Abstract
During herpes simplex virus 1 (HSV-1) infection there is a loss of the serine-2 phosphorylated form of RNA polymerase II (RNAP II) found in elongation complexes. This occurs in part because RNAP II undergoes ubiquitination and proteasomal degradation during times of highly active viral transcription, which may result from stalled elongating complexes. In addition, a viral protein, ICP22, was reported to trigger a loss of serine-2 RNAP II. These findings have led to some speculation that the serine-2 phosphorylated form of RNAP II may not be required for HSV-1 transcription, although this form is required for cellular transcription elongation and RNA processing. Cellular kinase cdk9 phosphorylates serine-2 in the C-terminal domain (CTD) of RNAP II. To determine if serine-2 phosphorylated RNAP II is required for HSV-1 transcription, we inhibited cdk9 during HSV-1 infection and measured viral gene expression. Inhibition was achieved by adding cdk9 inhibitors 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB) or flavopiridol (FVP) or by expression of a dominant–negative cdk9 or HEXIM1, which in conjunction with 7SK snRNA inhibits cdk9 in complex with cyclin 1. Here we report that inhibition of cdk9 resulted in decreased viral yields and levels of late proteins, poor formation of viral transcription-replication compartments, reduced levels of poly(A)+ mRNA and decreased RNA synthesis as measured by uptake of 5-bromouridine into nascent RNA. Importantly, a global reduction in viral mRNAs was seen as determined by microarray analysis. We conclude that serine-2 phosphorylation of the CTD of RNAP II is required for HSV-1 transcription.
Highlights
The largest subunit of RNA polymerase II (RNAP II) in eukaryotes contains a highly conserved C-terminal domain that consists of tandem repeats of the heptapeptide YSPTSPS, which is repeated 52 times in humans
The decrease in RNAP II levels and in phosphoserine-2 levels may be attributed to proteasomal degradation of stalled elongating RNAP II transcription complexes during highly active transcription in WT herpes simplex virus 1 (HSV-1) infections
During HSV-1 infection there is a decrease in the phosphoserine-2 form of RNAP II C-terminal domain (CTD) at later times of infection when transcription of viral genes is very robust, and which results in part from proteasomal degradation of stalled elongating transcription complexes [24,27]
Summary
The largest subunit of RNA polymerase II (RNAP II) in eukaryotes contains a highly conserved C-terminal domain that consists of tandem repeats of the heptapeptide YSPTSPS, which is repeated 52 times in humans. While unphosphorylated RNAP II is recruited to promoters, after assembly of the pre-initiation complex, serine-5 becomes phosphorylated during initiation, primarily by the kinase cdk, which is associated with the general transcription factor TFIIH [1,2]. Transition into the elongation phase of RNAP II transcription requires phosphorylation of serine-2 by the kinase cdk, which acts in conjunction with cyclin 1 in mammalian cells and the complex is referred to as P-TEFb for positive transcription elongation factor [2,3]. Transcription is paused by the repressors DSIF and the negative elongation factor, NELF resulting in short transcripts that require the recruitment of cdk9 [3,4,5]. Phosphorylation of CTD serine-2 has been shown to be required for co-transcriptional mRNA processing including splicing and polyadenylation [2,14,15,16,17,18]
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