Programmed splicing derangement as new EBV host cell shut-off mechanism

程序性剪接紊乱作为新的 EBV 宿主细胞关闭机制

• 批准号: 10446536
• 负责人: ERIK K FLEMINGTON
• 金额: $ 42.65万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446536
• 关键词: AIDS population; AIDS/HIV problem; Address; Alternative Splicing; Autoimmune Diseases; Automobile Driving; Cell Nucleus; Cells; Chemicals; Cues; Cytoplasm; DNA Polymerase II; DNA Polymerase III; DNA Tumor Viruses; Development; Down-Regulation; EBV-associated disease; Elements; Epstein-Barr Virus-Related Lymphoma; Epstein-Barr Virus-Related Malignant Neoplasm; Event; Exhibits; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; HIV; Herpesviridae; Hodgkin Disease; Human Herpesvirus 4; Human Herpesvirus 8; Human Pathology; Individual; Link; Lytic; Mediating; Messenger RNA; Metabolism; Modeling; Mus; Nasopharynx Carcinoma; Non-Hodgkin' s Lymphoma; Nuclear; Nuclear Structure; Pathway interactions; Population; Predisposition; Process; Production; Protein Biosynthesis; Proteins; RNA; RNA Decay; RNA Degradation; RNA Splicing; RNA-Binding Proteins; Resources; Role; Short Interspersed Nucleotide Elements; Site; Specificity; Spliceosomes; Stimulus; Structural Protein; Testing; Transcript; Transcriptional Activation; Translations; Untranslated RNA; Viral; Viral Genes; Viral Proteins; Viral Structural Proteins; Virion; Virus; Virus Latency; Virus Replication; arm; base; co-infection; exon skipping; gammaherpesvirus; insight; lytic replication; malignant stomach neoplasm; novel; novel virus; nuclease; programs; promoter; response; transcriptome; viral RNA; virus host interaction

The Epstein Barr virus is a DNA tumor virus that is associated with human pathologies including Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer, nasopharyngeal carcinoma and autoimmune diseases. EBV is particularly problematic in the HIV/AIDS population where EBV associated lymphomas are especially prevalent. While more than 90% of the world's population carries EBV, the virus typically exists in a “latent” state with little impact on the host. In response to certain stimuli or local microenvironmental cues, however, EBV enters the viral lytic replication program, leading to viral spread both within and between hosts. Despite the known role of viral latency proteins in EBV associated cancers, there are well-established links between elevated lytic replication and the onset of EBV associated cancers. Further, general elevation of EBV lytic replication is observed in the context of HIV co-infection (+ or – ART), likely contributing to the increased susceptibility of HIV infected individuals to EBV associated lymphomas and autoimmune diseases. With minimal genetic content, viruses are highly dependent on host cell resources for their replication and they elicit extensive alterations of host cell metabolic processes to facilitate efficient virus replication. One of the most conserved and well studied virus-host interactions in herpesvirus replication is “host shut off” where virus encoded factors degrade host cell RNAs destined for translation, freeing up translation resources for dedicated production of high amounts of viral structural proteins. Recently, the Glaunsinger lab showed that despite inducing global Pol III activation of host B2 SINE elements, the murine γ-herpesvirus, MHV68, inhibits host Pol II transcription as a second arm of host shut off, further promoting preferential viral protein synthesis. Using EBV reactivation models that facilitate interrogation of transcriptome changes in pure reactivating cell populations, we have gained insights into remarkable and unexpected interactions between EBV and the host cell transcriptome. Unlike MHV68, we found that EBV sustains cell Pol II gene expression at canonical promoters during lytic replication and strikingly, causes transcription at >10,000 new Pol II initiation sites across the cell genome. While the reason for the broad induction of predominantly non-coding Pol II (EBV) or Pol III (MHV68) transcription across host genomes is unclear, it could relate to some role in remodeling nuclear structure or redistribution of nuclear resources. Our studies also revealed that EBV reactivation induces widespread, noncanonical exon skipping, the extent of which surpasses the degree of exon skipping observed upon severe depletion of most spliceosome components. Preliminary analysis of KSHV reactivation similarly revealed widespread induction of exon skipping indicating that splicing disruption is not unique to EBV. Previous studies have shown that exon skipping can cause either nuclear retention or cytoplasmic nucleolytic degradation by the cellular nonsense mediated RNA decay (NMD) pathway; and we show that nearly 50% of exon skipping events observed during reactivation are NMD candidates. We hypothesize that EBV (and KSHV) lytic replication induces extensive non-canonical exon skipping of cell transcripts resulting in either nuclear retention or degradation through the cytoplasmic NMD pathway as a second, new arm of host shut off. While classic host shut off has been studied for many years, how specificity for cell transcripts is achieved has been largely enigmatic. Notably, herpesviral lytic genes exhibit a remarkably consistent feature of being primarily mono-exonic (i.e. unspliced). We hypothesize that splicing derangement is a new arm of host shut off that facilitates selective targeting of spliced cell transcripts to free up resources for high-level production of viral proteins. In this proposal, we will test this hypothesis, we will begin to address the mechanisms through which EBV induces splicing derangement and we will begin to address the consequences of splicing derangement on host and viral gene expression.

