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.

卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌的γ疱疹病毒，可在人类中建立终身感染。KSHV导致免疫功能低下患者发生卡波西肉瘤、原发性渗出性淋巴瘤（PEL）和侵袭性多中心Castleman病。KSHV的生命周期有两个不同的阶段：潜伏期和裂解性复制。虽然潜伏感染传统上与KSHV肿瘤发生有关，但越来越多的证据表明，KSHV的裂解复制不仅对病毒的传播很重要，而且对KSHV相关的肿瘤发生也很重要。KSHV的裂解周期由KSHV编码的复制和转录激活因子（RTA）的表达启动。RTA是诱导KSHV从潜伏感染细胞中裂解再活化所必需的，也是足够的。尽管RTA的重要作用，它仍然是知之甚少RTA如何能够管理，以快速诱导大量的各种各样的靶基因的高度染色质化的病毒和宿主基因组上所需的裂解复制KSHV。为了揭示RTA介导的KSHV感染细胞中基因调控的机制，我们一直在研究RTA在经历再激活的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再活化期间病毒和宿主基因组上活化组蛋白修饰的富集。RNA-seq还用于分析病毒和宿主基因在PEL细胞中潜伏和裂解再活化之间的差异基因表达。这些分析揭示了RTA的直接基因靶点和与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延伸的调节中发挥作用。与此一致，使用再活化的PEL细胞进行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、宿主转录延伸因子、染色质修饰酶和核小体重塑因子相互作用。基于我们的初步结果，我们假设RTA利用染色质调节和转录延长宿主因子与病毒辅因子协同诱导其病毒和宿主靶基因，以促进KSHV从潜伏期重新激活并诱导病毒复制。总的来说，本提案的目标是确定RTA对病毒和宿主基因组上转录延伸的调控的全基因组效应（Aim 1），并剖析RTA如何利用染色质修饰和重塑宿主因子、转录延伸宿主因子、和病毒辅因子，以促进RNA聚合酶II在KSHV裂解再活化期间在RTA靶基因处的进展（目的2）。这是一个高度创新的建议，它结合了基因组学与生化检测，以帮助更好地了解从潜伏感染的B细胞裂解再激活的分子机制，这在KSHV发病机制中具有生物学意义。