KSHV Lytic DNA Replication and its Control Mechanism

KSHV裂解性DNA复制及其控制机制

• 批准号: 8577970
• 负责人: Subhash C Verma
• 金额: $ 33.52万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8577970
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Arginine; Autoimmune Process; B-Lymphocytes; Binding; Binding Sites; Biochemical Genetics; Biological Assay; Cells; Chromatin; Cleaved cell; Complex; DNA; DNA analysis; DNA biosynthesis; DNA replication origin; Data; Endothelial Cells; Epigenetic Process; Episome; Exonuclease; Ganciclovir; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Graft Rejection; HIV; HIV Infections; Health; Herpesviridae; Herpesvirus 1; Highly Active Antiretroviral Therapy; Histone H3; Histone H4; Histones; Human; Human Herpesvirus 8; Immune; Immunocompromised Host; Immunosuppression; Immunosuppressive Agents; Incidence; Individual; Infection; Intervention; Kaposi Sarcoma; Knowledge; Label; Laboratories; Left; Length; Life; Link; Lysine; Lytic; Lytic Phase; Malignant Neoplasms; Mediating; Methylation; MicroRNAs; Modification; Molecular; Molecular Analysis; Morbidity - disease rate; Multicentric Angiofollicular Lymphoid Hyperplasia; N-methanocarbathymidine; Organ Transplantation; Outcome; Pathogenesis; Pathology; Patients; Peptide Initiation Factors; Pharmaceutical Preparations; Play; Point Mutation; Procedures; Production; Proteins; Recruitment Activity; Regulation; Replication Initiation; Replication Origin; Research; Risk; Role; Simplexvirus; Site; Stimulus; Tail; Testing; Therapeutic; Therapeutic immunosuppression; Transcript; Transferase; Transplant Recipients; Viral; Viral Genome; Virion; Virus; Work; base; cell type; chromatin modification; histone modification; innovation; insight; latent infection; lytic replication; monocyte; mortality; pre-miRNA; prevent; primary effusion lymphoma; promoter; protein function; public health relevance; resistance factors; tumor; tumor growth; tumor progression; tumorigenic; viral DNA

DESCRIPTION (provided by applicant): There is a fundamental gap in the understanding of molecular mechanism involved in regulating lytic DNA replication of Kaposi's sarcoma associated herpesvirus (KSHV) also known as Human Herpesvirus 8 (HHV8). Lytic DNA replication is important in inducing KSHV mediated tumoriegenesis, evidenced by the fact that AIDS patients receiving anti-herpetic drugs, Ganciclovir (GCV), Fascarnet (PFA) and N- methanocarbathymidine, which blocks lytic DNA replication, had reduced risk for Kaposi's sarcoma (KS). KSHV is tightly linked to various human malignancies including Primary Effusion Lymphomas (PELs), Kaposi's sarcoma (KS) and Multicentric Castleman's Disease (MCDs) and cause tumors predominantly in immune compromised individuals including HIV infected individuals and patients receiving immune suppressive therapies to prevent graft rejection. KSHV induced tumors are one of the major causes of morbidity and mortality of HIV/AIDS patients. This study is important because the incidence of immunosuppression is still a concern due to HIV infection and the use of immunosuppressive drugs in organ transplant/autoimmune patients. Introduction of HAART (highly active anti-retroviral therapy) have reduced the incidence of KS tumors but there are no specific treatment for KS tumors. The long-term goal of this project is to define the mechanism of lytic DNA replication and to identify the factors regulating DNA replication, which can be exploited for devising strategies to block KSHV pathogenesis. The objective of this application is to determine the mechanism used by a functional lytic origin and the role of viral exonuclease in genome amplification. This study will also determine the role of viral microRNA and epigenetic modifications in regulating replication initiation and virion production. Our preliminary data shows that single origin of replication (oriLyt-L) is preferentially used for initiating replication in a bidirectional manner. Our central hypothesis is that replication initiations at the lytic origins are regulated by genetic and epigenetic factors. The hypothesis has been formulated based on the preliminary data produced in our laboratory on replication initiation using biochemical and genetic approaches. The rationale for the proposed research is that determining molecular mechanism of lytic DNA replication will provide potential therapeutic avenues for thwarting the virus from infected cells o treat KSHV associated malignancies, which are a major health problems for HIV infected and organ transplant patients undergoing immunosuppressive therapies to prevent graft rejection. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identification of the functional replication origin and the mechanism of lytic DNA replication using SMARD, 2) Determining the control mechanism of oriLyt-mediated replication, and 3) Determining the dynamics of epigenetic modifications at oriLyt, which may dictate origin firing. Under the first aim, an already established approach, single molecular analysis of the replicated DNA (SMARD), will determine preferentially used replication initiation origin for viral genome replication in various cell backgrounds. Role of viral exonuclease will also be determined in generating unit length viral genome for packaging. Under the second aim, role of viral microRNA, binding to targets or transcription of its pre-miRNA through lytic origin, will be evaluated for replication initiation. Under the third aim, epigenetic modifications of chromatins a lytic origins will be investigated for their contribution in controlling origin usage. The approachis innovative, because we are using a powerful in-cell labeling of replicating DNA procedure, SMARD to determine origin usage, replication fork progression and branching in DNA after replication. We are also using innovative galK-Kan procedure to introduce point mutations in KSHV genes for determining specific protein functions to better understand their roles in the context of virus. The proposed research is significant, because it is expected to vertically advance and expand the understanding of lytic DNA replication in amplifying viral genome during viral reactivation. Ultimately, such knowledge could potentially be used for devising interventional strategies to block KSHV induced malignancies.

