HIV-1: microRNA interactions

描述(申请人提供)：摘要microRNAs(MiRNAs)是最近发现的一类小的、~22-nT的调节性RNA，现在已知在细胞分化和发育的调节中发挥关键作用。MiRNA表达的错误调节可能导致疾病的进展，特别是在癌症的情况下，最近的证据也涉及到miRNAs在病毒复制和发病机制方面的作用。包括许多疱疹病毒和多瘤病毒在内的几种病毒现在已知表达一系列病毒编码的miRNAs，而病毒感染也已知以促进病毒复制的方式干扰细胞miRNA的表达。尽管对HIV-1与细胞miRNA机制的相互作用知之甚少，但显然HIV-1感染可以改变细胞miRNA的表达模式，单个细胞miRNAs被认为可以促进或抑制HIV-1的复制。HIV-1也有可能编码一个或多个miRNAs，尽管这一点一直存在争议。在这项拨款申请中，我们建议利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ArgAerte特异的单抗恢复交联的RNA：RISC复合体，鉴定HIV-1感染细胞中表达的所有mRNAs上的所有RNA诱导沉默复合体(RISC)结合位点，然后我们将对其进行深度测序分析。然后，将对HIV-1感染细胞中表达的miRNAs所针对的细胞或HIV-1mRNAs进行突变分析，并结合功能分析，以确定mRNA：调节HIV-1复制效率的miRNA相互作用。最后，我们将确定药物特异性可卡因滥用是否改变了未感染和HIV-1感染的CD4+T细胞和巨噬细胞的miRNA表达谱，并将检查药物诱导的miRNA谱的任何变化是否可以解释从药物滥用患者分离的细胞中HIV-1复制的增强。总之，这一分析将导致对HIV-1感染如何修改miRNA表达谱的全面理解，并将提供对这些变化如何调节HIV-1复制效率的机械性理解。

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.