Studying the role of KSHV-encoded microRNAs

描述（由申请人提供）：MicroRNA是小的非编码调节RNA分子，其与mRNA的3 'UTR结合以阻止其翻译或诱导其降解。先前在从植物到哺乳动物的各种生物体中发现的miRNA，现在也已知由DNA病毒产生。人类？疱疹病毒Epstein-Barr病毒已显示编码可能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miRNA，我们从KSHV阳性的原发性渗出性淋巴瘤衍生细胞中克隆了小RNA。序列分析显示，11个分离的RNA的长度为19至23个碱基，完美地对齐KSHV。令人惊讶的是，所有候选miRNA都定位于KSHV潜伏相关区域内的单个基因组位置（Samols et al.，Journal of Virology 2005）。当我们的工作在审查中时，另外两个实验室报告了KSHV编码的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，通过该机制，KSHV可以在潜伏性和裂解性KSHV复制期间调节病毒和/或细胞基因表达。 在人类细胞中，已经鉴定出超过450种miRNAs。到目前为止，只有少数miRNA的靶点和功能已经通过实验确定，但一些miRNA如人hsa-miR-14和hsa-miR-181调节基本的生物学过程，如细胞凋亡、细胞增殖和造血，并且最近已经涉及肿瘤发生。 基于我们的初步研究结果，我们推测miRNAs在KSHV的生命周期中起着重要作用，并可能有助于KSHV的发病机制。为了直接解决这一假设，我们提出了以下具体目标： SA 1：不同来源的培养细胞和组织样本中潜伏和裂解复制期间KSHV miRNA表达的分析。 SA 2：由KSHV编码的miRNA调控的细胞和病毒靶标的鉴定。 SA 3：评估miRNA表达在病毒基因组中的作用。 为了支持我们的假设，我们提供了关于在稳定表达miRNA的293细胞中鉴定细胞miRNA靶标的新的初步数据（Samols et al.，PLoS Pathogens，2007年5月11日）。总之，在KSHV的潜伏相关区域内鉴定病毒编码的miRNA是新颖的并且非常令人兴奋的，因为它表明KSHV可以潜在地重编程宿主细胞环境的调节基因表达的全新水平。

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.