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: 8111861
• 负责人: JAISRI R LINGAPPA
• 金额: $ 37.97万
• 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/8111861
• 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; Health; 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

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（由申请方提供）：人胞苷脱氨酶APOBEC 3G和3F（A3 G/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。通过酶促作用，A3 G/F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，表明这种类型的超突变与疾病进展减少有关。然而，由于很少有研究集中在A3 G内源性表达的原代细胞，很少有人知道A3 G/F在原代细胞中的池是酶活性，A3 G/F活性是如何调节的原代细胞，以及如何生理相关的事件，如T细胞活化影响A3 G/F酶活性。使用用于测量A3 G酶活性的高通量测定，我们证明了在静息和完全活化的原代人T细胞中内源性表达的A3 G的脱氨酶活性被抑制，表明A3 G在原代T细胞中被负调控。我们的初步数据表明，在人外周血单核细胞（PBMC），A3 G是在T细胞受体刺激后短时间内激活。在本申请中，我们提出了在人PBMC和T细胞中的A3 G/F的系统研究。具体而言，我们将1）确定哪些信号通路激活和抑制T细胞中的A3 G酶活性; 2）检查作为感染靶点的PBMC中的A3 G/F是否有助于HIV DNA的超突变; 3）使用我们产生的特异性抗血清确定哪些A3蛋白被包装到由原代人T细胞和巨噬细胞产生的病毒中;和4）通过系统地测量由PBMC产生的病毒体的酶活性和感染性，以及由这些病毒体感染的靶细胞中的HIV超突变和逆转录物，定量A3 G/F在这些病毒体中的酶促和非酶促作用。拟议的研究将大大有助于我们了解A3 G/F如何在原代人类细胞中发挥作用，并将大大增加我们对A3 G/F在感染患者中的不完全理解。公共卫生相关性：人胞苷脱氨酶APOBEC 3G和3F（A3 G/F）是一种抗病毒蛋白，通过DNA编辑引起HIV DNA的灾难性超突变来部分抑制病毒复制。由于感染患者中高水平的A3 G/F介导的HIV DNA超突变与较低的病毒载量和较高的CD 4计数相关，因此了解体内超突变是如何发生的非常重要;然而，原代人类细胞中的A3 G/F相对研究不足。拟议的研究将通过确定哪些A3 G/F库在HIV感染的PBMC及其产生的病毒体中具有酶活性，以及信号传导途径是否调节A3 G/F酶活性和感染的PBMC中HIV DNA的超突变，来解决A3 G/F在原代人外周血单核细胞（PBMC）中的功能。