Regulation of APOBEC3G enzymatic activity in HIV-infected primary human T cells

人胞苷脱氨酶APOBEC 3G（A3 G）是一种抗病毒蛋白，可引起DNA超突变， HIV-1 DNA通过脱氨基胞苷。A3 G介导的HIV DNA超突变与较低的病毒感染相关 负载，强调其在体内的重要性，但不幸的是，高度突变的前病毒序列发现，只有在 10-20%的患者。因此，增加体内HIV脱氨基作用的干预措施可能在治疗上有用。 然而，关于表达A3 G的原代T细胞中A3 G酶活性的调节知之甚少 内源性的并且在体内被HIV感染。最近，我们建立了一个高通量，非放射性的 用于测量A3 G酶活性的测定。使用该测定，我们证明了脱氨酶活性 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细胞中负调控。我们的初步数据表明，在原代T细胞中，A3 G 脱氨酶活性受信号通路调节，这增加了信号通路可以 影响在感染期间发生的HI超突变的量。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APOBEC 3（A3）蛋白的调节以及这种调节是如何发生的。 监管影响艾滋病毒感染。 具体而言，我们建议1）确定哪些信号通路激活原代T细胞中的A3 G; 2） 确定磷酸化状态或与已知A3 G相互作用蛋白的关联是否与A3 G相关 3）检测A3 G酶活性和A3 G在活化过程中不同时间引起的超突变; HIV生命周期; 4）了解原代细胞中弱活性A3蛋白的调节， 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 A3 G在原代人T细胞中的脱氨酶活性，并确定这种调节如何影响生成 艾滋病病毒基因组的突变。

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.