HIV-1: microRNA interactions

描述（由申请人提供）：摘要微小RNA（miRNAs）是最近发现的一类小的、~22-nt的调节RNA，现在已知其在细胞分化和发育的调节中起关键作用。miRNA表达的失调可能导致疾病进展，特别是在癌症的情况下，最近的证据也表明miRNA与病毒复制和发病机制有关。现在已知几种病毒，包括许多疱疹病毒和多瘤病毒物种，表达一系列病毒编码的miRNA，并且还已知病毒感染以可能促进病毒复制的方式干扰细胞miRNA表达。虽然对HIV-1如何与细胞miRNA机制相互作用知之甚少，但似乎很清楚HIV-1感染可以改变细胞miRNA表达的模式，并且已经提出单个细胞miRNA可以促进或抑制HIV-1复制。HIV-1也可能编码一个或多个miRNA，尽管这一直存在争议。在这项资助申请中，我们建议使用微阵列和深度测序技术系统地分析HIV-1感染对原代CD 4 + T细胞和巨噬细胞中miRNA表达模式的影响。然后，我们将使用交联免疫沉淀（CLIP）技术，通过使用Argonaute特异性单克隆抗体来识别HIV-1感染细胞中表达的所有mRNA上的所有RNA诱导沉默复合物（RISC）结合位点，以恢复交联的mRNA：RISC复合物，然后我们将通过深度测序进行分析。然后将HIV-1感染细胞中表达的miRNA靶向的细胞或HIV- 1 mRNA进行突变分析，结合功能测定，以鉴定调节HIV-1复制效率的mRNA：miRNA相互作用。最后，我们将确定药物特异性可卡因滥用是否会改变未感染和HIV-1感染的CD 4 + T细胞和巨噬细胞中的miRNA表达谱，我们将检查是否有任何药物诱导的miRNA谱变化可以解释从药物滥用患者中分离的细胞中HIV-1复制的增强。总之，这种分析将导致对HIV-1感染如何改变miRNA表达谱的全面理解，并将提供对这些变化如何调节HIV-1复制效率的机制理解。公共卫生相关性：相关性微RNA（miRNA）是一类小的调控RNA，被认为调节广泛的细胞过程。我们将确定HIV-1感染如何改变miRNA表达模式以及HIV-1是否产生自己的miRNA。然后，我们将确定这些变化是否会增强病毒复制，并确定在正常和药物滥用患者中这种增强的机制。这项研究不仅有可能为HIV-1的发病机制提供新的线索，而且还提出了抑制病毒复制的新方法。

DESCRIPTION (provided by applicant): Abstract MicroRNAs (miRNAs) are a recently discovered class of small, ~22-nt regulatory RNAs that are now known to play key roles in the regulation of cellular differentiation and development. Misregulation of miRNA expression can contribute to disease progression, particularly in the case of cancer, and recent evidence also implicates miRNAs in aspects of viral replication and pathogenesis. Several viruses, including numerous herpesviruses and polyomavirus species, are now known to express a range of virally-encoded miRNAs, and virus infection is also known to perturb cellular miRNA expression in ways that may facilitate virus replication. Although relatively little is know about how HIV-1 interacts with the cellular miRNA machinery, it appears clear that HIV-1 infection can modify the pattern of cellular miRNA expression, and individual cellular miRNAs have been proposed to either facilitate or inhibit HIV-1 replication. It also remains possible that HIV-1 may encode one or more miRNAs, although this has been controversial. In this grant application, we propose to systematically analyze the effect of HIV-1 infection on the pattern of miRNA expression in primary CD4+ T cells and macrophages using microarray and deep sequencing technologies. We will then use cross-linking immunoprecipitation (CLIP) technologies to identify all the RNA induced silencing complex (RISC) binding sites on all the mRNAs expressed in HIV-1-infected cells by using an Argonaute-specific monoclonal antibody to recover cross-linked mRNA:RISC complexes, which we will then analyze by deep sequencing. Cellular or HIV- 1 mRNAs that are targeted by miRNAs expressed in HIV-1-infected cells will then be subjected to mutational analysis, combined with functional assays, to identify mRNA:miRNA interactions that modulate the efficiency of HIV-1 replication. Finally, we will determine whether drug-specifically cocaine-abuse modifies the miRNA expression profile in uninfected and HIV-1-infected CD4+ T cells and macrophages and we will examine whether any drug-induced changes in the miRNA profile can account for the reported enhancement in HIV-1 replication in cells isolated from drug-abusing patients. Together, this analysis will lead to a comprehensive understanding of how HIV-1 infection modifies the miRNA expression profile and will provide a mechanistic understanding of how these changes regulate the efficiency of HIV-1 replication. PUBLIC HEALTH RELEVANCE: Relevance MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate a wide range of cellular processes. We will determine how HIV-1 infection changes the pattern of miRNA expression and whether HIV-1 makes its own miRNAs. We will then determine whether these changes enhance viral replication and define the mechanisms underlying this enhancement in both normal and drug-abusing patients. This research has the potential to not only shed new light on HIV-1 pathogenesis but also suggest new approaches to inhibit virus replication.