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: 7657913
• 负责人: JAISRI R LINGAPPA
• 金额: $ 37.35万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7657913
• 关键词: 1,2-diacylglycerol; Abbreviations; Acute; Affect; Altretamine; Antiviral Agents; Biochemistry; Biological Assay; CD4 Positive T Lymphocytes; Cell Line; Cells; Chronic; Complex; Cytidine; Cytidine Deaminase; DNA; Data; Deaminase; Deamination; Diglycerides; Endogenous Retroviruses; Endoribonucleases; Enzymes; Evolution; Generations; Genome; HIV; HIV Infections; HIV-1; Hela Cells; Human; Immune Sera; Immunoprecipitation; Infection; Interferon-alpha; Interferons; Intervention; Knowledge; Life Cycle Stages; Measures; Mediating; Mitogens; Mutation; Pancreatic ribonuclease; Patients; Peptides; Peripheral Blood Mononuclear Cell; Personal Satisfaction; Phosphorylation; Phytohemagglutinins; Plasmids; Protein Kinase C; Proteins; Purpose; Radioactive; Regulation; Rest; Reverse Transcription; Ribonucleases; Ribonucleoproteins; Signal Pathway; T cell regulation; T-Lymphocyte; Tetradecanoylphorbol Acetate; Therapeutic; Time; Viral; Viral Load result; Virion; Virus; enzyme activity; follow-up; in vivo; insight; molecular mass; tool

The human cytidine deaminases APOBEC3G (A3G) are antiviral proteins that cause DNA hypermutation in HIV-1 DNA by deaminating cytidines. A3G-mediated hypermutation of HIV DNA correlates with lower viral loads, emphasizing its importance in vivo, but unfortunately hypermutated proviral sequences are found in only 10-20% of patients. Thus, interventions to increase deamination of HIV in vivo could be useful therapeutically. However, little is known about the regulation of A3G enzymatic activity in primary T cells, which express A3G endogenously and are infected by HIV in vivo. Recently we established a high-throughput, non-radioactive assay for measuring A3G enzymatic activity. Using this assay 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 primary T cells, A3G deaminase activity is regulated by signaling pathways raising the possibility that signaling pathways can influence the amount of HI hypermutation that occurs during infection. Here we propose to use our deaminase activity assay to understand the regulation of APOBEC3 (A3) proteins in primary T cells and how this regulation affects HIV infection. Specifically, we propose to 1) determine which signaling pathways activate A3G in primary T cells; 2) determine if phosphorylation state or association with known A3G interacting proteins correlates with A3G activation; 3) examine A3G enzymatic activity and hypermutation caused by A3G at different times during the HIV life cycle; and 4) understand the regulation of weakly active A3 proteins in primary cells that may promote HIV evolution. Thus, the studies proposed here will delineate how signaling pathways regulate endogenous deaminase activity of A3G in primary human T cells, and determine how this regulation influences generation of mutations in the HIV genome.

人类胞苷脱氨酶apobec3g（A3G）是抗病毒蛋白，在 HIV-1 DNA通过脱氨基胞苷。 HIV DNA的A3G介导的超数与较低的病毒相关 负载，强调其在体内的重要性，但不幸的是，仅在 10-20％的患者。因此，增加体内HIV脱氨的干预措施在治疗上可能是有用的。 然而，对原代T细胞中A3G酶活性的调节知之甚少，该酶活性表达A3G 内源性，并被体内HIV感染。最近，我们建立了一个高通量，非放射性活性 测量A3G酶活性的测定。使用此测定，我们证明了脱氨酶活性 抑制了在静止和完全活化的原代人T细胞中内源表达的A3G，这表明 A3G在原代T细胞中受负调节。我们的初步数据表明，在原代T细胞中A3G 脱氨酶活性通过信号通路来调节信号通路可以 影响感染期间发生的HI超数量。在这里我们建议使用脱氨酶 活性测定以了解原代T细胞中APOBEC3（A3）蛋白的调节，以及如何 调节会影响HIV感染。 具体而言，我们建议1）确定哪种信号通路激活原代T细胞中的A3G； 2） 确定磷酸化状态或与已知A3G相互作用蛋白的关联是否与A3G相关 激活; 3）检查A3G酶活性和在不同时间A3G引起的A3G酶活性和过度突击 艾滋病毒生命周期； 4）了解原代细胞中弱活性A3蛋白的调节，以促进 艾滋病毒的进化。因此，这里提出的研究将描述信号通路如何调节内源性 A3G在原代人T细胞中的脱氨酶活性，并确定该调节如何影响产生 HIV基因组中的突变。