KSHV Genome Replication during Primary Infection

原发感染期间 KSHV 基因组复制

• 批准号: 8691751
• 负责人: Subhash C Verma
• 金额: $ 28.71万
• 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: 2013-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8691751
• 关键词: Binding; Cell Cycle; Cell division; Cells; ChIP-seq; Chromatin; Complex; DNA; DNA biosynthesis; Data; Episome; Event; Functional RNA; Gene Expression; Genes; Genome; Goals; Graft Rejection; HIV; Health; Human Herpesvirus 8; Immunocompromised Host; Individual; Infection; Intervention; Knowledge; Label; Latent Virus; Life; Location; Lytic; Malignant Neoplasms; Methods; Mission; Molecular; Molecular Profiling; Morbidity - disease rate; Organ Transplantation; Outcome; Patients; Preventive Intervention; Proteins; Public Health; RNA; RNA purification; Recruitment Activity; Replication Initiation; Replication Origin; Research; Role; Site; Testing; Therapeutic; Therapeutic Intervention; Therapeutic immunosuppression; Trans-Activators; Transcript; Transplant Recipients; Viral; Viral Genes; Viral Genome; Virus; base; chromatin immunoprecipitation; expectation; innovation; insight; latent infection; latent persistent infection; lytic replication; mortality; next generation sequencing; prevent; protein expression; public health relevance; scaffold; single molecule; tumor; tumorigenesis

DESCRIPTION (provided by applicant): There is fundamental gap in our understanding of KSHV genome replication during primary infection. Infected cells go on to establish latent infection with multiple copies of viral episomes even after being infected with single multiplicity of infection (moi). Interestingly, latent virus replicates only once per cell cycle thus suggests tat viral genome amplifies during primary infection before establishing latency. However, the mechanism of genome replication during primary infection is not known. Understanding the mechanism of viral genome replication including the involvement of proteins will be important in identifying targets, which can be exploited to block KSHV primary infection and thus suppress latency establishment. The long-term goal of this project is to use interventional strategies to target primary infection for thwarting the virus from infected cells and thus treat KSHV associated malignancies, which are a major health problem for HIV infected and organ transplant patients undergoing immunosuppressive therapies. The objective of this application is to identify the roles of viral origins utilized for viral genome replication during primary infectin and the involvement of viral and cellular factors assisting DNA replication. Our next generation sequencing data of chromatin immunoprecipitated (ChIP-seq) with a lytic replication protein, RTA suggested the usage of lytic origin during primary infection. Additionally, next-generation sequencing of chromatin bound by a long non- coding RNA, PAN identified its binding to oriLyt during primary infection suggesting its role as scaffold to recruit replication complex at the origin. Our central hypothesis is that lytic replication origins are primarily activated during intial infection, which helps in synthesizing multiple copies of the viral genome through rolling circle replication. This hypothesis has been formulated based on our preliminary data produced by using Single Molecule Analysis of the Replicated DNA (SMARD), and next generation sequencing of Chromatin Immunoprecipitation (ChIP-seq) and Chromatin Isolation by RNA Purification (ChIRP-seq) approaches. The rationale for the proposed research is that, once it is known how KSHV replicates by using specific viral and cellular proteins, expression of those targets can be altered, which may open new and innovative avenues to prevent KSHV primary infection. This proposal will provide a better understanding of molecular events of genome replication during primary infection, thus will determine the critical targets which can be exploited for therapeutic benefits. Guided by strong preliminary data, this hypothesis will be tested by three specific aims: 1) Determining the roles of latent and lytic origins during primary infection using single molecule analysis of the replicated DNA (SMARD), 2) Determining the accumulation of replication complexes at the origins will identify the replication mechanism used for genome amplification and 3) Determining the role of PAN RNA in recruiting replication proteins at origins in regulating DNA replication during primary infection. The preliminary data of SMARD and next generation sequencing strongly suggests that viral genes are synthesized during primary infection which accumulate at viral origins most likely with the help of PAN RNA to initiate DNA replication. The approach is innovative, because we are utilizing a powerful single molecule analysis of the replicated DNA approach and next generation sequencing, which analyzes DNA replication and gene expression, respectively in an unbiased approach at single copy levels. The proposed research is significant because it is expected to vertically advance and expand the understanding of viral genome replication and the establishment of latent infection after primary infection. Ultimately, such knowledge has the potential to determine critical targets, which can be exploited for suppressing KSHV infection and associated malignancies.

描述（由申请人提供）：我们对初次感染期间KSHV基因组复制的理解存在基本差距。感染的细胞继续以多种副本的病毒发作来建立潜在感染 感染（MOI）。有趣的是，潜在病毒每个细胞周期仅复制一次，这表明在建立潜伏期之前，在原发性感染期间，TAT病毒基因组会放大。然而，尚不清楚原发性感染期间基因组复制的机制。了解包括蛋白质参与的病毒基因组复制的机制对于识别靶标很重要，这些靶标可以被利用以阻止KSHV原发性感染，从而抑制潜伏期的建立。该项目的长期目标是利用介入策略来靶向原发性感染，以阻止感染细胞中的病毒，从而治疗KSHV相关的恶性肿瘤，这对于接受免疫抑制疗法的HIV感染和器官移植患者来说是一个主要的健康问题。该应用的目的是确定原发性感染过程中用于病毒基因组复制的病毒起源的作用，以及辅助DNA复制的病毒和细胞因子的参与。我们使用裂解复制蛋白的染色质免疫沉淀（CHIP-SEQ）的下一代测序数据表明，在原发性感染过程中使用裂解起源。此外，PAN通过长的非编码RNA结合的染色质的下一代测序确定了其在原发性感染期间与Orilyt结合的结合，这表明其作为支架的作用，是募集起源的募集复制复合物的作用。 我们的中心假设是，裂解复制起源主要是在Intial感染过程中激活的，这有助于通过滚动圆复制来合成病毒基因组的多个副本。通过使用复制DNA（SMARD）的单分子分析以及染色质免疫沉淀（CHIP-SEQ）的下一代测序以及通过RNA纯化（CHIRP-SEQ）方法对下一代测序（CHIP-SEQ）和染色质素分离，该假设是根据我们的初步数据提出的。拟议的研究的理由是，一旦知道KSHV如何通过使用特定的病毒和细胞蛋白来复制，就可以改变这些靶标的表达，这可能会打开新的和创新的途径以防止KSHV原发性感染。该提案将更好地理解原发性感染期间基因组复制的分子事件，因此将确定可以利用用于治疗益处的关键靶标。 在强大的初步数据的指导下，该假设将通过三个特定目的测试：1）使用单分子分析在初次感染过程中确定潜在和透明源的作用，使用单分子分析（SMARD），2）确定在起源中的复制复制络合物的积累，以确定重复机制在重复机制中的作用，以确定基因的作用，以确定基因的作用，以确定基因的作用。调节原发性感染期间的DNA复制。的初步数据 Smard和Next Generation测序强烈表明，在原发性感染过程中合成了病毒基因，这些基因在病毒起源时很可能在PAN RNA的帮助下启动DNA复制。该方法具有创新性，因为我们正在利用复制的DNA方法和下一代测序的强大单分子分析，该测序分别分析了DNA复制和基因表达，分别以单拷贝水平的无偏方法进行了分析。拟议的研究之所以重要，是因为预计它将垂直提高和扩展对病毒基因组复制的理解，并在原发性感染后建立潜在感染。最终，这种知识有可能确定 可以利用来抑制KSHV感染和相关恶性肿瘤的关键目标。