SAMHD1诱导的dNTP池调制直接影响巨噬细胞中HIV-1的重组频率，但不影响T细胞中的HIV-1重组频率。(3)在前病毒DNA合成过程中加入致突变的非规范dUTP：我们发现巨噬细胞中的dTTP水平(低)与非规范dUTP的水平相差60倍，而在T细胞中则不是。我们进一步证明，细胞内dUTP/TTP的比率决定了HIV-1RT介导的dUTP掺入的频率(这是可以突变的)。因此，我们假设，VPX可以提高细胞内dNTP水平(包括dTTP，但不包括dUTP)，可以最大限度地减少潜在突变dUTP在巨噬细胞中的掺入。我们将研究与dNTP底物专一性有关的SAMHD1活性部位的结构和酶学特征。

DESCRIPTION (provided by applicant): We previously reported that human primary macrophage have approximately 100 times lower dNTP concentrations (20-40nM) than activated CD4+ T cells (2~5¿M). The limited dNTP pools impose a strong restriction on HIV reverse transcription in macrophages. Several recent papers in Nature reported that Vpx, a co-packaged accessory protein of HIV-2 and SIVs, enhances viral infectivity in macrophages by degrading a myeloid cell specific, host viral restriction factor, SAMHD1, which suppresses HIV-1 replication in macrophages. Two more follow-up studies revealed that SAMHD1 is a dGTP-dependent dNTP hydrolase, which can hydrolyze and inactivate cellular dNTPs, suggesting that SAMHD1 restricts HIV-1 infection in nondividing macrophages by lowering cellular dNTP pools, which is logically consistent with our observation that the dNTP concentration in macrophages is extremely low. Indeed, our recently accepted report in Nature Immunology revealed that while SAMHD1 expression reduces cellular dNTP pools, Vpx efficiently elevates cellular dNTP concentrations by degrading SAMHD1 in macrophages, supporting that Vpx enhances HIV infectivity by promoting proviral DNA synthesis kinetics in myeloid cell types. We predict that cellular dNTP availability mechanistically influences three important events of HIV biology that occur during the early step of HIV life cycle: (1) Viral sensitivity to NRTIs: The EC50 for NRTI- mediated inhibition of virus replication in acutely infected macrophages is approximately 10-fold lower than that in T cells. We hypothesize that Vpx reduces viral sensitivity to NRTIs by increasing cellular dNTP pools while SAMHD1 expression enhances viral sensitivity to NRTIs by hydrolyzing cellular dNTPs, in macrophages, but not in activated CD4+ T cells. (2) Viral recombination frequency: HIV-1 RT pausing, which is induced by lowering dNTP concentration during DNA synthesis, initiates RNA template switching and recombination. Thus, we hypothesize that dNTP pool modulation induced by Vpx and SAMHD1 directly affects HIV-1 recombination frequency in macrophages, but not in T cells. (3) Incorporation of mutagenic noncanonical dUTP during proviral DNA synthesis: We have shown that there is a 60-fold disparity between levels of dTTP (low) and the noncanonical dUTP in macrophages, but not in T cells. We further showed that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation (which can be mutagenic). Thus we hypothesize that Vpx, which elevates cellular dNTP levels (including dTTP, but not dUTP), can minimize the incorporation of potentially mutagenic dUTP in macrophages. We will investigate the structural and enzymological features of the SAMHD1 active site involved in dNTP substrate specificity.