HIV-1: microRNA interactions

描述（由申请人提供）：摘要MicroRNAs （miRNAs）是最近发现的一类小的，~22-nt的调控rna，目前已知在细胞分化和发育的调控中起关键作用。miRNA表达的错误调控可能导致疾病进展，特别是在癌症的情况下，最近的证据也表明miRNA在病毒复制和发病机制方面具有重要作用。一些病毒，包括许多疱疹病毒和多瘤病毒，现在已知表达一系列病毒编码的miRNA，并且病毒感染也已知以可能促进病毒复制的方式扰乱细胞miRNA的表达。尽管人们对HIV-1如何与细胞miRNA机制相互作用知之甚少，但很明显，HIV-1感染可以改变细胞miRNA表达模式，并且已经提出单个细胞miRNA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。在本次拨款申请中，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CD4+ 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）技术鉴定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，使用argonaute特异性单克隆抗体恢复交联mRNA:RISC复合物，然后我们将通过深度测序对其进行分析。被HIV-1感染细胞中表达的miRNA靶向的细胞或HIV-1 mRNA随后将进行突变分析，并结合功能测定，以确定调节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CD4+ T细胞和巨噬细胞中的miRNA表达谱，我们将检查药物诱导的miRNA表达谱的任何变化是否可以解释从吸毒患者分离的细胞中报道的HIV-1复制增强。总之，这一分析将导致对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MicroRNAs （miRNAs）是一类小的调控rna，被认为可以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是否制造自己的miRNA。然后，我们将确定这些变化是否会增强病毒复制，并确定正常和滥用药物患者中这种增强的机制。这项研究不仅有可能揭示HIV-1的发病机制，而且还可能提出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.