KSHV Abortive Replication Following De Novo Infection

项目总结（见说明）： KSHV感染B淋巴细胞并建立主要的潜伏感染。潜伏感染建立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，病毒进行DNA复制，这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。关于病毒复制失败的模式和调控机制知之甚少。ORF KS编码bZip家族的核蛋白。我们使用K8-null重组病毒的研究证明了KS在KSHV从头感染后的初始病毒DNA复制中的作用。当感染293 T、HFF和HMVEC细胞时，KS无效病毒与野生型病毒相比表现出低得多的病毒基因组拷贝数。K8在新生感染早期阶段的作用提供了对所谓的“流产病毒复制”的见解。在本研究中，我们将跟踪K8在从头感染的早期阶段的功能作用，以研究导致建立潜伏期的病毒复制失败的过程以及控制该过程的机制。具体而言，（1）探讨K8在KSHV原发感染早期的作用。我们将测试KB在该事件中的作用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募集失败的裂解DNA复制所需的蛋白质，包括病毒DNA聚合酶在内的病毒或细胞;（iii）K8通过KSHV基因组的表观遗传调控抑制一些基因并激活其他基因，以促进有效的失败病毒复制。(2)我们将研究KSHV初次感染早期的裂解性复制失败的性质。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是否参与了失败的DNA复制？(ii)在失败的裂解复制中，采用了什么样的DNA复制模式（3）？ 我们将用KSHV感染人PBMC，并分析K8和其他病毒因子对导致B细胞中潜伏期建立的失败复制的贡献。本研究可能为干预KSHV的生命周期提供新的策略，以有效治疗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.