Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7

Abstract

Transcriptional activity of RNA polymerase II (Pol II) is orchestrated by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes Simplex Virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) of the Pol II CTD by targeting CDK9. The functional implications of this are poorly understood. Here, we report that HSV-1 also induces a global loss of serine 7 phosphorylation (pS7). This effect was dependent on the expression of the two viral immediate-early proteins, ICP22 and ICP27. While lytic HSV-1 infection results in efficient Pol II degradation late in infection, we show that pS2/S7 loss precedes the drop in Pol II level. Interestingly, mutation of the RPB1 polyubiquitination site mutation K1268, which prevents proteasomal RPB1 degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained much higher overall RPB1 protein levels even at late times of infection, indicating that this pathway mediates bulk Pol II protein loss late in infection but is not involved in early CTD dysregulation. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for production of viral proteins, with Ser2 facilitating viral immediate-early gene expression and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments by immunofluorescence. These data expand the known means that HSV-1 employs to create pro-viral transcriptional environments at the expense of host responses.

A Review of the Multipronged Attack of Herpes Simplex Virus 1 on the Host Transcriptional Machinery

“…Recently, it was found that phospho-Ser7 (pS7) is down-regulated in addition to pS2 while the other major CTD modifications are preserved. Both ICP22 and ICP27 seemed to contribute to this loss in fibroblasts [50]. This may occur by the same means as the reduction of pS2 in infection, though pS7 loss was not observed with transient expression of ICP22 in HeLa cells [51].…”

“…Interestingly, these studies found prevention of pS2 loss with proteasome inhibitors. While the majority of pS2/7 could be residing on a small proportion of hyperphosphorylated IIo in the cell, phospho-CTD reductions occur before a comparable drop in total RPB1 protein levels in multiple cell types indicating that remodeling is regulated beyond bulk turnover [47,50]. In contrast to HeLa cells [72], total RPB1 protein levels were stable through the peak of viral transcription in primary fibroblasts, while an almost complete loss was observed by 24 hpi.…”

“…At the beginning of mRNA transcription, CDK7 phosphorylates CTD Ser5, and this helps recruit capping machinery to nascent RNA. Studies did not observe a decrease in levels of phospho-Ser5 (pS5) by Western blotting [50,72], and ChIP studies using Pol II antibodies that were specific for pS5 or the N-terminus of RPB1 observed similar trends on both the host and viral genome [29,30,42,73]. CDK7 also copurifies with Pol II isolated from infected cells [27], and genes with reduced pS2 in ICP22-expressing cells maintained an equal fraction of pS5 [51].…”

“…Relevant to this discussion is that Pol II can be trafficked to VICE domains where it has been proposed to undergo ubiquitinmediated degradation [74]. Proteasome inhibition, which prevents pS2/7 loss [49,50], also prevented the formation of these domains [74]. It remains to be determined whether trafficking to VICE domains facilitates CTD remodeling by localizing RPB1 to viral proteins or other cellular factors or if RPB1 is fated only for degradation.…”