Role of protein methylation in HIV latency

前病毒潜伏期仍然是从感染者身上根除艾滋病毒的一个重要障碍。表观遗传过程是调控HIV基因表达的关键。我们假设精氨酸和赖氨酸甲基化在病毒前潜伏期中起着重要的沉默作用，甲基转移酶的小分子抑制剂可以在治疗上促进潜伏感染的T细胞中HIV的诱导/根除（l/E）。这一假设得到了文献数据和我们自己初步未发表的研究的支持，在这些研究中，我们发现了几种甲基转移酶（MTs）在潜伏感染的T细胞中具有抑制HIV转录的活性。项目1.5的目标是全面研究mt在持续性HIV感染中的作用，并与Martin Deianey协作实验室的研究人员密切合作，验证mt在HIV前潜伏期的l/E中作为潜在的药物靶点。特异性目的1：综合分析J-Lat细胞和HIV潜伏期原代细胞培养模型中针对赖氨酸和精氨酸甲基转移酶的小发夹rna。我们将对11个已知的人类精氨酸MTs、24个已知的人类赖氨酸MTs和38个基于与赖氨酸MTs保守SET结构域特征的结构域同源性鉴定的新蛋白进行高度靶向的shRNA筛选，以确定一组强化HIV潜伏期的MTs作为l/E治疗的潜在药物靶点。专项目标2：研究甲基转移酶控制HIV潜伏期的分子机制。我们将研究在前期工作中发现的4个抑制mt （PRMT1、PRMT6、Set1和SETDB1）的转录活性。我们将研究这些因子在潜伏的HIV LTR中募集的情况，并在潜伏感染的T细胞（组蛋白）中鉴定这些酶的底物。即P-TEFb或NF-KB)。特异性目的3：测试甲基转移酶抑制剂对潜伏感染T细胞中HIV转录再激活的影响。我们将评估已知的精氨酸和赖氨酸MTs小分子抑制剂对J-Lat细胞和原代T细胞潜伏期模型中HIV前病毒潜伏期的影响。我们将对这些抑制剂主要针对的酶进行表征，并利用这些信息开发有效和特异性的HIV的l/E。总的来说，这些研究将为控制HIV前病毒潜伏期的基本机制提供新的分子见解，并支持本实验室的共同目标，即生产用于在动物和患者持续HIV感染的l/E中进行测试的化合物。

Proviral latency remains an important barrier to eradication of HIV from infected individuals. Epigenetic processes are critical in the regulation of HIV gene expression. We hypothesize that arginine and lysine methylation plays an important silencing role in proviral latency and that small molecule inhibitors of methyltransferases can contribute therapeutically to the induction/eradication (l/E) of HIV in latently infected T cells. This hypothesis is supported by data in the literature and our own preliminary unpublished studies, in which we identified several methyltransferases (MTs) with suppressive activity against HIV transcription in latently infected T cells. The goal of Project 1.5 is to comprehensively study the role of MTs in persistent HIV infection and in close collaboration with investigators across the Martin Deianey Collaboratory to validate MTs as potential drug targets in the l/E of HIV proviral latency. Specific Aim 1: Comprehensively analyze small hairpin RNAs directed against lysine and arginine methyltransferases in J-Lat cells and primary cell culture models of HIV latency. We will perform a highly targeted shRNA screen of 11 known human arginine MTs, 24 known human lysine MTs and 38 new proteins identified based on domain homology with a conserved SET domain characteristic for lysine MTs to identify a set of MTs that enforce HIV latency as potential drug targets for l/E therapy. Specific Aim 2: Study the molecular mechanisms how methyltransferases control HIV latency. We will study the transcriptional activities of four suppressive MTs (PRMT1, PRMT6, Set1 and SETDB1) identified by our preliminary work. We will study the in wVo recruitment of these factors to the latent HIV LTR and identify substrates of these enzymes in latently infected T cells (histones. Tat, P-TEFb or NF-KB). Specific Aim 3: Test the effect of methyltransferase inhibitors on the reactivation of HIV transcription in latently infected T cells. We will evaluate the effect of known small molecule inhibitors of arginine and lysine MTs on HIV proviral latency in J-Lat cells and primary T cell models of latency. We will characterize the enzymes primarily targeted by these inhibitors and use this information for the development of effective and specific l/E of HIV, Collectively, these studies will provide novel molecular insight into the basic mechanisms governing HIV proviral latency and support the collective goal of this Collaboratory to produce compounds to be tested in the l/E of persistent HIV infection in animals and patients.