Manipulation of kinase signaling by the human oncovirus KSHV

项目总结 卡波西肉瘤相关疱疹病毒(KSHV)是原发性渗出性淋巴瘤的病原体，是一种 多中心性Castleman病和Kaposi肉瘤的子集，Kaposi肉瘤是HIV/AIDS中最常见的恶性肿瘤 病人。KSHV ORF45是一种即刻早期基因和被膜蛋白，它激活细胞激酶RSK， 这对于裂解基因的最佳表达和后代病毒粒子的产生是必不可少的。准确的角色(S) ORF45诱导的RSK激活有助于KSHV的有效裂解复制仍有待确定。这个 长期目标是阐明KSHV促进病毒基因高效表达的机制(S)。 通过侵占宿主细胞机制进行高效的病毒复制，以及这如何促进KSHV 发病机制。近期的目标是确定ORF45激活的RSK在KSHV裂解中的作用 复制，包括ORF45/RSK底物对病毒表观基因组修饰、转录的贡献 还有翻译。中心假设是KSHV ORF45激活的RSK改变了几个 KSHV裂解复制过程中的不同底物，导致表观基因组修饰的调节， 转录和翻译。这一假说是在回顾主要文献和 申请人提供的初步资料。这项拟议的研究的基本原理是它将 允许假设驱动的方法来揭示对表面上的调控的机械解释 KSHV的表观基因组修饰和病毒/细胞基因表达。核心假设将通过以下方式进行检验 追求以下具体目标：1)确定KSHV ORF45激活的RSK在调节 组蛋白修饰及其对病毒和细胞转录的影响；以及2)表征这些影响 ORF45诱导的RSK激活对病毒和细胞翻译的影响。为了实现这些目标，一个高效的系统 并将采用可诱导的KSHV裂解再激活。对于第一个目标，染色质免疫沉淀和 下一代测序将用于准确测量KSHV裂解复制和 ORF45激活RSK对转录调控的影响。第二个目标将利用各种方法， 包括一种创新的核糖体图谱技术，以阐明ORF45- 激活了关于翻译控制的RSK。拟议研究的预期贡献是 证实了ORF45激活的RSK在KSHV裂解复制过程中的关键调控过程中的作用。 这些贡献将是重要的，因为它们将加深对KSHV诱导的 宿主细胞信号通路的失调，这可能有助于开发新的治疗方法 用于KSHV相关疾病。

PROJECT SUMMARY Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma, a subset of multicentric Castleman's disease, and Kaposi's sarcoma, the most common malignancy in HIV/AIDS patients. KSHV ORF45, an immediate-early gene and tegument protein, activates the cellular kinase RSK, which is essential for optimal lytic gene expression and progeny virion production. The precise role(s) of ORF45-induced RSK activation that contribute to efficient KSHV lytic replication remain to be determined. The long-term goal is to elucidate the mechanism(s) by which KSHV facilitates efficient viral gene expression and productive viral replication by appropriating the host cellular machinery, and how this contributes to KSHV pathogenesis. The immediate objective is to define the roles of ORF45-activated RSK throughout KSHV lytic replication, including contributions of ORF45/RSK substrates to viral epigenomic modifications, transcription and translation. The central hypothesis is that KSHV ORF45-activated RSK alters the activities of several diverse substrates during KSHV lytic replication, resulting in the regulation of epigenomic modifications, transcription and translation. This hypothesis was formulated upon reviewing the primary literature and preliminary data produced by the applicant. The rationale that underlies the proposed research is that it will allow for hypothesis-driven approaches to reveal a mechanistic explanation for the apparent regulation of epigenomic modifications and viral/cellular gene expression by KSHV. The central hypothesis will be tested by pursuing the following specific aims: 1) Determine the roles of KSHV ORF45-activated RSK in the regulation of histone modifications and the consequences on viral and cellular transcription; and 2) Characterize the effects of ORF45-induced RSK activation on viral and cellular translation. To achieve these aims, a system for efficient and inducible KSHV lytic reactivation will be employed. For the first aim, chromatin immunoprecipitation and next-generation sequencing will be used to accurately measure the effect(s) of both KSHV lytic replication and ORF45 activation of RSK on transcriptional regulation. The second aim will make use of various approaches, including an innovative ribosome profiling technique, to elucidate the functional significance of ORF45- activated RSK with regard to translational control. The expected contributions of the proposed research are to corroborate roles for ORF45-activated RSK in critical regulatory processes during KSHV lytic replication. These contributions will be significant because they will further the understanding of KSHV-induced dysregulation of the host cell signaling pathways, which may be useful in the development of novel treatments for KSHV-related diseases.