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

爱泼斯坦巴尔病毒是一种DNA肿瘤病毒，其与人类病理学相关，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。EBV在HIV/AIDS人群中尤其成问题，其中EBV相关的淋巴瘤尤其普遍。虽然世界上超过90%的人口携带EBV，但该病毒通常以“潜伏”状态存在，对宿主的影响很小。然而，在响应某些刺激或局部微环境线索时，EBV进入病毒裂解复制程序，导致病毒在宿主内和宿主之间传播。尽管已知病毒潜伏蛋白在EBV相关癌症中的作用，但在裂解性复制升高与EBV相关癌症的发作之间存在明确的联系。此外，在HIV共感染（+或- ART）的情况下观察到EBV裂解性复制的普遍升高，这可能有助于HIV感染个体对EBV相关淋巴瘤和自身免疫性疾病的易感性增加。由于具有最小的遗传含量，病毒高度依赖宿主细胞资源进行复制，并且它们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。疱疹病毒复制中最保守和研究最充分的病毒-宿主相互作用之一是“宿主关闭”，其中病毒编码的因子降解用于翻译的宿主细胞RNA，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，尽管诱导宿主B2 SINE元件的全局Pol III激活，但鼠γ-疱疹病毒MHV 68抑制宿主Pol II转录，作为宿主关闭的第二臂，进一步促进优先的病毒蛋白合成。使用EBV再活化模型，促进纯再活化细胞群体中转录组变化的询问，我们已经深入了解了EBV和宿主细胞转录组之间显着和意想不到的相互作用。与MHV 68不同，我们发现EBV在裂解复制期间维持细胞Pol II基因在典型启动子处的表达，并且引人注目地，在细胞基因组中在> 10，000个新的Pol II起始位点处引起转录。虽然在宿主基因组中广泛诱导主要非编码Pol II（EBV）或Pol III（MHV 68）转录的原因尚不清楚，但它可能与重塑核结构或核资源再分配中的某些作用有关。我们的研究还表明，EBV再激活诱导广泛的，非典型的外显子跳跃，其程度超过了大多数剪接体成分严重耗尽后观察到的外显子跳跃的程度。KSHV再活化的初步分析同样揭示了外显子跳跃的广泛诱导，表明剪接破坏不是EBV所独有的。以前的研究表明，外显子跳跃可以通过细胞无义介导的RNA衰变（NMD）途径引起细胞核滞留或细胞质溶核降解;我们发现，在再激活过程中观察到的近50%的外显子跳跃事件是NMD候选者。我们假设EBV（和KSHV）裂解性复制诱导细胞转录物的广泛的非典型外显子跳跃，导致核保留或通过细胞质NMD途径降解，作为宿主关闭的第二个新臂。虽然经典的宿主关闭已经研究了多年，但细胞转录物的特异性如何实现在很大程度上是谜。值得注意的是，疱疹病毒裂解基因表现出显著一致的主要是单外显子（即未剪接）的特征。我们假设剪接紊乱是宿主关闭的一个新分支，它有助于选择性靶向剪接的细胞转录本，以释放资源用于高水平的病毒蛋白质生产。在本提案中，我们将检验这一假设，我们将开始探讨EBV诱导剪接紊乱的机制，我们将开始探讨剪接紊乱对宿主和病毒基因表达的影响。

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.