Regulation of APOBEC3G enzymatic activity in HIV-infected primary human T cells

描述（由申请人提供）：人胞苷脱氨酶APOBEC3G和3F （A3G/F）是在人体组织中表达的抗病毒蛋白，通过酶和非酶机制抑制HIV复制。A3G/F通过酶作用编辑HIV DNA，导致灾难性的高突变。大型临床研究表明，高水平的A3G/ f介导的高突变与较低的病毒载量和较高的CD4计数相关，这表明这种类型的高突变与疾病进展减缓有关。然而，由于很少有研究关注A3G在原代细胞中的内源性表达，因此对于原代细胞中哪些A3G/F库具有酶活性，A3G/F活性如何在原代细胞中被调节，以及T细胞活化等生理相关事件如何影响A3G/F酶活性，我们知之甚少。利用高通量测定A3G酶活性的方法，我们证明了内源性表达的A3G在静息和完全激活的原代人T细胞中脱氨酶活性受到抑制，这表明A3G在原代T细胞中受到负调控。我们的初步数据表明，在人外周血单核细胞（PBMCs）中，A3G在T细胞受体刺激后的短时间内被翻译后激活。在这个应用中，我们建议系统地研究A3G/F在人pbmc和T细胞中的作用。具体来说，我们将1)确定哪些信号通路激活和抑制T细胞中的A3G酶活性；2)研究作为感染靶点的pbmc中的A3G/F是否与HIV DNA的高突变有关；3)利用我们制备的特异性抗血清，确定哪些A3蛋白被包装到人原代T细胞和巨噬细胞产生的病毒中；4)通过系统测量这些病毒粒子的酶活性和感染性，以及被这些病毒粒子感染的靶细胞中的HIV超突变和逆转录物，量化A3G/F在pbmc产生的病毒粒子中的酶促和非酶促作用。拟议的研究将极大地有助于我们了解A3G/F在原代人细胞中的功能，并将大大补充我们对感染患者中A3G/F的不完整理解。公共卫生相关性：人类胞苷脱氨酶APOBEC3G和3F （A3G/F）是抗病毒蛋白，部分通过DNA编辑引起HIV DNA的灾难性超突变来抑制病毒复制。由于感染患者中A3G/ f介导的HIV DNA高水平的高突变与较低的病毒载量和较高的CD4计数相关，因此了解体内高突变是如何产生的将是重要的；然而，A3G/F在人原代细胞中的研究相对较少。拟议的研究将通过确定感染HIV的外周血单核细胞及其产生的病毒粒子中哪些A3G/F池具有酶活性，以及信号通路是否调节感染外周血单核细胞中A3G/F的酶活性和HIV DNA的高突变，来研究A3G/F在原代人外周血单核细胞（PBMCs）中的功能。

DESCRIPTION (provided by applicant): The human cytidine deaminases APOBEC3G and 3F (A3G/F) are antiviral proteins that are expressed in human tissues and inhibit HIV replication by enzymatic and nonenzymatic mechanisms. Acting enzymatically, A3G/F edits HIV DNA, causing catastrophic hypermutation. Large clinical studies reveal that high levels of A3G/F-mediated hypermutation correlate with lower viral loads and higher CD4 counts, suggesting that this type of hypermutation is associated with reduced disease progression. However, because few studies have focused on A3G expressed endogenously in primary cells, little is known about which pools of A3G/F in primary cells are enzymatically active, how A3G/F activity is regulated in primary cells, and how physiologically relevant events like T cell activation affect A3G/F enzymatic activity. Using a high-throughput assay for measuring A3G enzymatic activity, we demonstrated that deaminase activity of endogenously expressed A3G in resting and fully activated primary human T cells is inhibited, suggesting that A3G is negatively regulated in primary T cells. Our preliminary data demonstrate that in human peripheral blood mononuclear cells (PBMCs), A3G is post-translationally activated at short times after T cell receptor stimulation. In this application, we propose a systematic study of A3G/F in human PBMCs and T cells. Specifically, we will 1) determine which signaling pathways activate and inhibit A3G enzymatic activity in T cells; 2) examine whether A3G/F in PBMCs that are the target of infection contributes to hypermutation of HIV DNA; 3) determine which A3 proteins are packaged into virus produced by primary human T cells and macrophages using specific antisera we have generated; and 4) quantify the enzymatic and nonenzymatic effects of A3G/F in virions produced by PBMCs by systematically measuring enzymatic activity and infectivity of these virions, as well as HIV hypermutation and reverse transcripts in target cells infected by these virions. The proposed studies should greatly contribute to our knowledge of how A3G/F functions in primary human cells and will add significantly to our incomplete understanding of A3G/F in infected patients. PUBLIC HEALTH RELEVANCE: The human cytidine deaminases APOBEC3G and 3F (A3G/F) are antiviral proteins that inhibit viral replication in part by causing catastrophic hypermutation of HIV DNA through DNA editing. Because high levels of A3G/F-mediated hypermutation of HIV DNA in infected patients correlate with lower viral loads and higher CD4 count, it will be important to understand how hypermutation arises in vivo; however, A3G/F in primary human cells is relatively understudied. The proposed studies will address how A3G/F functions in primary human peripheral blood mononuclear cells (PBMCs) by determining which pools of A3G/F are enzymatically active in HIV infected PBMCs and the virions they produce, and whether signaling pathways regulate A3G/F enzymatic activity and hypermutation of HIV DNA in infected PBMCs.