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，与人类病理有关，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在与EBV相关的淋巴瘤特别流行的艾滋病毒/艾滋病人群中尤其成问题。虽然世界上90%以上的人口携带EBV，但这种病毒通常以一种“潜伏”的状态存在，对宿主几乎没有影响。然而，作为对某些刺激或局部微环境线索的反应，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，但在升高的裂解复制和EBV相关癌症的发生之间存在着公认的联系。此外，在HIV合并感染(+或-ART)的背景下观察到EBV裂解复制的普遍增加，这可能导致HIV感染者对EBV相关淋巴瘤和自身免疫性疾病的易感性增加。由于遗传含量极低，病毒的复制高度依赖宿主细胞资源，它们会引起宿主细胞代谢过程的广泛变化，以促进病毒的有效复制。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”，即病毒编码因子降解预定用于翻译的宿主细胞RNA，释放翻译资源，专门生产大量病毒结构蛋白。最近，格劳辛格的实验室发现，尽管小鼠γ疱疹病毒MHV68诱导宿主B2氨基酸的POLIII全球激活，但当宿主的第二臂关闭时，MHV68抑制宿主POLII的转录，进一步促进优先病毒蛋白的合成。使用EBV重新激活模型，有助于询问纯粹重新激活的细胞群体中转录组的变化，我们已经获得了对EBV和宿主细胞转录组之间显著和意想不到的相互作用的见解。与MHV68不同，我们发现EBV在裂解复制过程中在规范的启动子上维持细胞POL II基因的表达，并显著地在整个细胞基因组中引起10,000个新的POL II启动子的转录。虽然在宿主基因组中广泛诱导以非编码为主的POL II(EBV)或POL III(MHV68)转录的原因尚不清楚，但它可能与重塑核结构或核资源重新分配的某些作用有关。我们的研究还表明，EBV的重新激活导致了广泛的、非规范的外显子跳跃，其程度超过了大多数剪接体成分严重耗尽时观察到的外显子跳跃的程度。对KSHV重新激活的初步分析同样显示了广泛的外显子跳跃诱导，这表明剪接破坏并不是EBV独有的。先前的研究表明，外显子跳跃可以通过细胞无义介导的RNA衰变(NMD)途径导致核滞留或细胞质溶核降解；我们还表明，在重新激活过程中观察到的外显子跳跃事件中，近50%是NMD候选事件。我们假设，EBV(和KSHV)的裂解复制诱导了广泛的非规范外显子跳过细胞转录，导致核保留或通过细胞质NMD途径降解，因为宿主的第二个新臂关闭。虽然经典的宿主关闭已经被研究了很多年，但细胞转录本的特异性是如何实现的在很大程度上是个谜。值得注意的是，疱疹病毒裂解基因表现出一个非常一致的特征，即主要是单外显子(即未剪接)。我们假设剪接错位是一种新的宿主臂关闭，有助于选择性靶向剪接细胞转录，以释放资源，以高水平生产病毒蛋白。在这个提案中，我们将检验这一假说，我们将开始解决EBV诱导剪接错位的机制，并开始解决剪接错位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.