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

Abstract

Several observations indicate that late-G1/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 G0/G1 but not in the G1/S phase of the cell cycle, (ii) several late-G1/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 G1/S phase of the cell cycle. To test the hypothesis that late-G1/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 G1/S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G1). 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 G1 onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.

Binding of Herpes Simplex Virus Type-1 Virions Leads to the Induction of Intracellular Signalling in the Absence of Virus Entry

“…CDK2 is involved in the progression of the cell cycle from G1 through to S phase. Transient activation of CDK2 was shown to occur early in HSV-2 infection at two hours post-infection, and is crucial in early HSV-1 infection [45] [46]. Kinase action by the cyclin A/CDK2 complex liberates the bound transcription factor E2F from Rb, a transcription factor that has previously been shown to be active during HSV-1 infection [47].…”

Novel Cell Type–Specific Antiviral Mechanism of Interferon γ Action in Macrophages

“…A third potential mediator of IFN-γ effects in BMMϕ is IFN-γ regulation of cell cycle. Cell cycle regulation by herpes viruses is a critical part of the viral life cycle (for reviews, see references 89 and 90919293949596). IFN-γ treatment of BMMϕ with IFN-γ results in expression of p21 waf-1 , leading to arrest at the G1/S boundary 97.…”

Phosphorylation of the Herpes Simplex Virus Type 1 Origin Binding Protein

“…Three micrograms of DNA was vacuum slot blotted onto a nylon membrane (GeneScreen; New England Nuclear Research Products, Boston, Mass.) as described previously (43). After UV cross-linking, the membrane was prehybridized for 1 h at 55°C in ExpressHyb solution (Clontech) and hybridized with a radiolabeled probe (3 ϫ 10 6 cpm/ml) specific for the HSV-1 UL26 gene (encoding capsid protein VP24) for 3 h at 55°C.…”