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.

Epstein Barr 病毒是一种与人类病理相关的 DNA 肿瘤病毒，包括霍奇金淋巴瘤、非霍奇金淋巴瘤、胃癌、鼻咽癌和自身免疫性疾病，EB 病毒在 EB 病毒/艾滋病人群中尤其常见，其中 EB 病毒相关淋巴瘤尤其常见。虽然世界上 90% 以上的人口携带 EBV，但该病毒通常以“潜伏”状态存在，对宿主的某些刺激几乎没有影响。然而，EBV 进入病毒裂解复制程序，导致病毒在宿主内部和宿主之间传播，尽管已知病毒潜伏蛋白在 EBV 相关癌症中的作用，但裂解复制升高与宿主之间存在明确的联系。此外，在 HIV 合并感染（+ 或 – ART）的情况下观察到 EBV 裂解性复制普遍升高，这可能导致感染 EBV 相关癌症的 HIV 个体的易感性增加。病毒的遗传含量极低，其复制高度依赖于宿主细胞资源，它们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。疱疹病毒复制是“宿主关闭”，病毒编码因子会降解用于翻译的宿主细胞 RNA，从而释放翻译资源用于专门生产大量病毒结构蛋白。最近，Glaunsinger 实验室表明，尽管诱导了宿主的全局 Pol III 激活。 B2 SINE 元件，即鼠 γ-疱疹病毒 MHV68，作为宿主第二臂关闭而抑制宿主 Pol II 转录，进一步促进优先病毒蛋白合成。利用 EBV 再激活模型，有助于研究纯再激活细胞群中的转录组变化。与 MHV68 不同，我们发现 EBV 在裂解复制过程中维持细胞 Pol II 基因在典型启动子处的表达，并且令人惊讶的是，虽然在宿主基因组中广泛诱导非编码 Pol II (EBV) 或 Pol III (MHV68) 转录的原因尚不清楚，但可能与某些因素有关。我们的研究还表明，EBV 重新激活会引起广泛的非典型外显子跳跃，其程度超过了严重耗尽时观察到的外显子跳跃程度。对 KSHV 重新激活的初步分析同样揭示了外显子跳跃的广泛诱导，表明剪接破坏并非 EBV 所独有。先前的研究表明，外显子跳跃可通过细胞无义介导的 RNA 衰变导致核滞留或细胞质溶核降解。 (NMD) 途径；我们发现，在重新激活过程中观察到的外显子跳跃事件中有近 50% 是 NMD 候选者。裂解复制会诱导细胞转录物发生广泛的非规范外显子跳跃，导致细胞核滞留或通过细胞质 NMD 途径降解，作为宿主的第二条新臂关闭。虽然经典宿主关闭已被研究多年，但其特异性如何。值得注意的是，疱疹病毒裂解基因表现出一种罕见的一致特征，即主要是单外显子（即未剪接）。关闭宿主的新臂，有助于选择性靶向剪接的细胞转录本，从而释放资源用于病毒蛋白的高水平生产。在本提案中，我们将测试这一假设，我们将开始解决 EBV 的机制。诱导剪接紊乱，我们将开始解决剪接紊乱对宿主和病毒基因表达的影响。