Studying the role of KSHV-encoded miRNAs

描述（由申请人提供）：卡波西肉瘤相关疱疹病毒（KSHV）编码12个在KS病变和KSHV相关淋巴增生性恶性肿瘤中高度表达的pre- mirna。异位表达和miRNA抑制方法，结合基因表达谱，已经确定了参与关键生物过程调控的靶mrna，如血管生成、细胞凋亡、内皮细胞分化和免疫监视。此外，一些mirna可能通过靶向病毒mrna来调节潜伏期。利用HITS-CLIP技术富集risc相关rna，对PEL细胞中KSHV miRNAs靶向的mRNA进行了初步分析，发现了数百个新的mRNA靶点，并证实了先前发现的靶点。此外，我们的分析表明，PEL细胞中高比例的RISC复合物含有KSHV编码的mirna。基于这些数据，我们假设KSHV mirna引起显著的组织特异性基因表达变化，有助于发病和肿瘤发生。为了直接解决这一假设，我们提出：1)利用HITS-CLIP在KSHV感染的PEL和内皮细胞中全面鉴定KSHV miRNA靶点；2)利用缺乏单个或多个mirna的KSHV突变体，表征其体外表型及其与病毒复制、潜伏期的建立和维持、细胞周期控制和细胞凋亡抗性相关的调控途径。总之，将miRNA靶点鉴定的核糖组学方法与病毒遗传学相结合，以及在培养细胞中确定病毒表型的能力，将提供对kshv编码的miRNA功能的详细分析，并可能导致抗病毒/抗肿瘤策略的发展。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs that are highly expressed in KS lesions and KSHV- associated lymphoproliferative malignancies. Ectopic expression and miRNA inhibition approaches, combined with gene expression profiling, have identified target mRNAs that are involved in the regulation of critical biological processes such as angiogenesis, apoptosis, endothelial cell differentiation, and immune surveillance. In addition, several miRNAs may regulate latency by targeting viral mRNAs. Preliminary analyses of the mRNAs targeted by KSHV miRNAs in PEL cells using HITS-CLIP technology to enrich RISC-associated RNAs have identified hundreds of new mRNA targets and confirmed previously identified targets. Moreover, our analysis showed that a high percentage of RISC complexes within PEL cells contain KSHV- encoded miRNAs. Based on these data, we hypothesize that the KSHV miRNAs cause significant and tissue-specific changes in gene expression that contribute to pathogenesis and tumorigenesis. To directly address this hypothesis we propose to 1) Comprehensively identify KSHV miRNA targets by HITS-CLIP in KSHV-infected PEL and endothelial cells; 2) Characterize in vitro phenotypes and their associated regulatory pathways with respect to viral replication, the establishment and maintenance of latency, cell cycle control and resistance to apoptosis by using KSHV mutants lacking single or multiple miRNAs. In summary, combining ribonomics approaches for miRNA target identification with viral genetics, and the ability to determine viral phenotypes in cultured cells, will provide a detailed analysis of KSHV-encoded miRNA functions and may lead to the development of antiviral/antitumor strategies.