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APOBEC 3G和3F（A3 G/F）是在大肠杆菌中表达的抗病毒蛋白。 并通过酶和非酶机制抑制HIV复制。代理 在酶促作用下，A3 G/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A3 G/F介导的超突变与较低的病毒载量和较高的CD 4计数相关， 这表明这种类型的超突变与疾病进展减缓有关。然而，在这方面， 由于很少有研究关注A3 G在原代细胞中的内源性表达， 原代细胞中A3 G/F的哪些库具有酶活性，原代细胞中A3 G/F活性如何调节， 细胞，以及生理相关事件如T细胞活化如何影响A3 G/F酶活性。使用 通过高通量测定A3 G酶活性，我们证明了A3 G脱氨酶活性 在静息和完全活化的原代人T细胞中内源性表达的A3 G被抑制，表明 A3 G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（PBMC），A3 G在T细胞后短时间内被激活， 细胞受体刺激在本申请中，我们提出了对人PBMC中A3 G/F的系统研究 和T细胞。具体来说，我们将1）确定哪些信号通路激活和抑制A3 G酶 2）检查作为感染靶点的PBMC中的A3 G/F是否有助于T细胞中的A3 G/F活性; HIV DNA的超突变; 3）确定哪些A3蛋白被包装到由初级生产的病毒中 人T细胞和巨噬细胞使用我们已经产生的特异性抗血清;和4）定量酶促免疫反应。 A3 G/F在PBMC产生的病毒体中的酶促和非酶促作用 这些病毒粒子的活性和感染性，以及HIV在靶细胞中的超突变和逆转录 被这些病毒体感染建议的研究将大大有助于我们了解A3 G/F 在原代人类细胞中的功能，并将大大增加我们对A3 G/F的不完全理解， 感染的病人。