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）：了解与调节Kaposi肉瘤相关疱疹病毒（KSHV）的分子机制有关的分子机制的基本差距，也称为人类疱疹病毒8（HHV8）。裂解DNA复制对于诱导KSHV介导的肿瘤发生很重要，这证明了接受抗基因药物的患者，Ganciclovir（GCV），fascarnet（PFA）和N-甲状腺伴膜氨酸，这阻碍了KapoSI的sarmecosi（Kaposi sarmea）（Kaposi的风险）。 KSHV与各种人类恶性肿瘤紧密相关，包括原发性积液淋巴瘤（PELS），Kaposi的肉瘤（KS）和多中心castleman病（MCDS），并主要引起免疫受损的个体肿瘤，包括HIV感染的个体和患者接受免疫抑制治疗，以防止免疫抑制治疗疗法，以防止植入术。 KSHV诱导的肿瘤是HIV/AIDS患者发病和死亡率的主要原因之一。这项研究很重要，因为免疫抑制的发生率仍然是由于HIV感染以及在器官移植/自身免疫性患者中使用免疫抑制药物的问题。引入HAART（高度活跃的抗逆转录病毒疗法）已降低了KS肿瘤的发生率，但对于KS肿瘤没有特定的治疗方法。该项目的长期目标是定义裂解DNA复制机制，并确定调节DNA复制的因素，可以利用这些因素来制定阻断KSHV发病机理的策略。该应用的目的是确定功能性裂解起源的机制以及病毒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率产生中的作用。我们的初步数据表明，复制的单一来源（Orilyt-L）优先用于双向启动复制。我们的中心 假设是，裂解起源的复制启动受遗传和表观遗传因素的调节。该假设是根据我们实验室中关于使用生化和遗传方法的复制起始产生的初步数据提出的。拟议研究的基本原理是，确定裂解DNA复制的分子机制将为挫败受感染细胞的病毒a治疗KSHV相关的恶性肿瘤提供潜在的治疗途径，这是HIV感染和器官移植患者的主要健康问题，可预防植入治疗疗法，以防止接受植入术。在强大的初步数据的指导下，将通过追求三个具体目的来检验该假设：1）使用Smard使用SMARD识别功能复制起源和裂解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 oferilyt of Orilyt的动力学的动力学，以确定原来的原点。在第一个目标下，已经建立的方法是对复制的DNA（SMARD）的单分子分析，将确定在各种细胞背景中的病毒基因组复制中优先使用的复制起始来源。还将确定病毒外切核酸酶的作用在生成包装的单位长度病毒基因组中。在第二个目标下，将评估病毒microRNA的作用，与靶标的靶标的或转录通过裂解起源，以进行复制启动。在第三个目标下，将研究染色质的表观遗传修饰，以控制其控制起源用法的贡献。这种方法是创新的，因为我们使用了复制DNA程序的强大的内部标记，以确定来源的使用，复制叉进程和复制后DNA的分支。我们还使用创新的Galk-Kan程序来引入KSHV基因中的点突变，以确定特定的蛋白质功能，以更好地理解其在病毒背景下的作用。拟议的研究很重要，因为预计它将在病毒再激活过程中垂直提高和扩展对扩增病毒基因组的裂解DNA复制的理解。最终，这种知识可能有可能用于制定介入策略以阻止KSHV引起的恶性肿瘤。