爱泼斯坦Barr病毒是一种DNA肿瘤病毒，与包括霍奇金淋巴瘤，非霍奇金淋巴瘤，失速癌，鼻咽癌和自身免疫性疾病在内的人类病理有关。 EBV在EBV相关淋巴瘤特别普遍的艾滋病毒/艾滋病人群中尤其有问题。尽管世界上有90％以上的人口具有EBV，但该病毒通常存在于“潜在”状态，对宿主的影响很小。然而，为了响应某些刺激或局部微环境提示，EBV进入了病毒裂解复制程序，从而导致宿主内部和宿主之间的病毒扩散。尽管病毒潜伏期蛋白在EBV相关的癌症中的已知作用，但在升高的裂解复制与EBV相关癌症的发作之间存在良好的联系。此外，在HIV共同感染（+或 - ART）的背景下观察到EBV裂解复制的一般升高，可能导致HIV感染的个体对EBV相关的淋巴瘤和自身免疫性疾病的敏感性增加。由于遗传含量最少，病毒高度依赖于宿主细胞资源的复制，并且会引起宿主细胞代谢过程的广泛改变，以促进有效的病毒复制。在疱疹病毒复制中，最保存和研究的病毒 - 宿主相互作用之一是“宿主关闭”，在该病毒中编码的因子降解了宿主细胞RNA，释放了用于翻译的宿主细胞RNA，从而释放了翻译资源，以专门生产大量的病毒结构蛋白。最近，Glaunsinger实验室表明，尽管诱导了宿主B2正弦元件的全球POL III激活，但鼠γ-鞘内病毒MHV68抑制了宿主Pol II转录，作为宿主关闭的第二组，进一步促进了优先的病毒蛋白合成。使用EBV重新激活模型，这些模型促进了纯净细胞种群中转录组变化的询问，我们对EBV与宿主细胞转录组之间的显着和意外相互作用有了深入的了解。与MHV68不同，我们发现EBV在裂解复制过程中持续了细胞II基因的表达，并引人注目的是在整个细胞基因组上引起> 10,000个新的Pol II倡议部位的转录。尽管尚不清楚跨宿主基因组的广泛诱导主要诱导主要非编码POL II（EBV）或POL III（MHV68）转录的原因，但它可能与重塑核结构或核资源重新分布中的某些作用有关。我们的研究还表明，EBV的重新激活诱导宽度，非规范外显子跳过，在严重部署大多数剪接体成分时，其效果表现出了外显子跳过的程度。 KSHV重新激活的初步分析类似地揭示了外显子跳过的宽度诱导，表明剪接破坏不是EBV所独有的。先前的研究表明，外显子跳过会导致细胞胡说八道介导的RNA衰变（NMD）途径引起核保留或细胞质核降解。我们表明，在重新激活期间观察到的外显子跳过事件中，几乎50％是NMD候选物。我们假设EBV（和KSHV）裂解复制会诱导细胞转录物的广泛非传统外显子跳过，从而导致通过细胞质NMD途径作为第二个宿主关闭的新手臂，从而导致核保留或降解。尽管经典的主机关闭了很多年，但如何实现细胞转录本的特异性在很大程度上是神秘的。值得注意的是，疱疹病毒裂解基因暴露了原发性外病的非常一致的特征（即未填充）。我们假设拼接演化是宿主关闭的新部门，它有助于选择性靶向剪接的细胞转录物，以释放资源以高级生产病毒蛋白。在该提案中，我们将检验该假设，我们将开始解决EBV诱导剪接演化的机制，我们将开始解决剪接进化对宿主和病毒基因表达的后果。