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 人体组织并通过酶促和非酶促机制抑制 HIV 复制。演戏 A3G/F 通过酶作用编辑 HIV DNA，导致灾难性的超突变。大型临床研究表明 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疾病进展的减缓有关。然而， 由于很少有研究关注原代细胞中内源表达的 A3G，因此我们对 A3G 知之甚少。 原代细胞中哪些 A3G/F 库具有酶活性，原代细胞中 A3G/F 活性如何调节 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 通过高通量测定 A3G 酶活性，我们证明了脱氨酶活性 静息和完全激活的原代人 T 细胞中内源性表达的 A3G 受到抑制，表明 A3G 在原代 T 细胞中受到负调节。我们的初步数据表明，在人类中 外周血单核细胞 (PBMC)，A3G 在 T 后短时间内被翻译后激活 细胞受体刺激。在此应用中，我们提出对人类 PBMC 中的 A3G/F 进行系​​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 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 超突变； 3)确定哪些A3蛋白被包装到初级产生的病毒中 使用我们生成的特定抗血清的人类 T 细胞和巨噬细胞； 4) 量化酶促反应 通过系统测量酶促作用，研究 A3G/F 对 PBMC 产生的病毒体的非酶促作用 这些病毒粒子的活性和感染性，以及靶细胞中的 HIV 超突变和逆转录 被这些病毒粒子感染。拟议的研究应该极大地有助于我们了解 A3G/F 如何 在原代人类细胞中发挥作用，并将显着增加我们对 A3G/F 的不完全理解 感染的患者。