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

HIV 感染的原代人 T 细胞中 APOBEC3G 酶活性的调节

• 批准号: 8137533
• 负责人: JAISRI R LINGAPPA
• 金额: $ 21.38万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8137533
• 关键词: Address; Affect; Antiviral Agents; CD4 Lymphocyte Count; CD4 Positive T Lymphocytes; Cell Line; Cells; Clinical Research; Complex; Cytidine Deaminase; DNA; Data; Deaminase; Disease Progression; Epithelial; Event; Genome; HIV; HIV Infections; HIV-1; Hela Cells; Human; Human Cell Line; Immune Sera; Infection; Knowledge; Light; Measures; Mediating; Patients; Peripheral Blood Mononuclear Cell; Plasmids; Proteins; Regulation; Relative (related person); Reporting; Research; Rest; Retroviridae; Reverse Transcription; Ribonucleoproteins; Signal Pathway; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Time; Transcript; Transformed Cell Line; Viral; Viral Load result; Virion; Virus; high throughput screening; human tissue; in vivo; macrophage; novel; protein function; public health relevance

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.

描述（由申请人提供）：人类胞苷脱氨酶APOBEC3G和3F（A3G/F）是在人体组织中表达的抗病毒蛋白，并通过酶促和非酶机制抑制HIV复制。酶促作用，A3G/F编辑HIV DNA，从而导致灾难性的过度突变。大型临床研究表明，高水平的A3G/F介导的高含量与较低的病毒载量和较高的CD4计数相关，这表明这种类型的超突变与疾病进展降低有关。但是，由于很少有研究集中在原代细胞中内源性表达的A3G上，因此对主要细胞中哪些A3G/F的池几乎不知名，在原代细胞中如何调节A3G/F活性，以及​​如何在生理相关的事件中调节t（例如T）细胞活化会影响A3G/F酶促活性。使用高通量测定法来测量A3G酶活性，我们证明了内源性表达A3G的脱氨酶活性在静止和充分活化的原代人T细胞中受到抑制，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，在人类外周血单核细胞（PBMC）中，A3G在T细胞受体刺激后短时间在短时间内激活。在此应用中，我们提出了对人PBMC和T细胞中A3G/F的系统研究。具体而言，我们将1）确定哪些信号通路激活并抑制T细胞中的A3G酶活性； 2）检查是感染靶标的PBMC中的A3G/F是否有助于HIV DNA的超数； 3）确定使用我们已经产生的特定抗血清，将哪些A3蛋白包装到由原代人T细胞和巨噬细胞产生的病毒中；和4）通过系统地测量这些病毒体的酶活性和感染性以及HIV超突变以及在这些病毒感染感染的靶细胞中，量化PBMC产生的A3G/F的酶促和非酶作用。拟议的研究应极大地有助于我们对原代人细胞中A3G/F的功能的了解，这将显着增加我们对受感染患者A3G/F的不完全理解。公共卫生相关性：人类细胞丁烷脱氨酶APOBEC3G和3F（A3G/F）是抗病毒蛋白，通过通过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HIV DNA的高水平A3G/F介导的HIV DNA的超数与较低的病毒载量和较高的CD4计数相关，因此了解体内如何出现过度成绩是重要的。但是，原代人细胞中的A3G/F相对低估了。拟议的研究将通过确定哪些A3G/F的池在HIV感染的PBMC及其产生的病毒体中酶活性，以及​​是否调节A3G/F酶促途径是否在HIV感染的PBMC中确定哪些A3G/F的池在HIV感染的PBMC中酶活性，以解决A3G/F的功能如何通过确定哪些A3G/F的池酶活性来调节A3G/F的功能。 HIV DNA在感染的PBMC中的活性和超数。