Role of MHV68 miRNAs in latencyand pathogenesis

 描述(申请人提供)：转化的人类伽马疱疹病毒EBV和KSHV在B细胞中建立稳定的潜伏感染，提供终生的病毒储存库，可能有助于恶性疾病的发展。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。由于在自然宿主中工作的困难，在人体内对伽马疱疹病毒的体内研究一直受到严重限制。小鼠伽马疱疹病毒68(MHV68)与EBV和KSHV相关，可引起小鼠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一种易于操作的小动物模型。与EBV和KSHV一样，MHV68表达一组定义的miRNAs，其在感染过程中的功能在很大程度上是未知的。由于这些病毒编码的miRNAs在体内长期潜伏期在B细胞中大量表达，在淋巴增生性疾病期间在增生性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发挥关键作用。为了验证这一假说，我们将1)确定单个MHV68 miRNAs在长期潜伏和淋巴增殖性疾病中的功能作用；2)使用尖端片段和深度测序方法结合体外和体内验证来确定关键miRNAs的特定病毒和宿主靶点；3)确定靶基因在体内的生物学相关性，并确定它们在B细胞成熟和存活中的作用。我们预计，在MHV68、KSHV和EBV miRNAs调控的mRNA靶标和细胞通路之间将有显著的重叠。对MHV68 miRNA突变体的体内系统分析，结合HITS-CLIP技术和对miRNA靶标的体内验证，为确定miRNAs在体内导致伽马疱疹病毒感染的分子机制提供了极其有力的手段，并应使我们能够确定这些共同靶标的抑制对伽马疱疹病毒潜伏期和致病机制的贡献。

 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.