Transcriptomic analysis of early HSV-1 transcription in the absence of the immediate early protein, ICP4 [Nanopore]. Transcriptomic analysis of early HSV-1 transcription in the absence of the immediate early protein, ICP4 [Nanopore]

Herpes simplex virus 1 (HSV-1) transcription is tightly regulated in a temporal cascade, utilizing cellular RNA polymerase (Pol). We previously observed that infection with HSV-1 mutants lacking immediate early (IE) genes a0, a4 and a22 exhibited unusually high levels of aberrant transcription across the viral genome at just 1.5 hpi. The strongest effect occurred in the absence of a4, which is both an essential transcriptional activator and repressor. The goal of the current study was to define the mechanism of ICP4-mediated early transcriptional repression on the viral genome. Using the transcriptomic tools PRO-Cap, PRO-Seq, GRO-Seq and Nanopore direct RNA sequencing we found that initiation was elevated at viral promoters of all temporal classes in the absence of ICP4. Despite higher levels of initiation, transcription of non-IE genes was stalled within gene bodies and did not lead to production of mature mRNA. We therefore posit that HSV-1 retains additional ICP4-independent mechanisms to limit expression of viral genes that initiate prematurely. The data also indicated rapid release from promoter proximal pausing and progression along HSV IE genes and revealed termination as an important rate -limiting regulatory step. These findings highlight multiple mechanisms that HSV-1 employs to regulate early transcription and identify ICP4’s repressive role is to restrict initiation on non IE genes, thereby ensuring correct progression of the temporal cascade. Overall design: To investigate the role of ICP4 in early HSV-1 transcription, HEp-2 cells infected with ICP4 HSV-1 mutant, n12, or its cognate repair were subjected to either PRO-Seq, PRO-Cap, GRO-Seq or Nanopore direct RNA sequencing.

单纯疱疹病毒1型（Herpes simplex virus 1, HSV-1）的转录以时序级联方式受到严格调控，其转录过程依赖宿主细胞RNA聚合酶（RNA polymerase, Pol）。
我们既往研究发现，缺失即刻早期基因（immediate early, IE）a0、a4与a22的HSV-1突变株在感染后1.5小时（hours post infection, hpi）即可在病毒全基因组范围内引发异常高水平的转录。其中缺失a4（该基因即为兼具核心转录激活与阻遏功能的感染细胞蛋白4（infected cell protein 4, ICP4））所产生的效应最为显著。
本研究旨在阐明ICP4介导的病毒基因组早期转录阻遏机制。
借助PRO-Cap、PRO-Seq、GRO-Seq与纳米孔直接RNA测序（Nanopore direct RNA sequencing）等转录组学技术，我们发现：在缺失ICP4的情况下，所有时序类别的病毒启动子处的转录起始水平均显著升高。尽管转录起始水平提升，但非即刻早期基因的转录却在基因体内发生停滞，无法产生成熟mRNA。
据此我们推测，HSV-1还存在不依赖ICP4的额外调控机制，用以限制过早启动的病毒基因表达。
本研究数据还显示，病毒即刻早期基因的启动子近端暂停现象可被快速解除，转录延伸过程得以顺利推进；同时发现转录终止是关键的速率限制性调控步骤。
上述发现揭示了HSV-1用于调控早期转录的多重机制，并证实ICP4的阻遏功能在于限制非即刻早期基因的转录起始，从而保障时序级联转录的正确进行。
总体实验设计：为探究ICP4在HSV-1早期转录中的功能，我们将感染了ICP4缺失型HSV-1突变株n12及其同源修复株的HEp-2细胞，分别开展PRO-Seq、PRO-Cap、GRO-Seq与纳米孔直接RNA测序。