Restriction of HIV infection by SAMHD1 protein

描述（由申请人提供）：巨噬细胞和树突状细胞在病毒感染中具有关键作用，提供经常抵抗抗病毒治疗的病毒库，并将先天性病毒检测与抗病毒适应性免疫应答联系起来。人类免疫缺陷病毒1（HIV-1）不能激活树突状细胞，并且由于有效的早期进入后限制阻止病毒cDNA的积累，其激活巨噬细胞的能力降低。这种限制在很大程度上是由细胞SAMHD 1蛋白介导的，其中的突变导致AGS综合征（AGS），这是一种与被认为是由内源性核酸引起的先天免疫应答的激活相关的自身免疫性疾病。重要的是，SAMHD 1被HIV-2/SIVsm病毒的Vpx蛋白降解，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，SAMHD 1介导了对HIV感染免疫反应的关键细胞类型的有效和相关限制，因此可以确定抗病毒治疗的长期结果。我们建议阐明控制和介导SAMHD 1限制HIV感染的细胞和分子机制。目标1中的实验将在基于病毒和细胞的测定的背景下定义SAMHD 1蛋白的功能组织，并评估SAMHD 1的病毒学和细胞功能之间的关系。目的2中的实验将评估SAMHD 1如何干扰HIV-1 cDNA合成。我们的数据表明，SAMHD 1脱氧核苷三磷酸三磷酸水解酶（dNTH 4）的催化活性调节在大肠杆菌中，是与其他细胞酶的情况下，本身是不足以限制HIV感染。因此，SAMHD 1在限制性与非限制性条件下的催化活性将是 表征，并评估SAMHD 1接近RTC对于限制发生的可能重要性。在目标3中，生物化学/蛋白质组学/反向遗传学方法的组合将用于鉴定控制SAMHD 1功能的细胞蛋白和翻译后修饰。目的4中的实验将表征在骨髓细胞从CD 34+祖细胞体外分化期间SAMHD 1介导的对骨髓细胞中HIV-1感染的抑制的发育概况，以及SAMHD 1可能参与非骨髓细胞中HIV-1感染的早期进入后阻断。还将评估在目的3中鉴定的SAMHD 1辅因子在原发性髓系白血病中的相对重要性。从我们的研究中获得的知识可能会导致新策略的概念，以改善对HIV的免疫反应和/或通过操纵SAMHD 1途径防止在组织巨噬细胞中形成长寿的HIV储库。

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.