Viral disruption of host transcriptome integrity

大多数病毒感染会引起宿主基因表达的重大变化，这是为病毒复制创造最佳环境的更广泛战略的一部分。病毒通过抑制宿主蛋白质的合成，重新利用宿主细胞的生物合成资源，最大限度地积累病毒蛋白质或核酸，从而最终提高新感染后代的产量。单纯疱疹病毒1型(HSV-1)就是这种重要的“宿主关闭”现象的典型例子。HSV-1感染伴随着宿主mRNA生物合成的大规模中断，其原因是RNA聚合酶II对转录的调控失调，并伴随着mRNA的稳定性、剪接、3‘端的形成和转录终止的变化。尽管已有多项研究表明ICP27在HSV-1阻断HSV-1的过程中起重要作用，但其确切机制及其相关作用仍有待阐明。最近，我们证明了HSV-1感染引起宿主核因子亚细胞定位的广泛变化，宿主核因子是安装、移除和识别内部RNA修饰所需的，包括腺苷(M6A)N6位的甲基化。这导致宿主和病毒RNA上存在的内部碱基修饰在全球范围内减少。重要的是，我们确定了重要的病毒调节蛋白ICP27对于这种惊人的效果是必要的和充分的。内部RNA修饰，如m6A，影响mRNA和lncRNA生物发生的许多方面，包括对硬连接剪接和切割/多聚腺苷信号的识别，m6A还调节成熟mRNAs的输出、稳定性和翻译。我们观察到，病毒基因的表达在感染周期的开始对m6A的丢失很敏感，但在以后的时间里受到的影响较小，这与RNA修饰频率的降低是一致的。综合这些观察结果，我们认为，人们对ICP27广泛干扰宿主基因表达的能力知之甚少，这至少部分是由它重新定义宿主转录组的表观遗传格局的能力所介导的。为了更好地理解这一点，我们将更全面地表征ICP27表达对负责RNA化学修饰和3‘端加工和转录终止的宿主机制的影响。最近的研究揭示了m6A的位置与附近的聚腺苷酸化和信号的使用之间的机械联系，我们假设ICP27利用这一点导致转录终止的大范围中断(DOTT)，从而优先影响HSV-1感染细胞中的宿主转录组。为了了解为什么HSV-1mRNAs的3‘端形成在很大程度上对ICP27介导的Dott不敏感，我们将分析m6A在病毒转录组中的安装情况，以确定m6A对病毒转录单位内的切割和多聚腺苷化位点使用的影响。

Most viral infections invoke major changes in host gene expression as part of a broader strategy to create an optimal environment for viral replication. By suppressing host protein synthesis, viruses repurpose the biosynthetic resources of the host cell to maximize the accumulation of viral proteins or nucleic acids and thus ultimately boost the yield of new infectious progeny. Herpes simplex virus type 1 (HSV-1) provides a prime example of this important ‘host shutoff’ phenomenon. HSV-1 infection is accompanied by widescale disruption of host mRNA biogenesis through dysregulation of transcription by RNA polymerase II coupled with changes in mRNA stability, splicing, 3’-end formation, and transcription termination. Although multiple studies have implicated the essential viral regulatory protein ICP27 in aspects of shutoff by HSV-1, the exact mechanisms and their relative contributions remain to be elucidated. Recently, we demonstrated that HSV-1 infection induces widespread changes in the subcellular localization of host nuclear factors required for the installation, removal and recognition of internal RNA modifications including methylation at the N6-position of adenosine (m6A). This results in global reductions in the internal base modifications present on both host and viral RNAs. Importantly, we identified the essential viral regulatory protein ICP27 as both necessary and sufficient for this striking effect. Internal RNA modifications such as m6A influence many aspects of mRNA and lncRNA biogenesis including recognition of hardwired splicing and cleavage/polyadenylation signals, and m6A also regulates the export, stability and translation of mature mRNAs. We observed that viral gene expression is sensitive to loss of m6A at the beginning of the infection cycle but is less impacted at later times, coincident with reduced RNA modification frequency. Drawing these observations together we propose that the poorly understood ability of ICP27 to broadly disrupt host gene expression is mediated, at least in part, by its ability to redefine the epigenetic landscape of the host transcriptome. To understand this better, we will more fully characterize the impact of ICP27 expression on the host machinery responsible for RNA chemical modification and for 3’-end processing and transcription termination. Recent studies reveal a mechanistic linkage between the sites of m6A placement and use of nearby polyadenylation and signals and we hypothesize that this is exploited by ICP27 to bring about a widescale disruption of transcription termination (DoTT) that preferentially impacts the host transcriptome in HSV-1 infected cells. To understand why 3’-end formation of HSV-1 mRNAs is largely insensitive to ICP27- mediated DoTT, we will profile m6A installation across the viral transcriptome to determine the impact of m6A on cleavage and polyadenylation site usage within viral transcription units.