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

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

The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation

Passage through the restriction point late in G(1) normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restriction-point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis

P53 is required for the checkpoint-dependent rescue of MIE gene expression in S/G2.

<p>HEL fibroblasts were stably transduced with lentiviruses expressing p53 or GFP-specific shRNAs. The resulting knock-down (KD) cells and a mock-infected control were treated with aphidicolin or doxorubicin as indicated. Immediately after treatment (0 h) or 24 h later cells were infected with HCMV-AD169 (A). Cells were harvested 4 h post infection and analyzed for expression of p53, p21 and GAPDH by immunoblotting (B) and for MIE expression versus DNA content by flow cytometry (C).</p

The long-term cellular response to genotoxic stress makes S/G2 cells permissive for MIE gene expression.

<p>(A) Experimental setup. Proliferating HEL fibroblasts were treated with sublethal doses of doxorubicin or UV-C radiation. At the indicated times after treatment, cells were infected with HCMV-AD169 and harvested 4 h later. (B) Flow cytometry analysis of DNA content and MIE gene expression, as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001096#ppat-1001096-g001" target="_blank">figure 1</a>.</p

Induction of MIE gene expression by CDK inhibition does not require nuclear localization of pp71.

<p>(A) Undifferentiated NT2 cells, differentiated NT2 cells (treated with RA for 7 days) and mouse bone marrow (BM) cells were analyzed for Cyclin A2 expression by immunoblot analysis. GAPDH expression was analyzed to control for equal loading. (B) Undifferentiated and differentiated NT2 cells were infected with HCMV-TB40/e (MOI = 5). Where indicated, SU9516 at 10 µM final concentration was added together with the virus. At 6 hpi cells were analyzed for subcellular localization of pp71 (green) by immunofluorescence microscopy. Nuclei were stained with DAPI fluorochrome (blue). Non-infected cells (right panel) were analyzed to control for background staining. Scale bars (red)  = 10 µm.</p

Sequential regulation of the HCMV replication cycle by different CDKs.

<p>After virus entry a cell cycle-regulated CDK is able to prevent the onset of IE gene expression. Once IE expression has started, CDK7 and 9 become an essential part of the viral transcription machinery. At early and late times of infection CDKs are needed for proper function and localization of viral substrates.</p

The roscovitine-mediated rescue of MIE gene expression in S/G2 cells is p53 independent.

<p>The indicated knock-down fibroblasts were treated with doxorubicin (+ Doxo) and infected by HCMV 24 later. Or cells were treated with 25 µM roscovitine (+ Rosco) and infected with HCMV shortly after the start of treatment, as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001096#ppat-1001096-g008" target="_blank">figure 8C</a>. Cells were harvested at 5 hpi and analyzed for MIE expression and DNA content, as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001096#ppat-1001096-g001" target="_blank">figure 1</a>.</p