Mechanisms regulating KSHV transcription elongation and termination

KSHV 转录延伸和终止的调节机制

• 批准号: 10619005
• 负责人: NICHOLAS K CONRAD
• 金额: $ 46.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-10 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10619005
• 关键词: Address; Affect; Binding; Binding Proteins; Biological Assay; CRISPR screen; Cell Nucleus; Cells; Complement; Coupled; DNA Viruses; Data; Disease; Elongation Factor; Gene Expression; Genes; Genetic Transcription; Goals; Herpesviridae; Human; Human Herpesvirus 8; Infection; Integration Host Factors; Introns; Kaposi Sarcoma; Life Cycle Stages; Light; Link; Lymphoma cell; Lytic; Lytic Phase; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Biology; Monitor; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Nuclear; Oncogenic Viruses; Pathogenesis; Patients; Phenotype; Phosphorylation; Play; Polymerase; Post-Transcriptional Regulation; Production; Protein Dephosphorylation; Protein phosphatase; Proteins; Publishing; RNA; RNA Binding; RNA Polymerase II; RNA Splicing; Regulation; Regulator Genes; Reporter Genes; Reporting; Role; Running; Signal Transduction; Small Interfering RNA; Speed; System; Testing; Transcription Elongation; Viral; Viral Genes; Viral Genome; Virion; Virus; Work; combat; comparative; density; experimental study; gammaherpesvirus; genome-wide; genome-wide analysis; human pathogen; insight; knock-down; lytic gene expression; lytic replication; novel; pathogen; pathogenic virus; posttranscriptional; primary effusion lymphoma; promoter; reactivation from latency; transcription termination; transcriptome sequencing; viral RNA

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.

项目总结/摘要 卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌病毒，可导致卡波西肉瘤，原发性 渗出性淋巴瘤（PEL）和多中心Castleman病（MCD）。像所有疱疹病毒一样，KSHV生命 一个周期包括潜伏期和裂解期，病毒依赖于复杂的基因表达级联 从潜伏期裂解激活。KSHV利用宿主细胞基因表达机制， 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（pol II）延伸的调节，如pol II在5 ′和3 ′末端的停顿所示。 基因. pol II延伸对宿主基因的几乎普遍存在的调节与KSHV对宿主的利用相结合 这表明病毒采用了类似的调节机制。尽管这种潜在的重要性， pol II通过延伸控制，KSHV转录延伸的调节在很大程度上尚未探索。使用 无偏见的全基因组CRISPR筛选，参与pol II延伸和mRNA 3 '末端形成的宿主因素 被鉴定为KSHV基因表达的负调节因子。这些因子在细胞中潜伏性的消耗 用KSHV感染性杆粒克隆（BAC 16）感染， 感染性病毒体裂解再活化。在所提出的工作中，连接伸长和3 ′-末端的机制， 将定义KSHV转录的形成因子。目标1将探讨这些延伸的重要性 因子在PEL细胞和溶解性γ疱疹病毒感染期间。Aim 2使用病毒基因报告基因构建体， 还原分子生物学来测试pol II暂停是否由病毒基因上的细胞因子诱导。 此外，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，将进行高通量方法的组合以检查宿主延伸的作用 影响病毒基因表达的因素。这些将包括RNA-seq测试基因表达水平，PAC-seq解决 mRNA 3 ′-末端形成和PRO-seq以确定病毒基因组上的pol II占有率。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细胞凋亡中起重要作用。 控制pol II暂停。在目的4中，延伸因子对KSHV基因的去磷酸化的靶点和作用 将被定义。成功完成拟议的研究将对该领域产生重大影响， 定义了一种新的宿主因子，该因子通过以前未描述的 机制。此外，所描述的机制很可能适用于其他DNA病毒， 人类基因表达也是如此。这些研究将使我们更深入地了解 这可能会导致深入了解如何打击KSHV相关疾病。