MicroRNA function in murine cytomegalovirus

该项目的总体目标是确定动物病毒利用和控制microRNA （miRNA）表达以建立感染并在其宿主中持续存在的机制。mirna是一类小RNA分子（约22个核苷酸长），在过去十年中被发现，通过靶向特定的信使RNA转录物来降解或抑制翻译来调节基因表达。据估计，人类中30%的基因可以被mirna调节，这意味着这些分子在调节转录组中的基本作用。病毒编码的mirna最近在许多不同的病毒中被发现，包括所有三个疱疹病毒亚科。最近的报道表明，病毒mirna可以靶向参与免疫反应的宿主基因。因此，这类分子代表了一类重要的潜在药物靶点。到目前为止，还没有在体内检测到病毒miRNA。了解mirna在完整生理系统中的功能作用至关重要；一项体内分析认为，病毒和细胞基因表达均可受到细胞环境以及抗病毒免疫反应程度的影响。我们将检测病毒miRNA在小鼠巨细胞病毒（CMV）中的功能。巨细胞病毒是乙型疱疹病毒家族的一员，是一种普遍存在的病毒，是临床发病的主要原因。巨细胞病毒具有高度的物种特异性；然而，小鼠MCMV的发病机制与人类HCMV的发病机制非常相似。因此，小鼠CMV是研究人类CMV的一个既定模型，并且可以使用细菌人工染色体（BAC）技术修饰MCMV基因组以产生miRNA缺失突变体。我们将使用MCMV miRNA缺失突变体和反义技术来检测病毒miRNA在体内的功能。微阵列技术将用于检测MCMV mirna靶向的宿主基因。这项工作与未来miRNA抑制剂作为抗病毒治疗药物的分析高度相关。最后，我们将建立一个体外检测病毒miRNA生物发生的系统。这需要深入了解病毒如何控制和利用这类分子，并应补充体内分析。这项工作产生的工具和知识将广泛适用于其他病毒系统。mirna也被证明在癌症形成、心血管和代谢疾病中发挥作用；因此，扩大对miRNA功能和机制的了解对人类健康至关重要。

The broad goal of this project is to determine the mechanism by which an animal virus utilizes and controls microRNA (miRNA) expression in order to establish an infection and persist in its host. MiRNAs are a class of small RNA molecules (~ 22 nucleotides long), discovered in the last decade, that regulate gene expression by targeting specific messenger RNA transcripts for degradation or inhibited translation. It is estimated that 30% of genes in humans could be regulated by miRNAs, implicating a fundamental role of these molecules in modulating the transcriptome. Viral-encoded miRNAs have been discovered recently in a number of different viruses, including all three herpesvirus subfamilies. Recent reports have demonstrated that viral miRNAs can target host genes involved in the immune response. This class of molecules, therefore, represents an important new class of potential drug targets. To date, no viral miRNA has been examined in vivo. Understanding the functional role of miRNAs in an intact physiological system is of vital importance; an in vivo analysis considers that both viral and cellular gene expression can be influenced by cell-context as well as the extent of the anti-viral immune response. We will examine viral miRNA function in murine cytomegalovirus (CMV). Cytomegalovirus, a member of the betaherpesvirus family, is a ubiquitous virus that is a major cause of morbidity in the clinical setting. CMV is highly species-specific; however, the pathogenesis of MCMV in mice is remarkably similar to that of HCMV in humans. Murine CMV is therefore an established model for studying human CMV and the MCMV genome can be modified to generate miRNA deletion mutants using bacterial artificial chromosome (BAC) technology. We will use both MCMV miRNA deletion mutants and antisense technology to examine viral miRNA function in vivo. Microarray technology will be used to examine the host genes that are targeted by MCMV miRNAs. This work is highly relevant to future analysis of miRNA inhibitors as anti-viral therapeutics. Finally we will establish a system for examining viral miRNA biogenesis in vitro. This is required to gain an in-depth understanding of how the virus controls and exploits this class of molecules and should complement the in vivo analysis. The tools and knowledge resulting from this work will be widely applicable in other viral systems. MiRNAs have also been shown to play a role in cancer formation and cardiovascular and metabolic diseases; expanding the understanding of miRNA function and mechanism, therefore, is of paramount importance to human health.