Cao Subing, Moss Walter, O'Grady Tina, Concha Monica, Strong Michael J, Wang Xia, Yu Yi, Baddoo Melody, Zhang Kun, Fewell Claire, Lin Zhen, Dong Yan, Flemington Erik K
Department of Structural and Cellular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
J Virol. 2015 Jul;89(14):7120-32. doi: 10.1128/JVI.00608-15. Epub 2015 Apr 29.
We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.
Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.
我们之前已经表明，爱泼斯坦-巴尔病毒（EBV）可能编码数百种在病毒激活过程中表达的病毒长链非编码RNA（vlncRNA）。在此我们表明，EBV潜伏复制起点（oriP）在激活过程中进行双向转录，向左（oriPtLs）和向右（oriPtRs）的转录本大多定位于细胞核中。虽然oriPtLs很可能是非编码的，但至少一些oriPtRs包含BCRF1/vIL10开放阅读框。尽管如此，具有长5'非翻译区的oriPtR转录本可能部分发挥非编码功能。oriPtL和oriPtR转录本均以晚期动力学表达，且它们的表达受到膦甲酸的抑制。RNA测序（RNA-seq）分析表明，oriPtLs和oriPtRs在其重复序列家族（FR）区域表现出广泛的“超编辑”。RNA二级结构预测显示，oriPtLs和oriPtRs的FR区域可能形成大型的、进化上保守且热力学稳定的发夹结构。发现双链RNA结合蛋白和RNA编辑酶ADAR与oriPtLs结合，可能促进FR发夹结构的编辑。此外，发现多功能旁斑蛋白NONO与oriPt转录本结合，这表明oriPts与基于旁斑的固有抗病毒免疫途径相互作用。对oriPtLs进行敲低和异位表达表明，它有助于全局病毒裂解基因表达和病毒DNA复制。总之，这些结果表明，这些新的vlncRNA与细胞固有免疫途径相互作用，并有助于促进病毒裂解级联反应的进展。
最近的研究表明，裂解性疱疹病毒转录组的复杂性显著高于之前的认识，最近发现了数百种病毒长链非编码RNA（vlncRNA）。过去几年对细胞lncRNA的研究才刚刚开始让我们初步了解它们在细胞中复杂形成和调控过程中所发挥的一系列功能。新鉴定的疱疹病毒lncRNA同样可能发挥多种不同功能，尽管这些功能可能是针对病毒感染周期的特定需求而定制的。在此我们描述了源自EBV潜伏复制起点的新型转录本。我们表明它们经过超编辑，与一种相对较新认识的抗病毒途径相互作用，并且在促进病毒裂解基因表达中发挥作用。这些研究是解开疱疹病毒裂解复制中vlncRNA功能复杂领域的起点。
