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

描述(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在酶的作用下编辑HIV DNA，导致灾难性的超突变。大型临床研究显示，高水平的A3G/F介导的超突变与较低的病毒载量和较高的CD4计数相关，表明这种类型的超突变与减缓疾病进展有关。然而，由于很少有研究关注A3G在原代细胞中的内源性表达，人们对原代细胞中哪些A3G/F池具有酶活性，A3G/F活性在原代细胞中如何调节，以及T细胞激活等生理相关事件如何影响A3G/F酶活性知之甚少。用高通量方法检测A3G的酶活性，我们证明内源性表达的A3G在静息和完全激活的原代T细胞中的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，在人外周血单个核细胞(PBMCs)中，A3G在T细胞受体刺激后的短时间内被翻译后激活。在这一应用中，我们提出了对人PBMCs和T细胞中A3G/F的系统研究。具体地说，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)检测作为感染目标的PBMCs中的A3G/F是否导致HIV DNA的高度突变；3)使用我们制备的特定抗血清确定哪些A3蛋白被包装到由原代人类T细胞和巨噬细胞产生的病毒中；以及4)通过系统地测量这些病毒粒子的酶活性和感染性，以及在感染这些病毒粒子的靶细胞中HIV的超突变和反向转录，来定量A3G/F在PBMC产生的病毒粒子中的酶和非酶效应。拟议的研究将极大地有助于我们了解A3G/F在原代人类细胞中的功能，并将显著增加我们对感染患者中A3G/F的不完全理解。与公共卫生相关：人类胞苷脱氨酶APOBEC3G和3F(A3G/F)是抗病毒蛋白，通过DNA编辑导致艾滋病毒DNA的灾难性过度突变，部分地抑制病毒复制。由于感染患者体内高水平的A3G/F介导的HIV DNA超突变与较低的病毒载量和较高的CD4计数相关，了解体内如何发生超突变将是重要的；然而，对原代人类细胞中的A3G/F的研究相对较少。拟议的研究将通过确定哪些A3G/F池在HIV感染的PBMC中具有酶活性及其产生的病毒粒子，以及信号通路是否调节感染的PBMC中A3G/F的酶活性和HIV DNA的高度突变来研究A3G/F在原代人外周血单个核细胞(PBMC)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.