Inhibitors of host kinases as molecular targets for immune defense against HIV-1

描述（由申请人提供）：精英控制者是在没有HAART的情况下病毒载量检测不到的HIV-1感染者，在这些患者中识别免疫防御机制可能揭示在更广泛的HIV-1患者人群中增加对HIV-1的免疫防御的关键新信息。精英控制者先前的研究主要集中在经典的T细胞和B细胞介导的抗HIV-1的免疫反应。然而，已经表明，这种适应性免疫活性可能不是这些患者中针对HIV-1的有效免疫保护的充分的，有时甚至不是必要的组分。在初步研究中，我们发现来自精英控制者的CD 4 T细胞对HIV-1复制的敏感性大大降低。来自精英控制者的CD 4 T细胞的这种部分抗性由细胞周期蛋白依赖性激酶抑制剂p21（waf-1/cip-1）的强烈上调介导，其能够独立地抑制HIV-1逆转录和mRNA转录。据我们所知，这些数据代表了第一个描述的分子HIV-1限制因子，积极抑制HIV-1复制在体内精英控制器，因此作为一个直接的分子靶点，提高宿主对HIV-1的抗性。在这里，我们建议通过战略性地关注三个具体目标来扩展我们对p21介导的HIV-1限制的研究：为了分析负责精英控制者的CD 4 T细胞中p21保护性高水平表达的分子途径，我们将研究miRNA如何调节CD 4 T细胞中p21基因的转录，使用从HIV-1精英控制者的独特队列中收集的纵向样品，对于这些控制者，PBMC样品从急性感染时起就可获得（具体目标1）。在具体目标2中，我们建议从机制上研究p21如何影响HIV-1逆转录，这是HIV-1最早和最重要的复制步骤之一。我们假设p21可以通过阻断激活逆转录酶的细胞周期蛋白依赖性激酶家族的宿主蛋白来间接抑制HIV-1逆转录酶。如果这些研究成功，我们将确定HIV-1宿主-病原体相互作用的一个新方面，可以拦截早期HIV-1复制步骤，因此可能对临床方法特别有吸引力，以增加宿主对HIV-1的抗性。最后，我们将机械地调查如何p21可能有助于HIV-1潜伏机制，通过影响宿主因子参与转录延长的HIV-1 mRNA转录本，这些调查可能提供重要的新信息，诱导长期无药物缓解HIV-1感染，防止病毒生长从潜伏感染的细胞（具体目标3）。总体而言，这些研究代表了一个高度跨学科的调查，整合免疫学，病毒学和生物化学方法的机制调查的一种新的HIV-1限制因子，是有效的限制易感性HIV-1感染体内精英控制者;如果成功，这些研究将揭示HIV-1发病机制重要新信息，HIV-1感染免疫防御和临床防治策略。

DESCRIPTION (provided by applicant): Elite controllers are HIV-1 infected persons with undetectable viral loads in the absence of HAART, and the identification of immune defense mechanisms in these patients may reveal critical new information for increasing immune defense against HIV-1 in a broader HIV-1 patient population. Previous studies in elite controllers have mostly focused on classical T- and B-cell mediated immune responses against HIV-1. However, it has been shown that such adaptive immune activity may not be a sufficient, and sometimes not even a necessary component of effective immune protection against HIV-1 in these patients. In preliminary studies, we found that CD4 T cells from elite controllers are substantially less susceptible to HIV-1 replication. This partial resistance of CD4 T cells from elite controllers was mediated by a strong upregulation of the cyclin- dependent kinase inhibitor p21 (waf-1/cip-1), which was able to independently suppress HIV-1 reverse transcription and mRNA transcription. To our knowledge, these data represent the first description of a molecular HIV-1 restriction factor that actively inhibits HIV-1 replication in vivo in elite controllers and an therefore serve as a direct molecular target for enhancing host resistance to HIV-1. Here, we propose to extend our investigations of p21-mediated HIV-1 restriction by strategically focusing on three specific aims: To analyze molecular pathways responsible for the protective high-level expression of p21 in CD4 T cells from elite controllers, we will investigate how miRNAs regulate p21 gene transcription in CD4 T cells, using longitudinal samples collected from a unique cohort of HIV-1 elite controllers for whom PBMC samples are available from the time of acute infection (specific aim 1). In specific aim 2, we propose to mechanistically investigate how p21 affects HIV-1 reverse transcription, one of the earliest and most important HIV-1 replication steps. We hypothesize that p21 can indirectly inhibit HIV-1 reverse transcriptase by blocking host proteins from the cyclin-dependent kinase family that activate reverse transcriptase. If these studies are successful, we will identify a new aspect of HIV-1 host-pathogen interactions that can intercept an early HIV-1 replication step and may therefore be particularly attractive for clinical approaches to increase host resistance to HIV-1. Finally, we will mechanistically investigate how p21 may contribute to mechanisms of HIV-1 latency by affecting host factors involved in transcriptional elongation of HIV-1 mRNA transcripts; these investigations may provide important new information for inducing a long-term drug-free remission of HIV-1 infection by preventing viral outgrowth from latently infected cells (specific aim 3). Overall, these studies represent a highly interdisciplinary investigation integrating immunologic, virologic and biochemical approaches for the mechanistic investigation of a novel HIV-1 restriction factor that is effective at limiting the susceptibility to HIV-1 infection in vivo in elite controllers; if sucessful, these studies will reveal important new information for HIV-1 pathogenesis, immune defense and clinical strategies for prevention and treatment of HIV-1 infection.