Role of MHV68 miRNAs in latencyand pathogenesis

 描述（由申请方提供）：转化型人γ疱疹病毒EBV和KSHV在B细胞中建立稳定的潜伏感染，提供可导致恶性疾病发展的病毒终身储存库。因此，确定控制B细胞长期潜伏期的机制对于设计合理的预防疾病的策略至关重要。人体γ疱疹病毒的体内研究由于在自然宿主中工作的困难而受到严重限制。鼠γ疱疹病毒68（MHV 68）与EBV和KSHV相关，并在小鼠中引起淋巴瘤和淋巴增生性疾病，为体内病毒/宿主关系的机制研究提供了易于操作的小动物模型。与EBV和KSHV一样，MHV 68表达一组确定的miRNA，其在感染期间的功能在很大程度上是未知的。由于这些病毒编码的miRNAs在体内长期潜伏期的B细胞和淋巴组织增生性疾病的增生性B细胞病变中大量表达，我们推测这些病毒miRNAs在潜伏期和发病机制中起关键作用。为了验证这一假设，我们将1）确定单个MHV 68 miRNA在长期潜伏期和淋巴组织增生性疾病中的功能作用; 2）使用尖端CLIP和深度测序方法结合体外和体内验证鉴定关键miRNA的特异性病毒和宿主靶标; 3）定义靶基因的体内生物学相关性并确定其在B细胞成熟和存活中的作用。我们预计，在mRNA靶点和由MHV 68、KSHV和EBV miRNA调控的细胞通路之间将存在显著重叠。MHV 68 miRNA突变体的系统体内分析，结合HITS-CLIP技术和miRNA靶点的体内验证，提供了一种非常强大的手段来确定miRNA在体内促进γ疱疹病毒感染的分子机制，并应使我们能够确定这些共享靶点的抑制对γ疱疹病毒潜伏期和发病机制的贡献。

 DESCRIPTION (provided by applicant): The transforming human gammaherpesviruses EBV and KSHV establish stable latent infections in B cells, providing a lifelong reservoir of virus tha can contribute to the development of malignant disease. Thus, defining the mechanisms that govern long-term latency in B cells is critical for designing rational strategies to prevent diseas. In vivo studies of gammaherpesviruses in humans have been severely limited by the difficulties of working in the natural host. Murine gammaherpesvirus 68 (MHV68) is related to EBV and KSHV and causes lymphomas and lymphoproliferative disease in mice, providing a readily manipulable small animal model for mechanistic studies of the virus/host relationship in vivo. Like EBV and KSHV, MHV68 expresses a defined set of miRNAs whose functions during infection are largely unknown. As these virus-encoded miRNAs are abundantly expressed in vivo in B cells during long-term latency and in hyperplastic B cell lesions during lymphoproliferative disease, we hypothesize that these viral miRNAs play key roles in latency and pathogenesis. To test this hypothesis, we will 1) Determine the functional role of individual MHV68 miRNAs in long-term latency and lymphoproliferative disease; 2) Identify specific virus and host targets of the key miRNAs using cutting-edge CLIP and deep sequencing approaches coupled with in vitro and in vivo validation; 3) Define the in vivo biological relevance of target genes and determine their roles in B cell maturation and survival. We anticipate that there will be significant overlap between the mRNA targets and cellular pathways regulated by MHV68, KSHV, and EBV miRNAs. The systematic in vivo analyses of MHV68 miRNA mutants, in conjunction with HITS-CLIP technology and in vivo validation of miRNA targets, provides an extremely powerful means to determine the molecular mechanism by which miRNAs contribute to gammaherpesvirus infection in vivo, and should allow us to define the contribution of repression of these shared targets to gammaherpesvirus latency and pathogenesis.