Mechanisms regulating KSHV transcription elongation and termination

项目摘要/摘要 卡波西肉瘤相关疱疹病毒(KSHV)是一种致癌病毒，可引起原发卡波西肉瘤。 渗出性淋巴瘤(PEL)和多中心性Castleman病(MCD)。像所有的疱疹病毒一样，KSHV的生命 周期由潜伏期和裂解期组成，病毒依赖于一系列复杂的基因表达。 在潜伏期的裂解重新激活期间。KSHV利用宿主细胞基因表达机制转录 并在转录后控制基因表达的时间和水平。对于宿主基因，适当的转录 涉及RNA聚合酶II(PolII)伸长的调节，如PolII在5‘端和3’端暂停 基因。Pol II对寄主基因的几乎普遍的调节与KSHV对寄主的利用 机械设备表明，该病毒采用了类似的调控机制。尽管存在这种潜在的重要性， POL II通过伸长调控，对KSHV转录伸长的调控在很大程度上还没有被探索。使用 无偏全基因组CRISPR筛选，参与PolII延伸和mRNA3末端形成的宿主因素 被鉴定为KSHV基因表达的负调控因子。这些因子在细胞中的潜伏期耗尽 感染KSHV感染性巴氏杆菌克隆(BAC16)可显著提高生产速度和总体产量 裂解后的感染性病毒粒子重新激活。在拟议的工作中，连接延伸率和3‘端的机构 KSHV转录的形成因素将被定义。目标1将探讨这些延伸的重要性 PEL细胞和溶血性伽马疱疹病毒感染过程中的因素。AIM 2使用病毒基因报告构建和 简化论分子生物学测试PolII停顿是否由病毒基因上的细胞因素引起。 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，将执行高通量方法的组合来检查主机延伸的作用 影响病毒基因表达的因素。这些将包括用于测试基因表达水平的rna-seq和用于解决的pac-seq。 信使核糖核酸3‘端形成，并用PRO-SEQ确定PolII在病毒基因组上的占有率。最后，初步的 已发表的数据表明，伸长因子的可逆磷酸化可能在 控制POL II暂停。在目标4中，KSHV基因上延伸因子去磷酸化的靶点和作用 将会被定义。成功完成拟议研究将在以下方面对外地产生相当大的影响 定义一种新的宿主因子，该宿主因子通过先前未描述的 机制(S)。此外，所描述的机制很可能适用于其他DNA病毒和 也可以告知人类的基因表达。这些研究将对这些机制有更深的理解。 这可能导致对如何抗击KSHV相关疾病的洞察。

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.