Restriction of HIV infection by SAMHD1 protein

描述（由申请人提供）：巨噬细胞和树突细胞在病毒感染中发挥关键作用，提供经常抵抗抗病毒治疗的病毒库，并将先天病毒检测与抗病毒适应性免疫反应联系起来。人类免疫缺陷病毒 1 (HIV-1) 无法转导树突状细胞，并且转导巨噬细胞的能力降低，因为早期进入后的有效限制阻止了病毒 cDNA 的积累。这种限制在很大程度上是由细胞 SAMHD1 蛋白介导的，这种突变会导致 Aicardi Goutieres 综合征 (AGS)，这是一种与先天免疫反应激活相关的自身免疫性疾病，被认为是由内源性核酸引起的。重要的是，SAMHD1 是 HIV-2/SIVsm 病毒 Vpx 蛋白降解的目标，这与其在抑制灵长类慢病毒感染中的关键作用一致。这些发现表明，SAMHD1 介导对关键细胞类型的有效且相关的限制，这些细胞类型协调对 HIV 感染的免疫反应，因此可以决定抗病毒治疗的长期结果。我们建议阐明 SAMHD1 控制和介导限制 HIV 感染的细胞和分子机制。目标 1 中的实验将在病毒和细胞检测背景下定义 SAMHD1 蛋白的功能组织，并评估 SAMHD1 的病毒学功能和细胞功能之间的关系。目标 2 中的实验将评估 SAMHD1 如何干扰 HIV-1 cDNA 合成。我们的数据表明，SAMHD1 脱氧核苷三磷酸三磷酸水解酶 (dNTPase) 催化活性在细胞中受到调节，与其他细胞酶的情况一样，其本身不足以限制 HIV 感染。因此，限制性条件与非限制性条件下的 SAMHD1 催化活性将是 将评估 SAMHD1 接近 RTC 对于发生限制的可能重要性。在目标 3 中，将结合使用生化/蛋白质组学/反向遗传学方法来识别控制 SAMHD1 功能的细胞蛋白和翻译后修饰。目标 4 中的实验将表征在 CD34 祖细胞体外分化过程中 SAMHD1 介导的髓系细胞中 HIV-1 感染抑制的发育特征，以及 SAMHD1 可能参与非髓系细胞中 HIV-1 感染的早期进入阻断。还将评估目标 3 中确定的 SAMHD1 辅助因子在原发性骨髓细胞中的相对重要性。从我们的研究中获得的知识可能会导致新策略的构想，通过操纵 SAMHD1 途径来改善对 HIV 的免疫反应和/或防止组织巨噬细胞中形成长寿命的 HIV 储存库。

DESCRIPTION (provided by applicant): Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist anti-viral therapies and linkin innate virus detection to anti-viral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to a potent early post-entry restriction preventing accumulation of viral cDNA. The restriction is to a large extent mediated by cellular SAMHD1 protein, mutations in which lead to Aicardi Goutieres Syndrome (AGS), an autoimmune disease associated with activation of the innate immune response thought to be provoked by endogenous nucleic acids. Importantly, SAMHD1 is targeted for degradation by the Vpx protein of HIV-2/SIVsm viruses, consistent with its key role in inhibiting primate lentivirus infection. These findings indicate that SAMHD1 mediates potent and relevant restriction in key cell types that orchestrate the immune response to HIV infection, and hence could determine long-term outcomes of antiviral therapies. We propose to elucidate the cellular and molecular mechanisms that control and mediate the restriction of HIV infection by SAMHD1. Experiments in aim 1 will define the functional organization of the SAMHD1 protein in the context of viral and cell-based assays, and assess the relationship between virologic and cellular functions of SAMHD1. Experiments in aim 2 will assess how SAMHD1 interferes with HIV-1 cDNA synthesis. Our data suggest that SAMHD1 deoxynucleoside triphosphate triphosphohydrolase (dNTPase) catalytic activity is regulated in cels, as is the case with other cellular enzymes, and per se is not sufficient to restrict HIV infection. Hence, SAMHD1 catalytic activity under restrictive vs non-restrictive conditions will be characterized and the possible importance of SAMHD1 proximity to the RTC for the restriction to occur will be assessed. In aim 3, a combination of biochemical/proteomic/reverse genetic approaches will be used to identify cellular proteins and post-translational modifications that control SAMHD1 function. Experiments in aim 4 wil characterize developmental profiles of SAMHD1-mediated inhibition of HIV-1 infection in myeloid cells during their in vitro differentiation from CD34+ progenitor cells, and the possible involvement of SAMHD1 in early post-entry blocks of HIV-1 infection in non-myeloid cells. The relative importance of SAMHD1 co-factors, identified in aim 3, in primary myeloid cels wil also be assessed. The knowledge gained from our studies could lead to the conception of new strategies to improve the immune response to HIV and/or prevent formation of long-lived HIV reservoirs in tissue macrophages, by manipulating the SAMHD1 pathway.