HIV-1: microRNA interactions

HIV-1：微小RNA相互作用

• 批准号: 8233429
• 负责人: BRYAN R. CULLEN
• 金额: $ 38.07万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233429
• 关键词: Affect; Affinity; Applications Grants; Binding; Binding Sites; Biological Assay; CD4 Positive T Lymphocytes; Cell physiology; Cells; Cocaine; Cocaine Abuse; Complex; Development; Disease Progression; Drug abuse; Gene Expression; Genome; Grant; HIV-1; Health; Herpesviridae; Human; Immunoprecipitation; Individual; Infection; Lead; Light; Malignant Neoplasms; Messenger RNA; MicroRNAs; Molecular Profiling; Monoclonal Antibodies; Mutate; Pathogenesis; Patients; Pattern; Pharmaceutical Preparations; Play; Polyomavirus; Porifera; Positioning Attribute; Process; Production; RNA; RNA Interference; RNA-Induced Silencing Complex; Recovery; Report (account); Research; Ribonucleosides; Site; Small Interfering RNA; T-Lymphocyte; Technology; Testing; Translations; Viral; Virion; Virus; Virus Diseases; Virus Replication; Western Blotting; abstracting; cell growth regulation; crosslink; in vivo; interest; knock-down; mRNA Expression; macrophage; non-drug; novel strategies; particle; programs

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.

描述（由申请人提供）：抽象microRNA（miRNA）是最近发现的小型，〜22-NT调节性RNA类，现在已知在细胞分化和发育的调节中起关键作用。 miRNA表达的正调可能导致疾病进展，尤其是在癌症的情况下，最近的证据也暗示了病毒复制和发病机理方面的miRNA。现在已知几种病毒，包括许多疱疹病毒和多瘤病毒物种，可以表达一系列病毒编码的miRNA，并且病毒感染也以促进病毒复制的方式扰动细胞miRNA表达。尽管对HIV-1如何与细胞miRNA机械相互作用相对较少了解，但很明显，HIV-1感染可以改变细胞miRNA表达的模式，并提出了单个细胞miRNA，以促进或抑制HIV-1复制。尽管这是有争议的，但HIV-1也可能编码一个或多个miRNA。在此赠款应用中，我们建议使用微阵列和深层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，我们将使用交联的免疫沉淀（夹）技术来识别所有RNA诱导的沉默复合物（RISC）结合位点在HIV-1感染的细胞中表达的所有mRNA上，使用Argonaute特异性单克隆抗体，以恢复交叉链接的mRNA：RISC COMPLECTES，我们将通过深层测序进行分析，然后通过深层分析。然后，在HIV-1感染细胞中表达的miRNA靶向的细胞或HIV-1 mRNA将进行突变分析，并结合功能分析，以识别mRNA：调节HIV-1复制效率的miRNA相互作用。最后，我们将确定在未感染和HIV-1感染的CD4+ T细胞和巨噬细胞中修改了药物特异性可卡因的滥用，是否会修改MiRNA的表达谱，我们将检查MiRNA概况中的任何药物诱导的变化是否可以解释与药物抗药性患者分离的细胞中报告的HIV-1重复性增强。总之，这一分析将使人们对HIV-1感染如何修饰miRNA表达曲线有一个全面的理解，并将提供对这些变化如何调节HIV-1复制效率的机械理解。公共卫生相关性：相关性microRNA（miRNA）是一类小型调节性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表达的模式以及HIV-1是否制造了自己的miRNA。然后，我们将确定这些变化是否增强了病毒复制，并定义了正常和滥用药物的患者中这种增强的基础机制。这项研究不仅有可能对HIV-1发病机理开发新的启示，而且还提出了抑制病毒复制的新方法。