SAMHD1 controls dNTP pool and HIV sensitivity to NRTIs

描述（由申请人提供）：我们之前报道过，人原代巨噬细胞的dNTP浓度（20-40nM）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。《自然》杂志最近的几篇论文报道，Vpx是HIV-2和siv的共包装辅助蛋白，通过降解骨髓细胞特异性宿主病毒限制因子SAMHD1来增强巨噬细胞中的病毒感染性，SAMHD1抑制巨噬细胞中的HIV-1复制。另外两项后续研究发现，SAMHD1是一种dgtp依赖的dNTP水解酶，可以水解和灭活细胞内的dNTP，这表明SAMHD1通过降低细胞内dNTP池来限制非分裂巨噬细胞内HIV-1感染，这与我们观察到巨噬细胞内dNTP浓度极低的结果在逻辑上是一致的。事实上，我们最近在《自然免疫学》上发表的报告显示，虽然SAMHD1表达减少了细胞dNTP池，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通过促进髓细胞类型的前病毒DNA合成动力学来增强HIV传染性。我们预测细胞dNTP的可用性在机制上影响HIV生命周期早期阶段发生的三个重要的HIV生物学事件：(1)病毒对NRTI的敏感性：急性感染巨噬细胞中NRTI介导的病毒复制抑制的EC50比T细胞低约10倍。我们假设Vpx通过增加细胞dNTP池来降低病毒对nrti的敏感性，而SAMHD1表达通过水解细胞dNTP来增强病毒对nrti的敏感性，在巨噬细胞中，但在活化的CD4+ T细胞中没有。(2)病毒重组频率：DNA合成过程中降低dNTP浓度导致HIV-1 RT暂停，引发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重组频率，而对T细胞没有影响。(3)在原病毒DNA合成过程中诱变的非规范dUTP的掺入：我们已经证明巨噬细胞中dTTP（低）水平与非规范dUTP水平之间存在60倍的差异，但在T细胞中没有。我们进一步表明，细胞dUTP/TTP比率决定了HIV-1 rt介导的dUTP掺入的频率（这可能是致突变的）。因此，我们假设Vpx可以提高细胞dNTP水平（包括dTTP，但不包括dUTP），从而最大限度地减少巨噬细胞中潜在致突变dUTP的掺入。我们将研究与dNTP底物特异性相关的SAMHD1活性位点的结构和酶学特征。

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.