Inhibition of the Cyclin-Dependent Kinases at the Beginning of Human Cytomegalovirus Infection Specifically Alters the Levels and Localization of the RNA Polymerase II Carboxyl-Terminal Domain Kinases cdk9 and cdk7 at the Viral Transcriptosome

Abstract

We previously reported that defined components of the host transcription machinery are recruited to human cytomegalovirus immediate-early (IE) transcription sites, including cdk9 and cdk7 (S. Tamrakar, A. J. Kapasi, and D. H. Spector, J. Virol. 79:15477-15493, 2005). In this report, we further document the complexity of this site, referred to as the transcriptosome, through identification of additional resident proteins, including viral UL69 and cellular cyclin T1, Brd4, histone deacetylase 1 (HDAC1), and HDAC2. To examine the role of cyclin-dependent kinases (cdks) in the establishment of this site, we used roscovitine, a specific inhibitor of cdk1, cdk2, cdk7, and cdk9, that alters processing of viral IE transcripts and inhibits expression of viral early genes. In the presence of roscovitine, IE2, cyclin T1, Brd4, HDAC1, and HDAC2 accumulate at the transcriptosome. However, accumulation of cdk9 and cdk7 was specifically inhibited. Roscovitine treatment also resulted in decreased levels of cdk9 and cdk7 RNA. There was a corresponding reduction in cdk9 protein but only a modest decrease in cdk7 protein. However, overexpression of cdk9 does not compensate for the effects of roscovitine on cdk9 localization or viral gene expression. Delaying the addition of roscovitine until 8 h postinfection prevented all of the observed effects of the cdk inhibitor. These data suggest that IE2 and multiple cellular factors needed for viral RNA synthesis accumulate within the first 8 h at the viral transcriptosome and that functional cdk activity is required for the specific recruitment of cdk7 and cdk9 during this time interval.

Role of cdk9 in the Optimization of Expression of the Genes Regulated by ICP22 of Herpes Simplex Virus 1

“…This modification is associated with changes in the abundance, activity, and localization of cdk9 and cdk7, as shown previously by S. Tamrakar et al (40). This same group has also shown that the addition of cdk inhibitors at the start of infection alters the levels and localization of cdk9 and cdk7 (15). More recently, the Kaposi's sarcoma-associated herpesvirus K-cyclin protein has been shown to interact with cdk9 (6).…”

Tegument Proteins of Human Cytomegalovirus

“…Thus, it was not surprising to find that UL69 binds to RNA nonspecifically, but what was surprising was that this ability to bind RNA was not required for UL69-mediated nuclear RNA export (187). However, the recent finding of UL69 localization to sites of viral gene expression (86) indicates that UL69 selects viral mRNAs for export because it accumulates at subnuclear sites where viral transcripts are synthesized. This mechanism may also explain why UL69 stimulates the expression of reporter constructs that contain an simian virus 40 intron (201).…”

Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

“…While several studies have localized IE transcription to the viral transcriptosomes, which are subnuclear foci that contain the input viral genome as well as several cellular transcription factors, cyclin-dependent kinases, and RNAP II that form adjacent to POD/ND10 structures (24,25,30,57), the site of viral late gene transcription has not been established, although it has been hypothesized to occur around the replication center, given the localization of phosphorylated RNAP II there at later times of infection (57). We have further identified H3K4 and newly labeled BrU transcripts to localize outside the viral replication center, further indicating the perireplication center region to be transcriptionally active.…”