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的情况下病毒载量无法检测到的HIV-1感染者，这些患者中免疫防御机制的识别可能会揭示在更广泛的HIV-1患者群体中加强对HIV-1的免疫防御的关键新信息。以前对精英控制者的研究主要集中在经典的T和B细胞介导的针对HIV-1的免疫反应上。然而，已经表明，这种适应性免疫活动可能不是这些患者有效免疫保护HIV-1的充分组成部分，有时甚至不是必要的组成部分。在初步研究中，我们发现精英控制员的CD4T细胞对HIV-1复制的敏感性大大降低。精英控制者的CD4T细胞的这种部分抵抗是由细胞周期蛋白依赖的激酶抑制物p21(WAF-1/CIP-1)的强烈上调所介导的，该抑制物能够独立地抑制HIV-1的逆转录和mRNA转录。据我们所知，这些数据首次描述了在精英控制者体内主动抑制HIV-1复制的分子HIV-1限制因子，从而成为增强宿主对HIV-1抵抗力的直接分子靶点。在这里，我们建议通过策略性地专注于三个特定目标来扩展我们对p21介导的HIV-1限制的研究：为了分析精英控制者CD4T细胞中p21保护性高水平表达的分子途径，我们将使用从急性感染时起就可获得PBMC样本的HIV-1精英控制者的独特队列中收集的纵向样本，来研究miRNAs如何调节CD4T细胞中的p21基因转录(特定目标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复制的最早和最重要的步骤之一。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的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宿主与病原体相互作用的新方面，可以拦截HIV-1早期复制步骤，因此可能特别适合于临床方法来增加宿主对HIV-1的抵抗力。最后，我们将从机制上研究p21如何通过影响参与HIV-1mRNA转录延长的宿主因素来影响HIV-1潜伏期的机制；这些研究可能为通过防止潜伏期感染细胞中的病毒生长来诱导HIV-1感染的长期无药物缓解提供重要的新信息(特定目标3)。总体而言，这些研究代表了一项高度跨学科的研究，整合了免疫学、病毒学和生化方法，以机制研究一种新的HIV-1限制因子，该因子有效地限制了精英控制者体内对HIV-1感染的易感性；如果成功，这些研究将揭示HIV-1发病机制、免疫防御以及预防和治疗HIV-1感染的临床策略的重要新信息。

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.