Studying the role of KSHV-encoded microRNAs

描述(申请人提供)：microRNAs是一种小的非编码调节性RNA分子，它与mRNAs的3‘UTRs结合，以阻止它们的翻译或诱导它们的降解。以前在从植物到哺乳动物的各种生物中都发现了miRNAs，现在也知道它是由DNA病毒产生的。人类疱疹病毒Epstein-Barr病毒已被证明编码miRNAs，它可能同时调节病毒和细胞基因。为了确定Kaposi肉瘤相关疱疹病毒(KSHV)是否编码miRNAs，我们从KSHV阳性的原发渗出性淋巴瘤来源的细胞中克隆了小RNA。序列分析显示，11个分离的RNA长度为19至23个碱基，与KSHV完美比对。令人惊讶的是，所有候选miRNAs都映射到KSHV潜伏期相关区域内的单个基因组位置(Samols等人，《病毒学杂志》2005年)。在我们的工作进行审查的同时，另外两个实验室报告了KSHV编码的miRNAs的鉴定。因此，这些数据表明，病毒编码的miRNAs代表了一种新的机制，通过这种机制，KSHV可以在潜伏和裂解复制过程中调节病毒和/或细胞基因的表达。 在人类细胞中，已经鉴定出超过450个miRNAs。到目前为止，只有几个miRNAs的靶点和功能被实验确定，但一些miRNAs，如人的hsa-miR-14和hsa-miR-181，调节着基本的生物学过程，如细胞凋亡、细胞增殖和造血，最近被认为与肿瘤的发生有关。 根据我们的初步结果，我们推测miRNAs在KSHV的生活周期中发挥重要作用，并可能参与KSHV的致病过程。为了直接解决这一假设，我们提出了以下具体目标： SA1：分析KSHV miRNA在不同来源的培养细胞和组织样本中潜伏和裂解复制过程中的表达。 SA2：识别受KSHV编码的miRNAs调控的细胞和病毒靶标。 SA3：评估miRNA表达在病毒基因组环境中的作用。 为了支持我们的假设，我们提供了在稳定表达miRNA的293细胞中识别细胞miRNA靶标的新的初步数据(Samols等人，PLoS Pathogens，2007年5月11日)。总之，在KSHV潜伏期相关区域内识别病毒编码的miRNAs是新颖和非常令人兴奋的，因为它暗示了一个全新的基因表达调控水平，KSHV可以通过它潜在地重新编程宿主细胞环境。

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.