KSHV Lytic DNA Replication and its Control Mechanism

KSHV裂解性DNA复制及其控制机制

• 批准号: 8662192
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8662192
• 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）：在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上存在根本性的空白。裂解性DNA复制在诱导KSHV介导的肿瘤发生中起重要作用，事实证明，艾滋病患者接受抗疱疹药物更昔洛韦（GCV）、Fascarnet （PFA）和N-甲氨基甲烷（N- methanocarbathmidine），阻断裂解性DNA复制，降低了卡波西肉瘤（KS）的风险。KSHV与多种人类恶性肿瘤密切相关，包括原发性积液淋巴瘤（PELs）、卡波西肉瘤（KS）和多中心Castleman病（MCDs），主要在免疫受损个体（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）中引起肿瘤。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，因为在器官移植/自身免疫患者中，由于HIV感染和免疫抑制药物的使用，免疫抑制的发生率仍然是一个值得关注的问题。高效抗逆转录病毒疗法（HAART）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明确溶解性DNA复制的机制，并确定调节DNA复制的因素，这可以用于制定阻断KSHV发病机制的策略。该应用程序的目的是确定功能性裂解起源和病毒核酸外切酶在基因组扩增中的作用的机制。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粒子产生中的作用。我们的初步数据表明，单起点复制（oriLyt-L）优先用于以双向方式启动复制。我们的中心