Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3 processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.ICP27 is a multifunctional regulatory protein that is required for herpes simplex virus 1 (HSV-1) productive infection. This 63-kDa phosphoprotein is expressed with immediate-early kinetics, and it is required for appropriate expression of viral early and late gene products; in addition, it contributes to the shutoff of host protein synthesis (for review, see reference 54). ICP27 has been demonstrated to function posttranscriptionally at the level of RNA processing and export (4,35,(49)(50)(51), and recently evidence for a role in translation initiation has been presented (15, 16). Further, ICP27 also appears to contribute to the transcriptional regulation of HSV-1 early and late genes (23, 61). Thus, ICP27 appears to function in all stages of viral gene expression from transcription through translation. The mechanisms of some of its effects on gene expression have been elucidated. At early times after infection, ICP27 interacts with several splicing factors (4, 51), including members of a family of essential splicing factors termed SR proteins, and affects their phosphorylation. This results in the blockage of prespliceosomal assembly, which in turn contributes to the shutoff of host protein synthesis because cellular pre-mRNAs cannot be properly processed (51). At later times, beginning at about 6 h after infection, ICP27 begins to shuttle between the nucleus and cytoplasm (6,38,42,49,56). ICP27 binds to viral mRNAs (37,49,55) and facilitates their export to the cytopla...

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BackgroundThe cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.Methodology/Principle FindingsHere we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.Conclusions/SignificanceWe propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.