K-Rta as a Novel SUMO and Epigenetic Regulator

描述（由申请人提供）：基因表达的表观遗传调控在真核生物的许多基本生物过程中起着关键作用。我们一直专注于定义表观遗传机制，涉及染色质蛋白的翻译后修饰，在卡波西肉瘤疱疹病毒（KSHV）的生命周期中，这是卡波西肉瘤（KS），艾滋病相关的恶性肿瘤的病原体。该病毒在感染细胞中表现出潜伏期，其中仅表达少数病毒基因。细胞信号通路可以通过一个时间级联反应重新激活潜伏细胞产生病毒，该级联反应调节近100个病毒基因的表达。因此，KSHV是一个非常有吸引力的模型来研究影响染色质结构和动力学的表观遗传机制，这是真核细胞中调控转录的关键过程。 小泛素样修饰物（SUMO）是一种通过共价连接（即SUMO化）到多种靶蛋白上来调节多种细胞过程（包括异染色质形成）的蛋白质。SUMO化，像磷酸化一样，作为翻译后信号分子将信号传递到含有SUMO相互作用基序（SIM）的下游靶标;这些靶标包括影响染色质结构的蛋白质，这些蛋白质通常使转录沉默。我们最近的研究表明，主要的病毒转录反式激活因子，K-Rta，是一个SUMO靶向泛素（Ub）连接酶，降解SUMO修饰的蛋白质。我们假设这种SUMO靶向的Ub连接酶功能对于K-Rta通过破坏潜伏感染细胞中病毒附加体周围的抑制环境来启动KSHV复制的裂解（生产）阶段是重要的。这种抑制性环境由病毒潜伏相关核抗原（拉娜）建立，LANA是一种SUMO结合蛋白，其将病毒附加体束缚到宿主细胞染色体的异染色质区域，从而维持病毒的潜伏状态。该提案将测试这一假设，即SUMO修饰的调节在KSHV生命周期中起着关键作用，通过调节病毒染色质的结构来建立潜伏期和重新激活该病毒。在第一个目标中，我们将研究K-Rta作为SUMO依赖性泛素连接酶在KSHV复制中的作用。在第二个目标中，将分析K-Rta以SUMO依赖性方式起作用并影响病毒染色质的表观遗传特征的潜在机制。总之，这些研究将确定K-Rta以及拉娜在KSHV复制中的新作用，并将提供对控制潜伏期和再活化的翻译后修饰和染色质重塑的见解。 公共卫生相关性：这项关于KSHV的R 01资助申请的研究将对基因表达调控和表观遗传控制产生新的理解。我们的研究将提供有关病毒再激活机制的基础知识，并可能产生治疗感染性疾病的创新方法，特别是由这种致瘤病毒引起的癌症。

DESCRIPTION (provided by applicant): Epigenetic regulation of gene expression plays a critical role in many fundamental biological processes in eukaryotes. We have focused on defining epigenetic mechanisms, involving post-translation modification of chromatin proteins, in the life cycle of Kaposi's sarcoma herpesvirus (KSHV), which is an etiological agent of Kaposi's sarcoma (KS), an AIDS-associated malignancy. This virus exhibits a latent phase in infected cells in which only a few viral genes are expressed. Cell signaling pathways can reactivate latent cells to produce virus through a temporal cascade that regulates expression of nearly 100 viral genes. Thus, KSHV is a very attractive model to study epigenetic mechanisms that affect chromatin structure and dynamics, which are key processes regulating transcription in eukaryotic cells. The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes, including heterochromatin formation, by covalent attachment (i.e. sumoylation) to a diverse array of target proteins. Sumoylation, like phosphorylation, serves as a post-translational signal molecule to transmit signals to down-stream targets containing a SUMO-interacting motif (SIM); these targets include proteins that impact chromatin structure that generally silence transcription. Our recent studies show that the main viral transcriptional transactivator, K-Rta, is a SUMO-targeting ubiquitin (Ub) ligase, which degrades SUMO- modified proteins. We hypothesize that this SUMO-targeting Ub ligase function is important for K-Rta to initiate the lytic (productive) phase of KSHV replication by disrupting the repressive environment surrounding of the viral episome in latently infected cells. This repressive environment is established by the viral latency- associated nuclear antigen (LANA), which is a SUMO-binding protein that tethers viral episomes to heterochromatic regions of host cell chromosomes and thereby maintains the latent state of the virus. This proposal will test the hypothesis that the regulation of SUMO modification plays a key role in the KSHV life cycle in both establishment of latency and reactivation of this virus by modulating the structure of viral chromatin. In the first Aim, we will investigate the role of K-Rta as a SUMO-dependent ubiquitin ligase in KSHV replication. In the second Aim, the potential mechanisms whereby K-Rta acts in a SUMO-dependent manner and affects the epigenetic signature of viral chromatin will be analyzed. Taken together, these studies will define a new role of K-Rta, as well as LANA, in KSHV replication, and will provide insights into the post- translational modifications and chromatin remodeling that govern latency and reactivation. PUBLIC HEALTH RELEVANCE: The research in this R01 grant application on KSHV will produce a new understanding of regulation of gene expression and epigenetic control. Our research will provide fundamental knowledge regarding the mechanisms of viral reactivation and could produce innovative approaches for the treatment of infectious disease, especially cancer caused by this tumorigenic virus.