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: 8305577
• 负责人: JAISRI R LINGAPPA
• 金额: $ 37.45万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305577
• 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; 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

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.

人胞苷脱氨酶APOBEC3G和3F(A3G/F)是在 通过酶机制和非酶机制抑制HIV复制。演技 在酶的作用下，A3G/F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， 这表明这种类型的超突变与疾病进展的减缓有关。然而， 由于很少有研究关注A3G在原代细胞中的内源性表达，因此人们对此知之甚少。 原代细胞中哪些A3G/F池具有酶活性，A3G/F活性在原代细胞中是如何调节的 细胞，以及T细胞激活等生理相关事件如何影响A3G/F酶活性。使用 高通量测定A3G酶活性，我们证明了A3G脱氨酶活性 在静息和完全激活的原代人类T细胞中内源性表达A3G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 外周血单个核细胞(PBMCs)，A3G在T细胞后短时间内被翻译后激活 细胞受体刺激。在这一应用中，我们提出了对人外周血单核细胞中A3G/F的系统研究 和T细胞。具体地说，我们将1)确定哪些信号通路激活和抑制A3G酶 T细胞的活性；2)检查作为感染目标的PBMC中的A3G/F是否有助于 HIV DNA的超突变；3)确定哪些A3蛋白被包装成病毒，由初级 使用我们制备的特异性抗血清检测人T细胞和巨噬细胞；4)定量检测酶 系统测定A3G/F对PBMCs产生的病毒粒子的非酶效应 这些病毒粒子的活性和感染性，以及靶细胞中艾滋病毒的超突变和逆转录本 被这些病毒粒子感染。拟议的研究将极大地帮助我们了解A3G/F 功能，这将大大增加我们对A3G/F的不完全理解 被感染的病人。