Mechanisms regulating KSHV transcription elongation and termination

项目概要/摘要 卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌病毒，可引起卡波西肉瘤，原发性 渗出性淋巴瘤（PEL）和多中心卡斯尔曼病（MCD）。与所有疱疹病毒一样，KSHV 的生命体 周期由潜伏期和裂解期组成，病毒依赖于复杂的基因表达级联 在从潜伏期裂解重新激活期间。 KSHV 使用宿主细胞基因表达机制进行转录 并在转录后控制基因表达的时间和水平。对于宿主基因，正确的转录 涉及 RNA 聚合酶 II (pol II) 延伸的调节，如 pol II 在 5´ 和 3´ 端暂停所示 基因。 pol II 延伸对宿主基因的几乎无处不在的调节与 KSHV 对宿主的使用相结合 机器表明该病毒采用类似的调节机制。尽管有这样的潜在重要性 pol II 通过延伸进行控制，KSHV 转录延伸的调节在很大程度上尚未被探索。使用 无偏见的全基因组 CRISPR 筛选、参与 pol II 延伸和 mRNA 3´ 末端形成的宿主因子 被鉴定为 KSHV 基因表达的负调节因子。细胞中这些因子的潜在耗尽 感染 KSHV 感染性杆粒克隆 (BAC16) 可显着提高生产速度和总体产量 裂解重新激活后具有传染性的病毒粒子。在拟议的工作中，连接伸长和 3' 末端的机制 KSHV 转录的形成因子将被定义。目标 1 将探讨这些伸长率的重要性 PEL 细胞中和裂解性伽马疱疹病毒感染期间的因素。目标 2 使用病毒基因报告构建体 还原分子生物学来测试 pol II 暂停是否是由病毒基因上的细胞因素引起的。 此外，还将确定病毒基因延伸控制的顺式作用和反式作用要求。 在目标 3 中，将结合使用高通量方法来检查宿主延伸的作用 影响病毒基因表达的因素其中包括用于测试基因表达水平的 RNA-seq、用于解决基因表达水平问题的 PAC-seq mRNA 3´ 末端形成，以及 PRO-seq 来确定病毒基因组上的 pol II 占据情况。最后，初步 已发表的数据表明，延伸因子的可逆磷酸化可能在 控制 pol II 暂停。目标4，KSHV基因延伸因子去磷酸化的靶点和作用 将被定义。拟议研究的成功完成将对该领域产生相当大的影响 定义了一种新的宿主因子，该因子通过以前未描述的方式负调节病毒基因表达 机制。此外，所描述的机制很可能适用于其他 DNA 病毒和 也告知人类基因表达。这些研究将使人们更深入地了解其机制 致病病毒的研究可能有助于深入了解如何对抗 KSHV 相关疾病。

PROJECT SUMMARY/ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Like all herpesviruses, the KSHV life cycle consists of latent and lytic phases, and the virus relies on a sophisticated cascade of gene expression during lytic reactivation from latency. KSHV uses the host cell gene expression machinery to transcriptionally and posttranscriptionally control the timing and levels of gene expression. For host genes, proper transcription involves regulation of RNA polymerase II (pol II) elongation illustrated by pol II pausing at the 5´ and 3´ ends of genes. The near ubiquitous regulation of pol II elongation on host genes coupled with KSHV’s use of the host machinery suggest that the virus employs similar mechanisms of regulation. Despite this potential importance of pol II control by elongation, regulation of KSHV transcription elongation has been largely unexplored. Using an unbiased genome-wide CRISPR screen, host factors involved in pol II elongation and mRNA 3´ end formation were identified as negative regulators of KSHV gene expression. Depletion of these factors in cells latently infected with a KSHV infectious bacmid clone (BAC16) robustly increases the speed and overall production of infectious virions upon lytic reactivation. In the proposed work, the mechanisms linking elongation and 3´-end formation factors to KSHV transcription will be defined. Aim 1 will explore the importance of these elongation factors in PEL cells and during lytic gammaherpesvirus infection. Aim 2 uses viral gene reporter constructs and reductionist molecular biology to test whether pol II pausing is induced by cellular factors on viral genes. Moreover, the cis-acting and trans-acting requirements for elongation control of viral genes will be determined. In Aim 3, a combination of high-throughput methods will be performed to examine the roles of host elongation factors on viral gene expression. These will include RNA-seq to test gene expression levels, PAC-seq to address mRNA 3´-end formation, and PRO-seq to determine pol II occupancy on the viral genome. Finally, preliminary and published data suggest that reversible phosphorylation of elongation factors may play essential roles in the control of pol II pausing. In Aim 4, the targets and role of dephosphorylation of elongation factors on KSHV genes will be defined. Successful completion of the proposed studies will have considerable impact on the field by defining a novel host factor that negatively regulates viral gene expression by a previously undescribed mechanism(s). Moreover, it is likely that the mechanisms described will apply to additional DNA viruses and inform human gene expression as well. These studies will generate a deeper understanding of the mechanisms of a pathogenic virus which may lead to insights into how to combat KSHV related diseases.