Viral and host strategies for regulation of KSHV infection

KSHV 感染调节的病毒和宿主策略

• 批准号: 10165473
• 负责人: Zsolt Toth
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10165473
• 关键词: Acetylation; Address; Affect; Agreement; Apoptosis; Binding; Biochemical; Biological; Biological Assay; Cell Cycle; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Remodeling Factor; Complex; DNA; DNA Binding; DNA biosynthesis; DNA metabolism; Data; Deposition; Development; Ensure; Enzymes; Epigenetic Process; Etiology; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Herpesviridae Infections; Histones; Human; Human Herpesvirus 8; Immunocompromised Host; Infection; Integration Host Factors; Kaposi Sarcoma; Lead; Life; Life Cycle Stages; Link; Lymphoma cell; Lytic; Lytic Phase; Malignant Neoplasms; Mediating; Methylation; Molecular; Multicentric Angiofollicular Lymphoid Hyperplasia; NURF; Pathogenesis; Pathway interactions; Phase; Play; Polycomb; Prevention; Production; Proteins; RNA Polymerase II; Regulation; Repressor Proteins; Resources; Role; Structure; Testing; Tissue-Specific Gene Expression; Transactivation; Transcription Coactivator; Transcription Elongation; Transcription Initiation; Transcriptional Elongation Factors; Transcriptional Regulation; Viral; Virus; Virus Replication; base; chromatin remodeling; gammaherpesvirus; genome-wide; global run on sequencing; histone acetyltransferase; histone demethylase; histone modification; infected B cell; innovation; latent infection; lytic gene expression; lytic replication; mutant; notch protein; primary effusion lymphoma; promoter; reactivation from latency; recruit; transcription factor; transcriptome; transcriptome sequencing; tumorigenesis; viral DNA

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing gamma herpesvirus, which establishes life-long infection in humans. KSHV accounts for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and aggressive forms of multicentric Castleman's disease in immunocompromised patients. Life cycle of KSHV has two distinct phases: latency and lytic replication. While latent infection has been traditionally linked to KSHV tumorigenesis, accumulating evidence suggests that lytic replication of KSHV is important for not only the spreading of the virus but also KSHV-associated oncogenesis. The lytic cycle of KSHV is initiated by the expression of the replication and transcription activator (RTA) encoded by KSHV. RTA is required and sufficient for inducing lytic reactivation of KSHV from latently infected cells. Despite the essential role of RTA, it is still poorly understood how RTA can manage to rapidly induce a large variety of its target genes on the highly chromatinized viral and host genomes that are required for lytic replication of KSHV. To uncover the mechanisms of RTA-mediated gene regulation in KSHV-infected cells, we have been studying the transcriptional function of RTA in PEL cells undergoing reactivation. We performed ChIP-seq analyses to determine the binding of RTA and the enrichment of activating histone modifications on both viral and host genomes during latency and lytic reactivation. RNA-seq was also used to analyze the differential gene expression of viral and host genes between latency and lytic reactivation in PEL cells. These analyses revealed the direct gene targets of RTA and the chromatin changes associated with RTA-binding. The functional study of an RTA mutant showed that RTA can play a role in the regulation of RNA polymerase II elongation. In agreement with this, RTA complex purification using reactivated PEL cells showed that RTA interacts with several viral factors associated with DNA replication and transcription, host transcription elongation factors, chromatin modifying enzymes, and nucleosome remodeling factors. Based on our preliminary results, we hypothesize that RTA utilizes the chromatin regulatory and transcription elongation host factors in concert with viral co-factors to induce its viral and host target genes for promoting KSHV reactivation from latency and induce viral replication. Collectively, the goal of this proposal is to determine the genome-wide effect of RTA on the regulation of transcription elongation on both the viral and host genomes (Aim 1), and dissect the mechanisms of how RTA utilizes the chromatin modifying and remodeling host factors, the transcription elongation host factors, and viral co-factors to facilitate the progression of RNA polymerase II at RTA target genes during lytic reactivation of KSHV (Aim 2). This is a highly innovative proposal, which combines genomics with biochemical assays to help better understand the molecular mechanisms of lytic reactivation from latently infected B cells, which has biological importance in KSHV pathogenesis.

卡波西（Kaposi）与肉瘤相关的疱疹病毒（KSHV）是一种引起癌症的伽马疱疹病毒，它在人类中建立了终生感染。 KSHV解释了Kaposi的肉瘤，原发性淋巴瘤（PEL）和免疫功能低下患者中多中心骑员病的侵略性形式。 KSHV的生命周期有两个不同的阶段：潜伏期和裂解复制。传统上，潜在感染与KSHV肿瘤发生有关，但积累的证据表明，KSHV的裂解复制对于不仅对病毒的扩散，而且对KSHV相关的肿瘤发生也很重要。 KSHV的裂解周期是由KSHV编码的复制和转录激活剂（RTA）的表达引发的。需要RTA，足以诱导潜在感染细胞的KSHV裂解重新激活。尽管RTA具有至关重要的作用，但仍然鲜为人知的是RTA如何在高度染色质的病毒和宿主基因组上迅速诱导其大量靶基因，这是KSHV裂解复制所需的。为了发现KSHV感染细胞中RTA介导的基因调节的机制，我们一直在研究RTA在接受重新激活的PEL细胞中的转录功能。我们进行了CHIP-SEQ分析，以确定RTA的结合以及在潜伏期和裂解重新激活期间对病毒和宿主基因组的激活组蛋白修饰的富集。 RNA-seq还用于分析pel细胞中病毒和溶液重新激活之间病毒和宿主基因的差异基因表达。这些分析揭示了RTA的直接基因靶标和与RTA结合相关的染色质变化。 RTA突变体的功能研究表明，RTA可以在RNA聚合酶II伸长率的调节中发挥作用。与此一致，使用重新激活的PEL细胞的RTA复合物纯化表明，RTA与与DNA复制和转录相关的几种病毒因子相互作用，宿主转录伸长因子，染色质修饰酶和核小体重塑因子。基于我们的初步结果，我们假设RTA与病毒共同因素一起利用染色质调节和转录伸长宿主因子来诱导其病毒和宿主靶基因来促进潜伏期的KSHV重新激活并诱导病毒复制。总的来说，该提案的目的是确定RTA对跨基因组对病毒和宿主基因组调节的调节（AIM 1）的调节（AIM 1），并阐明RTA如何利用染色质修饰和重塑宿主因素，转录延长宿主因素和病毒式依赖于pryse的机制。 KSHV的重新激活（AIM 2）。这是一项高度创新的建议，它结合了基因组学与生化测定法，以帮助更好地了解延迟感染的B细胞裂解重新激活的分子机制，在KSHV发病机理中具有生物学重要性。