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.
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.
Kapasi
Anokhi J
AJ
Skaggs School of Pharmacy and Pharmaceutical Sciences, Room 3254, Mail Code 0712, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093-0712, USA.
Spector
Deborah H
DH
D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D003587:Cytomegalovirus / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000378:metabolism; D000077546:Roscovitine; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014764:Viral Proteins / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase; D000092468:Bromodomain Containing Proteins
D018797:Cell Cycle Proteins; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D004279:DNA, Viral; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011687:Purines; D012367:RNA, Viral; D012097:Repressor Proteins; D000077546:Roscovitine; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C503684:CCNT1 protein, human; C446019:pUL69 protein, Human herpesvirus 5; C467969:CDK9 protein, human; C110550:HDAC1 protein, human