ICP22 of Herpes Simplex Virus 1 Decreases RNA Polymerase Processivity

Abstract

To determine the role of ICP22 in transcription we performed precise nuclear run-on followed by deep sequencing (PRO-Seq) and Global nuclear run-on with sequencing (GRO-seq) in cells infected with a viral mutant lacking the entire ICP22-encoding α22 (US1/US1.5) gene, and a virus derived from this mutant bearing a restored α22 gene. At 3 hours post infection (hpi), the lack of ICP22 reduced RNA polymerase (Pol) promoter proximal pausing (PPP) on the immediate early α4, α0, and α27 genes. Diminished PPP at these sites accompanied increased Pol processivity across the entire HSV-1 genome in GRO-seq assays, resulting in substantial increases in anti-sense and intergenic transcription. The diminished PPP on α gene promoters at 3hpi was distinguishable from effects caused by treatment with a viral DNA polymerase inhibitor at this time. The ICP22 mutant had multiple defects at 6 hpi, including lower viral DNA replication, reduced Pol activity on viral genes, and increased Pol activity on cellular genes. The lack of ICP22 also increased PPP release from most cellular genes, while a minority of cellular genes exhibited decreased PPP release. Taken together these data indicate that ICP22 acts to negatively regulate transcriptional elongation on viral genes in part to limit anti-sense and intergenic transcription on the highly compact viral genome. This regulatory function directly or indirectly helps to retain Pol activity on the viral genome later in infection. Importance The longstanding observation that ICP22 reduces RNA polymerase II (Pol II) serine 2 phosphorylation which initiates transcriptional elongation is puzzling because this phosphorylation is essential for viral replication. The current study helps explain this apparent paradox because it demonstrates significant advantages in negatively regulating transcriptional elongation, including the reduction of anti-sense and intergenic transcription. Delays in elongation would be expected to facilitate the ordered assembly and functions of transcriptional initiation, elongation, and termination complexes. Such limiting functions are likely to be important in herpesvirus genomes that are otherwise highly transcriptionally active and compact, comprising mostly short, intron-less genes near neighboring genes of opposite sense, and containing numerous 3’-nested sets of genes that share transcriptional termination signals but differ at transcriptional start sites on the same template strand.

Immediate early proteins of herpes simplex virus transiently repress viral transcription before subsequent activation

“…5F). However, ΔICP22 had a significant reduction in the promoter peak on these genes, matching its previously noted phenotype at 3 hpi (11). Analysis of the relative read density across the repair-repressed genes indicated that n212 maintained strong PPP on these genes, similar to n12 (Fig 5G).…”

“…6G). This differed from ΔICP22 which had reduced Pol activity on IE genes at 3 hpi and loss of Pol activity from most genes at 6 hpi (11).…”

“…HEp-2 cells harvested at 3 and 6 hpi were chosen for comparison to previous HSV-1 PRO-Seq studies (5,13). We included the 1.5 hpi time point to measure transcription prior to the onset of viral DNA replication (5).…”

“…The result with ΔICP22 likely reflects ICP22's known role in reducing processivity of RNA Polymerase (11), yet it was surprising that this occurred over the entire viral genome and was not limited to IE genes. One possible explanation is that transcription is initiated at VP16 sites, but that the increased Pol processivity in the absence of ICP22 leads to substantial read through into neighbouring genes (37).…”

“…ICP22 negatively regulates Pol processivity and release to elongation through reduction of serine 2 phosphorylation on the Pol C-terminal domain (CTD) (12). This negative elongation activity of ICP22 is important for enhancing promoter proximal pausing (PPP) on viral IE genes, and controlling anti-sense transcription on the compact HSV-1 genome (13).…”