KSHV Genome Replication during Primary Infection

原发感染期间 KSHV 基因组复制

• 批准号: 8836907
• 负责人: Subhash C Verma
• 金额: $ 29.6万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8836907
• 关键词: Binding; Cell Cycle; Cell division; Cells; ChIP-seq; Chromatin; Complex; DNA; DNA biosynthesis; Data; Episome; Event; 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; Untranslated RNA; 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KSHV在初次感染过程中的基因组复制的理解存在根本差距。被感染的细胞即使在感染了单一多发性病毒后，也会继续通过多拷贝的病毒表观染色体建立潜伏感染 感染率(MOI)。有趣的是，潜伏病毒在每个细胞周期内只复制一次，因此表明TAT病毒基因组在初次感染期间在建立潜伏期之前会放大。然而，基因组在初次感染过程中的复制机制尚不清楚。了解病毒基因组复制的机制，包括蛋白质的参与，对于确定靶点将是重要的，这些靶点可以被用来阻止KSHV的初次感染，从而抑制潜伏期的建立。该项目的长期目标是使用介入性策略来针对原发感染，以阻止感染细胞中的病毒，从而治疗与KSHV相关的恶性肿瘤，这是艾滋病毒感染和接受免疫抑制治疗的器官移植患者的主要健康问题。这项应用的目的是确定病毒起源在初次感染期间用于病毒基因组复制的作用，以及帮助DNA复制的病毒和细胞因子的参与。我们的下一代染色质免疫沉淀(CHIP-SEQ)与裂解复制蛋白的RTA测序数据表明，在初次感染期间使用裂解起源。此外，下一代染色质测序发现，PAN与一个长的非编码RNA结合，在初次感染期间与oriLyt结合，表明它作为支架在起始处招募复制复合体。我们的中心假设是，裂解复制起点主要在初始感染期间被激活，这有助于通过滚环复制合成病毒基因组的多个副本。这一假设是基于我们的初步数据，这些数据是通过使用复制DNA的单分子分析(SMARD)、染色质免疫沉淀的下一代测序(CHIP-SEQ)和RNA纯化的染色质分离(CHIRP-SEQ)方法产生的。这项拟议研究的基本原理是，一旦知道KSHV是如何通过使用特定的病毒和细胞蛋白复制的，这些靶标的表达就可以改变，这可能会开辟新的创新途径来预防KSHV的初次感染。这一建议将有助于更好地了解初次感染过程中基因组复制的分子事件，从而确定可用于治疗效果的关键靶点。在强大的初步数据的指导下，这一假说将受到三个特定目标的检验：1)使用复制DNA的单分子分析(SMARD)确定潜在和裂解来源在初次感染中的作用；2)确定复制复合体在起始处的积累将确定用于基因组扩增的复制机制；3)确定PAN RNA在原点招募复制蛋白在调节初次感染过程中DNA复制的作用。第一季度的初步数据 SMARD和下一代测序有力地表明，病毒基因在初次感染期间合成，这些基因在病毒起源处积累，很可能是在PAN RNA的帮助下启动DNA复制。这种方法是创新的，因为我们利用了复制DNA方法和下一代测序的强大单分子分析，它们分别在单拷贝水平上以无偏见的方法分析DNA复制和基因表达。这项拟议的研究具有重要意义，因为它有望在垂直方向上推进和扩大对病毒基因组复制和初次感染后潜伏感染建立的理解。最终，这样的知识有可能确定 关键靶点，可用于抑制KSHV感染和相关恶性肿瘤。