KSHV Genome Replication during Primary Infection

描述（由申请人提供）：我们对KSHV基因组在初次感染期间复制的理解存在根本性差距。感染细胞继续建立潜伏感染与多个拷贝的病毒附加体，即使在被感染的单一多样性 感染（MOI）。有趣的是，潜伏病毒每个细胞周期仅复制一次，因此表明在建立潜伏期之前达特病毒基因组在初次感染期间扩增。然而，原发感染期间基因组复制的机制尚不清楚。了解病毒基因组复制的机制，包括蛋白质的参与将是重要的，在确定目标，可以利用阻断KSHV原发感染，从而抑制潜伏建立。该项目的长期目标是使用干预策略来靶向原发性感染，以阻止病毒从受感染细胞中释放，从而治疗KSHV相关的恶性肿瘤，这是HIV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。本申请的目的是确定在原发性感染期间用于病毒基因组复制的病毒起点的作用以及辅助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RTA的测序数据表明，在原发感染期间使用裂解起点。此外，由长的非编码RNA结合的染色质的下一代测序，PAN鉴定了其在原发性感染期间与oriLyt的结合，表明其作为在起点募集复制复合物的支架的作用。 我们的中心假设是裂解性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拷贝。这一假设是基于我们通过使用复制DNA的单分子分析（SMARD）、染色质免疫沉淀的下一代测序（ChIP-seq）和通过RNA纯化的染色质分离（ChIRP-seq）方法产生的初步数据而制定的。这项研究的基本原理是，一旦知道KSHV如何通过使用特定的病毒和细胞蛋白进行复制，这些靶点的表达就可以改变，这可能为预防KSHV原发性感染开辟新的创新途径。这一建议将提供一个更好的了解基因组复制的分子事件在原发性感染，从而将确定关键的目标，可以利用治疗的好处。 在强有力的初步数据的指导下，这一假设将通过三个具体目标进行检验：1）使用复制DNA的单分子分析（SMARD）确定原发感染期间潜伏和裂解起源的作用，2）确定复制复合物在起点处的积累将鉴定用于基因组扩增的复制机制，以及确定PAN RNA在原发感染期间在调节DNA复制的起点招募复制蛋白中的作用。初步数据 SMARD和下一代测序强烈表明，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，最有可能在PAN RNA的帮助下启动DNA复制。该方法是创新的，因为我们正在利用复制DNA方法和下一代测序的强大单分子分析，其分别以单拷贝水平的无偏倚方法分析DNA复制和基因表达。拟议的研究是重要的，因为它有望垂直推进和扩大对病毒基因组复制和初次感染后潜伏感染的建立的理解。最终，这些知识有可能决定 关键的目标，可用于抑制KSHV感染和相关的恶性肿瘤。

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.