Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

Herpes simplex virus 1 (HSV-1) transcription is tightly regulated in a temporal cascade, utilizing cellular RNA polymerase. We previously observed that infection with HSV-1 mutants lacking immediate early (IE) genes α0, α4, and α22 exhibited high levels of aberrant transcription across the viral genome at just 1.5 hpi. The strongest effect occurred in the absence of full-length ICP4 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 PRO-Cap, PRO-Seq, GRO-Seq, and Nanopore direct RNA sequencing to analyze viral transcription, 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. Thus, ICP4-independent mechanisms limit expression of viral genes that initiate prematurely. GRO-seq data also indicated rapid release from promoter proximal pausing, and rapid progression along HSV IE gene bodies, with termination as an important rate-limiting regulatory step. These findings highlight multiple mechanisms that HSV-1 employs to regulate early transcription and shows that ICP4's repressive role is to restrict initiation, thereby ensuring correct progression of the temporal cascade. IMPORTANCE Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.