Herpes Simplex Virus 1 (HSV-1) ICP22 Protein Directly Interacts with Cyclin-Dependent Kinase (CDK)9 to Inhibit RNA Polymerase II Transcription Elongation

<div><p>The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II <i>in vitro</i>. Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.</p></div

Transient ectopic expression of ICP22 causes specific loss of pol II CTD Ser2 and Tyr1 phosphorylation.

<p>(<b>A</b>) Myc epitope-tagged ICP22 and U_S1.5 were ectopically expressed in HeLa cells from a transfected expression vector containing the ICP22 open-reading frame followed by three Myc epitope tags. pcDNA3 was used as the control in this and all subsequent transfections. An anti-Myc tag antibody was used for the western blot analysis and the positions of full-length ICP22 and U_S1.5 are noted. α-tubulin was used as a loading control (<b>B</b>) Western blot analysis was performed as described in (A). The antibody used is indicated on the right. (<b>C</b>) Western blot analysis was carried out using antibodies to the CTD phosphorylation marks indicated on the right. CTCF was used as a loading control.</p

Ectopic expression of ICP22 or the 193–256 subdomain causes loss of Ser2 phosphorylation from pol II transcribing host cell protein-coding genes.

<p>(<b>A</b>) Diagrams of the PLK2 and EIF2S3 genes, with the position of chromatin immunoprecipitation (ChIP) primer pairs indicated. (<b>B</b>), (<b>C</b>), (<b>E</b>) The results of ChIP analysis using the antibodies indicated on the left after transfection of vectors expressing the Myc-tagged proteins indicated. (<b>D</b>), (<b>F</b>) The ratio of the CTD modification to pol II as indicated at the left.</p

Sequence of primer sets used for qRT-PCR analysis.

<p>Sequence of primer sets used for qRT-PCR analysis.</p

Model for the role of ICP22 in inhibition of pol II CTD Ser2 phosphorylation.

<p>In uninfected cells (top panel), the negative elongation factor (NELF) and the DRB-sensitivity-inducing factor (DSIF) enhance pol II stalling. Subsequent recruitment of P-TEFb allows phosphorylation of DSIF, NELF and Ser2 of the pol II CTD, which leads to productive elongation. In the context of HSV-1-infected cells (bottom panel), ICP22 associates with P-TEFb and inhibits the kinase activity of CDK9 at the site of transcription, as indicated by the loss of phosphorylation of Ser2 of the pol II CTD, NELF and DSIF. As a consequence, the transition to productive elongation is inhibited. Interaction between ICP22 and pol II is not necessary to recruit ICP22 to genes or inhibit CDK9 when ICP22 is ectopically expressed in cells on its own. However, the interaction between pol II and ICP22 may be necessary to recruit ICP22 to host cell genes in HSV1-infected cells. Alternatively, interaction between ICP22 and pol II may play a role in regulation of viral gene expression by ICP22.</p

Ectopic expression of ICP22 or the 193–256 subdomain causes loss of pol II CTD Ser2 phosphorylation.

<p>Alignment of the conserved core motif within ICP22 of the alphaherpesviruses showing the degree of conservation according to the scheme above. Alignments were generated using PRALINE (<a href="http://www.ibi.vu.nl/programs/pralinewww/" target="_blank">http://www.ibi.vu.nl/programs/pralinewww/</a>). Asterisks indicate identical residues. Accession Nos. AEDO2597, AEV91400, AAA46092, CAA54262, AEL30878 (<b>B</b>) Top, diagram of full-length ICP22 and 193–256 showing the position of the alphaherpesvirus conserved core motif and the epiptope tags (Myc). Bottom, the results of western blot analysis of extracts from HeLa cells transfected with the constructs indicated using antibodies to the Myc tag. α-tubulin was used as a loading control. (<b>C</b>) Western blot analysis of whole-cell extract from HeLa cells transfected with the constructs indicated using antibodies to the Ser2P CTD phosphorylation mark. RPAP2 was used as a loading control.</p

ICP22 interacts with pol II and CDK9.

<p>(<b>A</b>) GST-mediated pull-down from HeLa nuclear extract with the recombinant proteins indicated at the top, followed by western blot analysis using anti-pol II and anti-CDK9 antibodies. On the right, a diagram of full-length ICP22 and 193–256 shows the position of the alphaherpesvirus conserved core motif and the GST tags. (<b>B</b>) GST-mediated pull-down of recombinant P-TEFb by the recombinant proteins indicated at the top followed by western blot analysis using an anti-CDK9 antibody.</p