KSHV Lytic DNA Replication and its Control Mechanism

KSHV裂解性DNA复制及其控制机制

• 批准号: 8839201
• 负责人: Subhash C Verma
• 金额: $ 35.66万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839201
• 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; genetic approach; histone modification; innovation; insight; latent infection; lytic replication; monocyte; mortality; pre-miRNA; prevent; primary effusion lymphoma; promoter; protein function; 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 tumorigenesis, 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）：在对卡波西肉瘤相关疱疹病毒（KSHV）（也称为人类疱疹病毒8型（HHV 8））的裂解DNA复制调控所涉及的分子机制的理解方面存在根本性的空白。裂解性DNA复制在诱导KSHV介导的肿瘤发生中是重要的，这由接受抗疱疹药物更昔洛韦（GCV）、Fascarnet（PFA）和阻断裂解性DNA复制的N-甲氧卡巴胸苷的AIDS患者降低了卡波西肉瘤（KS）的风险的事实证明。KSHV与各种人类恶性肿瘤紧密相关，包括原发性渗出性淋巴瘤（PEL）、卡波西肉瘤（KS）和多中心Castleman病（MCD），并主要在免疫受损个体（包括HIV感染个体和接受免疫抑制治疗以防止移植物排斥的患者）中引起肿瘤。KSHV引起的肿瘤是HIV/AIDS患者发病和死亡的主要原因之一。这项研究是重要的，因为免疫抑制的发生率仍然是一个问题，由于艾滋病毒感染和使用免疫抑制药物的器官移植/自身免疫患者。HAART（高效抗逆转录病毒疗法）的引入降低了KS肿瘤的发病率，但没有针对KS肿瘤的特异性治疗。本项目的长期目标是确定裂解DNA复制的机制，并确定调节DNA复制的因素，这可以用于设计阻断KSHV致病机制的策略。本申请的目的是确定功能性裂解起点使用的机制和病毒核酸外切酶在基因组扩增中的作用。本研究还将确定病毒microRNA和表观遗传修饰在调节复制起始和病毒体产生中的作用。我们的初步数据表明，单一复制起点（oriLyt-L）优先用于启动复制的双向方式。我们的中央 假设是裂解起点的复制起始受遗传和表观遗传因素调节。这一假设是根据我们实验室使用生物化学和遗传学方法对复制起始产生的初步数据制定的。提出的研究的基本原理是，确定裂解DNA复制的分子机制将提供潜在的治疗途径，用于阻止病毒从感染的细胞中传播，以治疗KSHV相关的恶性肿瘤，这是HIV感染和器官移植患者接受免疫抑制治疗以防止移植排斥反应的主要健康问题。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测试：1）使用SMARD鉴定功能性复制起点和裂解DNA复制的机制，2）确定oriLyt介导的复制的控制机制，以及3）确定oriLyt处的表观遗传修饰的动态，这可能决定起点发射。在第一个目标下，已经建立的方法，复制DNA的单分子分析（SMARD），将确定在各种细胞背景中用于病毒基因组复制的优先使用的复制起始起点。还将确定病毒核酸外切酶在产生用于包装的单位长度病毒基因组中的作用。在第二个目标下，将评估病毒microRNA的作用，即与靶结合或通过裂解起点转录其pre-miRNA，用于复制起始。根据第三个目标，染色质裂解起源的表观遗传修饰将被调查的贡献，在控制原产地的使用。该方法具有创新性，因为我们使用了一种强大的细胞内标记复制DNA的方法，SMARD来确定复制后DNA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，从而更好地了解它们在病毒中的作用。这项研究具有重要意义，因为它有望纵向推进和扩展对裂解DNA复制在病毒再活化过程中扩增病毒基因组的理解。最终，这些知识可能被用于设计干预策略，以阻止KSHV诱导的恶性肿瘤。