Restriction of HIV infection by SAMHD1 protein

描述(申请人提供)：巨噬细胞和树突状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和Linkin天生病毒检测，以对抗病毒适应性免疫反应。人类免疫缺陷病毒1型(HIV-1)不能转导树突状细胞，并且由于进入后的一种有效的限制，阻止了病毒DNA的积累，所以转导巨噬细胞的能力降低。这种限制在很大程度上是由细胞SAMHD1蛋白介导的，突变会导致Aicardi Goutieres综合征(AGS)，这是一种与激活天然免疫反应有关的自身免疫性疾病，被认为是由内源性核酸引发的。重要的是，SAMHD1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方面的关键作用一致。这些发现表明，SAMHD1介导了对关键细胞类型的有效和相关的限制，这些细胞类型协调了对艾滋病毒感染的免疫反应，因此可能决定抗病毒治疗的长期结果。我们建议阐明控制和介导SAMHD1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基于细胞的分析的背景下确定SAMHD1蛋白的功能组织，并评估SAMHD1的病毒学和细胞功能之间的关系。AIM 2中的实验将评估SAMHD1如何干扰HIV-1cDNA的合成。我们的数据表明，SAMHD1脱氧核苷三磷酸水解酶(DNTPase)的催化活性在CELS中受到调控，与其他细胞酶一样，本身不足以限制HIV感染。因此，在限制性和非限制性条件下的SAMHD1催化活性将是 并将评估SAMHD1靠近RTC对限制发生的可能重要性。在目标3中，将使用生化/蛋白质组/反向遗传方法的组合来识别控制SAMHD1功能的细胞蛋白质和翻译后修饰。目的4的实验将描述SAMHD1在髓系细胞从CD34+祖细胞体外分化过程中抑制HIV-1感染的发育特征，以及SAMHD1可能参与非髓系细胞进入后早期阻断HIV-1感染的可能。目标3中确定的SAMHD1辅助因子在初级髓系细胞中的相对重要性也将被评估。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途径来提高对艾滋病毒的免疫反应和/或防止组织巨噬细胞中形成长期存活的艾滋病毒储存库。

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.