RNA Polymerase II Promoter-Proximal Pausing and Release to Elongation Are Key Steps Regulating Herpes Simplex Virus 1 Transcription

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (Pol II). Expression of viral immediate early (alpha) genes is followed sequentially by early (beta), late (gamma(1)), and true late (gamma(2)) genes. We used precision nuclear run-on with deep sequencing to map and to quantify Pol II on the HSV-1(F) genome with singl-enucleotide resolution. Approximately 30% of total Pol II relocated to viral genomes within 3 h postinfection (hpi), when it occupied genes of all temporal classes. At that time, Pol II on alpha genes accumulated most heavily at promoter-proximal pause (PPP) sites located similar to 60 nucleotides downstream of the transcriptional start site, while beta genes bore Pol II more evenly across gene bodies. At 6 hpi, Pol II increased on gamma(1) and gamma(2) genes while Pol II pausing remained prominent on alpha genes. At that time, average cytoplasmic mRNA expression from alpha and beta genes decreased, relative to levels at 3 hpi, while gamma(1) relative expression increased slightly and gamma(2) expression increased more substantially. Cycloheximide treatment during the first 3 h reduced the amount of Pol II associated with the viral genome and confined most of the remaining Pol II to alpha gene PPP sites. Inhibition of both cyclin-dependent kinase 9 activity and viral DNA replication reduced Pol II on the viral genome and restricted much of the remaining Pol II to PPP sites. IMPORTANCE These data suggest that viral transcription is regulated not only by Pol II recruitment to viral genes but also by control of elongation into viral gene bodies. We provide a detailed map of Pol II occupancy on the HSV-1 genome that clarifies features of the viral transcriptome, including the first identification of Pol II PPP sites. The data indicate that Pol II is recruited to late genes early in infection. Comparing alpha and beta gene occupancy at PPP sites and gene bodies suggests that Pol II is released more efficiently into the bodies of beta genes than alpha genes at 3 hpi and that repression of alpha gene expression late in infection is mediated by prolonged promoter-proximal pausing. In addition, DNA replication is required to maintain full Pol II occupancy on viral DNA and to promote elongation on late genes later in infection.