Requirement for Cellular Cyclin-Dependent Kinases in Herpes Simplex Virus Replication and Transcription

Abstract

Several observations indicate that late-G 1 /S-phase-specific cellular functions may be required for herpes simplex virus (HSV) replication: (i) certain mutant HSV strains are replication impaired during infection of cells in the G 0 /G 1 but not in the G 1 /S phase of the cell cycle, (ii) several late-G 1 /S-phase-specific cellular proteins and functions are induced during infection, and (iii) the activity of a cellular protein essential for expression of viral immediate-early (IE) genes, HCF, is normally required during the late G 1 /S phase of the cell cycle. To test the hypothesis that late-G 1 /S-phase-specific cellular functions are necessary for HSV replication, HEL or Vero cells were infected in the presence of the cell cycle inhibitors roscovitine (Rosco) and olomoucine (Olo). Both drugs inhibit cyclin-dependent kinase 1 (cdk-1) and cdk-2 (required for cell cycle progression into the late G 1 /S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G 1 ). We found that HSV replication was inhibited by Rosco and Olo but not by lovastatin (a cell cycle inhibitor that does not inhibit cdk activity), staurosporine (a broad-spectrum protein serine-threonine kinase inhibitor), PD98059 (an inhibitor specific for erk-1 and -2) or iso-Olo (a structural isomer of Olo that does not inhibit cdk activity). The concentrations of Rosco and Olo required to inhibit cell cycle progression and viral replication in both HEL and Vero cells were similar. Inhibition of viral replication was found not to be mediated by drug-induced cytotoxicity. Efforts to isolate Rosco- or Olo-resistant HSV mutants were unsuccessful, indicating that these drugs do not act by inhibiting a single viral target. Viral DNA replication and accumulation of IE and early viral RNAs were inhibited in the presence of cell cycle-inhibitory concentrations of Rosco or Olo. We therefore conclude that one or more cdks active from late G 1 onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.