Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

Abstract

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2–CDK-dependent sensor of the host cell cycle and differentiation state

“…Human embryonic lung (HEL) fibroblasts (Fi301), NT2, and lentivirus-transduced U373 cells were described previously (9,10). Where indicated, cells were treated with roscovitine (Santa Cruz), CVT313 (Merck), SU9516 (Santa Cruz), retinoic acid, or TSA (Sigma-Aldrich).…”

“…All other recombinant viruses were derived from TB40-BAC4 by BAC mutagenesis (see SI Materials and Methods). Viruses were propagated on HEL fibroblasts and titered as previously described (9). A multiplicity of infection (MOI) of 5-10 IE-protein forming units per cell was used for experiments.…”

“…This block is HDAC-independent and can be relieved by inhibition of cyclin-dependent kinases 1 and 2 (CDK1/2). Of note, CDK1/2 inhibition also promotes IE gene expression in undifferentiated cell lines of embryonic origin, suggesting a unifying mechanism of virus control (9). Cyclin A2 has recently been identified as the key regulator that recruits CDK1/2 for inhibition of the HCMV lytic cycle (10).…”

“…Flow cytometric analysis of DNA content, Oct3/4, and IE1/IE2 expression was carried out exactly as described (9).…”

“…HEL fibroblasts were grown on glass coverslips and infected with the indicated viruses. At 2 h postinfection, cells were fixed and processed for immunofluorescence staining essentially as described (9). The following primary antibodies were used: mouse-anti-GFP (ab1218, Abcam) and rabbit-anti-RFP (ab62341, Abcam).…”