KSHV Abortive Replication Following De Novo Infection

项目总结(见说明)： KSHV感染B淋巴细胞并建立一种主要的潜伏感染。建立潜伏感染的详细过程和机制仍然难以捉摸。在KSHV颗粒进入宿主细胞后，病毒经历DNA复制，导致病毒基因组在潜伏期建立之前积累到每个细胞50-100个拷贝。关于病毒复制流产的方式和调控机制，我们知之甚少。ORF KS编码bZip家族的核蛋白。我们利用K8缺失的重组病毒进行的研究证实了KS在新生KSHV感染后初始病毒DNA复制中的作用。KS缺失病毒感染293T、HFF和HMVEC细胞时，病毒基因组拷贝数明显低于野生型病毒。K8在新发感染的早期阶段的作用为我们提供了对所谓的“病毒流产复制”的洞察。在这项研究中，我们将遵循K8在从头感染早期阶段的功能作用来研究导致潜伏期建立的流产病毒复制过程以及控制这一过程的机制。特别是，(1)我们将探讨K8在KSHV原发感染早期的作用。我们将测试Kb在这一事件中扮演的角色的三个非排他性模型：(I)K8释放LANA介导的Ori|yt依赖的病毒DNA复制；(Ii)K8招募流产的裂解DNA复制所需的蛋白质，包括病毒DMA聚合酶；(Iii)K8通过对KSHV基因组的表观遗传调控抑制某些基因并激活其他基因，以促进有效的流产病毒复制。(2)我们将研究KSHV初次感染早期的流产裂解复制的性质。这可能代表了病毒DNA复制的第三种模式。我们将通过解决以下问题来研究这一关键事件：(I)KSHV Ori-Lyt是否参与了DNA复制的流产？(Ii)在流产的裂解复制中采用了什么DNA复制模式(3)？ 我们将用KSHV感染人PBMC，并分析K8和其他病毒因子在导致B细胞潜伏期建立的流产复制中的作用。这项研究可能导致干预KSHV生活史的新策略，旨在有效治疗KSHV相关疾病。

PROJECT SUMMARY (See instructions): KSHV infects B lymphocytes and establishes a predominantly latent infection. The detailed process and the mechanism underlying the establishment of latent infection remain elusive. After a KSHV particle enters a host cell, the virus undergoes DNA replication which results in accumulation of the viral genome to 50-100 copies per cell prior to establishment of latency. Little is known regarding the mode of the abortive viral replication and the regulation mechanism. ORF KS encodes a nuclear protein of the bZip family. Our study using K8-null recombinant virus demonstrated a role of KS in initial viral DNA replication following de novo KSHV Infection. KS-null viruses exhibit much lower viral genome copy numbers in comparison to wild type viruses when infecting 293T, HFF and HMVEC cells. The role of K8 in the early stage of de novo infection provides insights into the so-called "abortive viral replication". In this study, we will follow the functional role of K8 in the early stage of de novo infection to investigate the process of abortive viral replication that leads to establishment of latency and the mechanism controlling this process. In particular, (1) we will explore the role of K8 in the early stage of KSHV primary infection. We will test three non-exclusive models for the role of KB in this event: (i) K8 releases LANA-mediated ori-|yt-dependent viral DNA replication; (ii) K8 recruits the proteins required for abortive lytic DNA replication, viral or cellular inclusive of viral DMA polymerase; (iii) K8 represses some genes and activates other genes through epigenetic regulation of KSHV genome to facilitate effective abortive viral replication. (2) We will study the nature of abortive lytic replication in the early stage of KSHV primary infection. This may represent the third mode of viral DNA replication. We will study this crucial event by addressing the following questions: (i) Is the KSHV ori-Lyt involved in the abortive DNA replication? (ii) What mode of DNA replication is employed in the abortive lytic replication (3)? We will infect human PBMC with KSHV and analyze the contributions of K8 and other viral factors to the abortive replication that leads to establishment of latency in B cells. This study may leads to new strategy to interfere with KSHV life cycle aiming at efficacious treatment of KSHV-associated diseases.