Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication

Abstract

A process of pseudomitosis occurs during human cytomegalovirus infection that appears similar to cellular mitosis but involves the formation of multiple spindle poles, abnormal condensation, and mislocalization of chromosomal DNA. The relationship of this process to viral replication and cell cycle regulation during infection has been poorly understood. Pseudomitosis consistently peaks at late times of infection in all viral strains examined but at overall highest frequencies (30% to 35% of cells) using one common laboratory strain variant (AD169varATCC). Cyclin-dependent kinase 1 (Cdk1) plays a crucial role in pseudomitosis, mirroring its role in conventional mitosis. Dominant negative Cdk1 inhibits and wild-type Cdk1 stimulates this process; however, viral yields remain the same regardless of pseudomitosis levels. Broad inhibition of cell cycle-regulated kinases (Cdk1/Cdk2/Cdk5/Cdk9) with indirubin-3'-monoxime substantially decreases viral yields and synergizes with the viral UL97 kinase inhibitor, maribavir. Thus, Cdk1 is necessary and sufficient to drive pseudomitosis, whereas a combination of viral and cell cycle-regulated kinases is important during viral replication.

Figure 1. Pseudomitosis Levels Induced by Commonly Used CMV Strains

Confluent HFs infected with the indicated CMV strains for 50, 72, and 96 h were immunostained with monoclonal antibodies (mAb) against γ-tubulin 1 and against IE1/IE2. The percentage of IE1/IE2⁺ pseudomitotic cells was determined after counting at least 500 cells for each strain at each time point [7]. Mean percentages and standard deviation from a representative experiment are shown.

Figure 2. Impact of Cdk1 on Pseudomitosis and Replication

(A) Immunoblot analysis of Cdk1 expression in protein extracts from nontransduced (−) and GFP-, Cdk1wt-GFP−, or Cdk1dn-GFP−transduced HFs infected with AD169varATCC (AC) or AD169varDE (DE) for 72 h. The blot was incubated with an anti-Cdk1 monoclonal antibody (mAb) as described in Materials and Methods. Expected molecular weights: 34 kDa for Cdk1 and 61 kDa for Cdk1wt or dn-GFP. Black arrowhead, endogenous Cdk1; gray arrowhead, recombinant Cdk1. (B) GFP fluorescence images of HFs transduced with retroviruses expressing GFP, Cdk1wt-GFP, or Cdk1dn-GFP and infected with AD169varATCC for 72 h (original magnification ×50). Higher magnification images (×200) are displayed in the insets. Arrowheads indicate representative pseudomitotic cells; arrows indicate interphase cells. (C) Percentage of IE1/IE2⁺ and GFP⁺ pseudomitotic cells in GFP-, Cdk1wt-GFP−, or Cdk1dn-GFP−transduced HFs infected with AD169varATCC or AD169varDE for 72 h. Mean percentages and standard deviation from four independent experiments are shown. (D) Viral yields from nontransduced (−) and GFP-, Cdk1wt-GFP−, or Cdk1dn-GFP−transduced HFs infected with AD169varATCC for 50, 72, 96, and 120 h in one representative experiment. (E) Viral yields from nontransduced (−) and GFP-, Cdk1wt-GFP−, or Cdk1dn-GFP−transduced HFs infected with AD169varATCC or AD169varDE for 72 h in two independent experiments.

Figure 3. AD169varATCC and AD169varDE Yields Depend on the Activity of Cellular Kinases

(A) Mean viral yields with standard deviations (three independent experiments) from HFs infected with AD169varATCC or AD169varDE in the presence of 1% or 2% DMSO or in the presence of 5 μM or 10 μM IMO for 24, 48, 72, 96, 120, and 144 h. (B) Viral yields from GFP-, Cdk1wt-GFP−, or Cdk1dn-GFP−expressing HFs infected with AD169varATCC in the presence of 2% DMSO or 10 μM IMO for 24, 48, 72, 96, and 120 h from one representative experiment.

Figure 4. Checkerboard Analysis of Cellular Cdk Inhibitor IMO and VPK Inhibitor MBV on Viral Yields

Mean infectivity (with standard deviation of four replicates indicated by error bars) determined by plaque assay on culture supernatants of HFs at 24, 48, 72, 96, and 144 h postinfection with AD169varATCC in the presence of 0, 5, or 10 μM IMO (left) or in combination with different concentrations of MBV (0.025, 0.1, and 0.4 μM) from one experiment.