Human cytomegalovirus infection induces specific hyperphosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II that is associated with changes in the abundance, activity, and localization of cdk9 and cdk7.

Human cytomegalovirus infection in the presence of the cyclin-dependent kinase (cdk) inhibitor roscovitine leads to changes in differential splicing and the polyadenylation of immediate early IE1/IE2 and UL37 transcripts (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004). To determine if this was associated with specific phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit by cdk7/cyclin H and cdk9/cyclin T1, we examined the expression and localization of these kinases and the various phosphorylated forms of RNAP II. Infection resulted in increased RNAP II CTD phosphorylated on serines 2 and 5 and increased levels of activity of cdk7 and cdk9. At early times, cdk9 localizes with input viral DNA, and aggregates of cdk9 and cdk7 and a subset of Ser2-phosphorylated RNAP II colocalize with IE1/IE2 proteins adjacent to promyelocytic leukemia protein oncogenic domains. Later, cdk9 and Ser2-phosphorylated RNAP II form a nuclear punctate pattern; cdk7 resides in replication centers, and Ser5-phosphorylated RNAP II clusters at the peripheries of replication centers. Roscovitine treatment leads to decreased levels of hyperphosphorylated RNAP II (RNAP IIo) in infected cells and of hypophosphorylated RNAP II in mock-infected and infected cells. The RNAP IIo decrease does not occur if roscovitine is added 8 h postinfection, as was previously observed for processing of IE transcripts. These results suggest that accurate IE gene expression requires specific phosphorylation of the RNAP II CTD early in infection.

Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production.

Human cytomegalovirus (HCMV) infection leads to dysregulation of multiple cell cycle-regulatory proteins. In this study, we examined the effects of inhibition of cyclin-dependent kinase (cdk) activity on viral replication. With the drug Roscovitine, a specific inhibitor of cyclin-dependent kinases 1, 2, 5, 7, and 9, we have shown that during the first 6 h of infection, cyclin-dependent kinase-dependent events occurred that included the regulated processing and accumulation of the immediate-early (IE) UL122-123 transcripts and UL36-37 transcripts. Altered processing of UL122-123 led to a loss of IE1-72 and an increase in IE2-86. The ratio of spliced to unspliced UL37 transcripts also changed. These effects did not require de novo protein synthesis or degradation of proteins by the proteasome. Addition of Roscovitine at the beginning of the infection was also associated with inhibition of expression of selected viral early gene products, viral DNA replication, and late viral gene expression. When Roscovitine was added after the first 6 h of infection, the effects on IE gene expression were no longer observed and viral replication proceeded through the late phase, but viral titers were reduced. The reduction in viral titer was observed even when Roscovitine was first added at 48 h postinfection, indicating that cyclin-dependent kinase activity is required at both IE and late times. Flavopiridol, another specific inhibitor of cyclin-dependent kinases, had similar effects on IE and early gene expression. These results underscore the importance of accurate RNA processing and reiterate the significant role of cell cycle-regulatory factors in HCMV infection.