Studying the role of KSHV-encoded microRNAs

描述（由申请人提供）：MicroRNAs是一种小的非编码调节RNA分子，它与mrna的3' utr结合，以阻止mrna的翻译或诱导其降解。以前在从植物到哺乳动物的各种生物中发现的mirna，现在也知道是由DNA病毒产生的。人类？爱泼斯坦-巴尔疱疹病毒已被证明可以编码潜在地调节病毒和细胞基因的mirna。为了确定卡波西肉瘤相关疱疹病毒（KSHV）是否编码miRNAs，我们从KSHV阳性的原发性积液淋巴瘤来源细胞中克隆了小rna。序列分析显示，11个长度为19 ~ 23个碱基的分离rna与KSHV完全一致。令人惊讶的是，所有候选mirna都映射到KSHV潜伏期相关区域内的单个基因组位置（Samols等人，Journal of Virology 2005）。在回顾我们的工作时，另外两个实验室报告了kshv编码mirna的鉴定。因此，这些数据表明，病毒编码的mirna代表了一种新的机制，通过这种机制，KSHV可以在潜伏和裂解KSHV复制过程中调节病毒和/或细胞基因表达。

DESCRIPTION (provided by applicant): MicroRNAs are small non-coding regulatory RNA molecules that bind to 3'UTRs of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by DNA viruses. The human ?-herpesvirus Epstein-Barr Virus has been shown to encode miRNAs which potentially regulate both viral and cellular genes. To determine whether Kaposi's Sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV positive primary effusion lymphoma derived cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align to KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-associated region of KSHV (Samols et al., Journal of Virology 2005). While our work was in review, two other laboratories reported the identification of KSHV-encoded miRNAs. Hence, these data suggest that virally-encoded miRNAs represent a novel mechanism by which KSHV may regulate viral and/or cellular gene expression during both latent and lytic KSHV replication. In human cells, over 450 miRNAs have been identified. Targets and functions of only a few miRNAs have been experimentally determined thus far, yet some miRNAs such as human hsa-miR-14 and hsa-miR-181 regulate fundamental biological processes like apoptosis, cell proliferation, and hematopoiesis and very recently have been implicated in tumorigenesis. Based on our preliminary results we hypothesize that miRNAs play an important role in the KSHV life cycle and may contribute to KSHV pathogenesis. To directly address this hypothesis we propose the following specific aims: SA1: Analysis of KSHV miRNA expression during latent and lytic replication in cultured cells and tissues samples of different origins. SA2: Identification of cellular and viral targets regulated by KSHV-encoded miRNAs. SA3: Evaluate the role of miRNA expression in the context of the viral genome. In support of our hypothesis we provide new preliminary data on identifying cellular miRNA targets in stably miRNA-expressing 293 cells (Samols et al., PLoS Pathogens, 2007, May 11). In summary, the identification of virally encoded miRNAs within the latency-associated region of KSHV is novel and very exciting as it suggests an entirely new level of regulating gene expression by which KSHV can potentially reprogram the host cell environment.