描述（由申请人提供）：对于参与调节卡波西肉瘤相关疱疹病毒（KSHV）也称为人类疱疹病毒8（HHV8）的裂解性DNA复制的分子机制的理解存在根本性差距。裂解性 DNA 复制对于诱导 KSHV 介导的肿瘤发生非常重要，艾滋病患者接受抗疱疹药物更昔洛韦 (GCV)、Fascarnet (PFA) 和 N-甲碳胸苷（可阻断裂解性 DNA 复制）可降低患卡波西肉瘤的风险，这一事实证明了这一点。 （KS）。 KSHV 与多种人类恶性肿瘤密切相关，包括原发性渗出性淋巴瘤 (PEL)、卡波西肉瘤 (KS) 和多中心卡斯尔曼病 (MCD)，并主要在免疫功能低下的个体中引起肿瘤，包括 HIV 感染者和接受免疫抑制治疗以防止移植排斥的患者。 KSHV 诱发的肿瘤是 HIV/AIDS 患者发病和死亡的主要原因之一。这项研究很重要，因为由于艾滋病毒感染和器官移植/自身免疫患者使用免疫抑制药物，免疫抑制的发生率仍然是一个令人担忧的问题。 HAART（高效抗逆转录病毒疗法）的引入降低了KS肿瘤的发病率，但目前尚无针对KS肿瘤的特异性治疗方法。该项目的长期目标是确定裂解性 DNA 复制的机制并确定调节 DNA 复制的因素，从而可用于制定阻断 KSHV 发病机制的策略。本应用的目的是确定功能性裂解起源所使用的机制以及病毒核酸外切酶在基因组扩增中的作用。这项研究还将确定病毒 microRNA 和表观遗传修饰在调节复制起始和病毒粒子产生中的作用。我们的初步数据表明，单复制起点（oriLyt-L）优先用于以双向方式启动复制。我们的中央 假设是裂解起点的复制起始受到遗传和表观遗传因素的调节。该假设是根据我们实验室使用生化和遗传方法产生的复制启动的初步数据制定的。拟议研究的基本原理是，确定裂解性 DNA 复制的分子机制将为阻止病毒从受感染的细胞中传播或治疗 KSHV 相关恶性肿瘤提供潜在的治疗途径，这些恶性肿瘤是 HIV 感染者和接受免疫抑制治疗的器官移植患者的主要健康问题。防止移植排斥。在强有力的初步数据的指导下，这一假设将通过追求三个具体目标进行检验：1) 使用 SMARD 识别功能性复制起点和裂解性 DNA 复制机制，2) 确定 oriLyt 介导的复制的控制机制，以及 3)确定 oriLyt 表观遗传修饰的动态，这可能决定起源激发。在第一个目标下，一种已经建立的方法，即复制DNA的单分子分析（SMARD），将确定在各种细胞背景下病毒基因组复制的优先使用的复制起始起点。病毒核酸外切酶在生成用于包装的单位长度病毒基因组中的作用也将被确定。在第二个目标下，将评估病毒 microRNA 的作用，即与靶标结合或通过裂解起点转录其前体 miRNA，以启动复制。在第三个目标下，将研究染色质和溶解起源的表观遗传修饰在控制起源使用方面的贡献。该方法具有创新性，因为我们使用强大的复制 DNA 细胞内标记程序 SMARD 来确定复制后 DNA 中的起点使用、复制叉进展和分支。我们还使用创新的 galK-Kan 程序在 KSHV 基因中引入点突变，以确定特定的蛋白质功能，以更好地了解它们在病毒中的作用。拟议的研究意义重大，因为它有望垂直推进和扩大对病毒重新激活期间扩增病毒基因组的裂解DNA复制的理解。最终，这些知识有可能用于设计干预策略来阻止 KSHV 诱发的恶性肿瘤。