Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription

Abstract

Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G0, G1, and S). In all cases, infection led to cell cycle arrest. Cells infected in G0 or G1 phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G0, G1, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.

FIG. 1

Flow cytometry analysis of HFFs infected at various points in the cell cycle. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. Cells were infected at the time of replating (G₀) (A), 12 h postplating (G₁) (B), or 24 h post-plating (S phase) (C). Samples of mock- and HCMV-infected cells were harvested at defined times p.i. (indicated above the histograms) and analyzed for DNA content by flow cytometry following staining of the DNA with propidium iodide. The number above each histogram on the left side indicates relative cell number.

FIG. 2

Differential CPE as a result of infection of cells at different phases of the cell cycle. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. To control for the possibility of variability in the infections, the times of replating were staggered so that all infections (G₀, G₁, and S phase) could be performed concurrently with the same stock of virus. For each type of infection (G₀, G₁, and S phase), one representative flask of four was used for photography. Cells were photographed in situ with a 35-mm camera attached to a phase microscope. All photographs depict cells infected by HCMV at an MOI of 5. The cell cycle phases at the time of infection and the times p.i. when cells were photographed are as follows: G₀, 12 (A) and 36 (B) h; G₁, 12 (C) and 36 (D) h; S, 12 (E) and 36 (F) h. Magnification, ×93.

FIG. 3

Effect of cell cycle phase at the time of HCMV infection on viral protein expression. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. As described for Fig. 2, cells were concurrently infected at the time of replating (G₀) or 24 h postplating (S phase). At defined times p.i., cultures were harvested for Western analysis. Cell lysates (100 μg) from each time point were subjected to electrophoresis and subsequent immunoblotting analysis with Ab against the HCMV IE proteins IE1 72 and IE2 86 (A) or 2.2-kb early proteins U_L112-113 (B).

FIG. 4

Effect of cell cycle phase at the time of HCMV infection on IE protein expression in individual cells as determined by immunofluorescence. HFFs, grown on coverslips, were serum starved, followed by serum stimulation to generate plates containing cells either in G₀ or in S phase, thus allowing concurrent infection (see Materials and Methods). At defined times p.i., coverslips were then processed for immunofluorescence. Cells were stained with Ab to the IE viral proteins (B, D, F, H, and J), and Hoechst dye was used to illuminate the DNA in the cells (A, C, E, G, and I). Shown are G₀-phase mock-infected (A and B) and HCMV-infected (C and D) cells at 12 h p.i., S-phase mock-infected cells at 12 h p.i. (E and F), and S-phase HCMV-infected cells at 12 (G and H) and 36 (I and J) h p.i. Magnification, ×108. (K) Samples of mock- and HCMV-infected cells (from G₀- and S-phase infections) were harvested at 1 h p.i. and analyzed for DNA content by flow cytometry following staining of the DNA with propidium iodide.

FIG. 5

Cells infected in S phase display a preferential delay in viral protein expression during cellular DNA synthesis. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density onto coverslips. At 23 h postplating, cells were labeled with BrdU for 30 min, followed by infection at 24 h postplating (S phase) with HCMV. At 12 and 28 h p.i., coverslips were processed for immunofluorescence. Cells were double stained with Ab against BrdU and HCMV IE protein or against BrdU and HCMV pp65 tegument protein. In both cases, cells were stained with Hoechst dye to illuminate DNA. Two fields of HCMV-infected cells double-stained with Ab against BrdU and IE protein are shown. Magnification, ×333.

FIG. 6

Effect of HCMV infection on the steady-state kinetics of cyclin A. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. Cells were infected at the time of replating (G₀) (A), 12 h postplating (G₁) (B), or 24 h postplating (S phase) (C). At defined times p.i., cells were harvested for Western analysis. Cell lysates (100 μg) from each time point were subjected to electrophoresis and subsequent immunoblotting analysis with anti-cyclin A Ab.

FIG. 7

Effect of HCMV infection on steady-state kinetics of cyclin E. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. Cells were infected at the time of replating (G₀) (A) or 24 h postplating (S phase) (B). At defined times p.i., cells were harvested for Western analysis with anti-cyclin E Ab.

FIG. 8

Effect of HCMV infection on steady-state kinetics of cyclin B. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. Cells were infected at the time of replating (G₀) (A) or 24 h postplating (S phase) (B). At defined times p.i., cells were harvested for Western analysis with anti-cyclin B Ab.

FIG. 9

Effects of HCMV infection on mRNA levels of cyclins A, B, and E. HFFs were synchronized by contact inhibition and stimulated to cycle by replating at lower density. The cells were infected with HCMV or mock infected at the time of replating (G₀). At defined times p.i., 6 × 10⁷ cells were harvested from both mock- and HCMV-infected cell populations for the isolation of mRNA. The mRNA was then used for Northern analysis as described in Materials and Methods.