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. 2008 Oct;82(20):10188-98.
doi: 10.1128/JVI.01212-08. Epub 2008 Jul 30.

Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells

Lüder Wiebusch  1 , Anke Neuwirth, Linus Grabenhenrich, Sebastian Voigt, Christian Hagemeier
Affiliations

Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells

Lüder Wiebusch et al. J Virol. 2008 Oct.

Abstract

The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G(0)/G(1) phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G(1) DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G(1). Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G(2). Moreover, MCMV can also replicate in G(2) cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G(1) or G(2). Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.

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Figures

FIG. 1.
FIG. 1.
MCMV inhibits proliferation by arresting cells at two points in the cell division cycle. (A) Experimental setup. Proliferating mouse fibroblasts were infected with MCMV (MOI = 5) the day after plating. (B and C) Infected and mock-infected control cells were harvested at the indicated time points and analyzed for cell number (B) and cell cycle distribution (C). Time points between 0 and 12 hpi are applicable only to the analysis of cell cycle distribution. (B) Shown are growth curves where the absolute number of cells per dish is given for each time point of harvest. Mean values and standard deviations represent the results of four independent experiments. (C) DNA histograms of 3T3 cells, in which the relative DNA content is plotted against cell number. A 2N DNA content is indicative of G0/G1 cells and a 4N DNA content of cells in either G2 or mitosis.
FIG. 2.
FIG. 2.
The late cell cycle block of MCMV-infected cells occurs after completion of S phase but prior to the onset of mitosis. (A) Experimental setup. Density-arrested NIH 3T3 fibroblasts were first replated at lower density and then treated with HU for synchronization at G1/S. Two hours after removal of HU, cells were MCMV or mock infected. (B) During the following 14 h, cell cycle progression was monitored at constant intervals of 1 h by flow cytometry. (C) In addition, the proportion of mitotic cells was determined for each sample by analyzing phosphorylation of histone H3 at serine 10. (D) To analyze chromatin condensation in a more direct way, cells arrested by MCMV with a G2 DNA content were stained with Giemsa's solution and examined by light microscopy. Nocodazole-treated cells were used as a positive control. Representative results are shown.
FIG. 3.
FIG. 3.
MCMV replicates both in cells arrested with a G1 DNA content and in cells arrested with a G2 DNA content. Asynchronously proliferating NIH 3T3 cells were MCMV infected (+MCMV) as described in the legend to Fig. 1. Where indicated, ganciclovir (+GCV) was added immediately after infection. Cells were harvested at the indicated time points, and their DNA content was analyzed by flow cytometry.
FIG. 4.
FIG. 4.
MCMV lacks the strict cell cycle dependence of HCMV major immediate-early gene expression. Proliferating 3T3 fibroblasts were infected with MCMV or a mock control. Cells were harvested at 4 hpi and double stained with propidium iodide and fluorescently labeled antibodies against the indicated MCMV immediate-early proteins. Cells were analyzed by flow cytometry for cellular DNA content and viral gene expression. Data are depicted as dot plots and histogram overlays.
FIG. 5.
FIG. 5.
Conservation of MCMV cell cycle characteristics in human fibroblasts. (A) Asynchronously proliferating HEL fibroblasts and 3T3 cells were infected with MCMV or HCMV as indicated. Cells were harvested at 4 hpi and analyzed for DNA content and viral immediate-early gene expression as detailed in the legend to Fig. 4. (B and C) HEL fibroblasts were serum starved for 3 days and then restimulated by serum readdition (0 h). Restimulated cells were infected by HCMV or MCMV or left noninfected. Infections were carried out at the beginning of serum stimulation or at 18 h after restimulation, when cells were just entering S phase. Cells were harvested at 2-h intervals between 20 and 36 h and analyzed for cell cycle distribution by flow cytometry.
FIG. 6.
FIG. 6.
De novo expression of viral genes is needed for the MCMV-mediated G1 and G2 cell cycle arrest. (A and B) Purified MCMV particles were irradiated with increasing doses of UV light and used to infect NIH 3T3 cells. Cells were harvested at 24 hpi to analyze the number of MCMV ie1-expressing cells by flow cytometry (A) or at 1 hpi to measure the extent of virus-induced NF-κB activation by electrophoretic mobility shift assay (B). (C and D) NIH 3T3 cells were released from the density arrest and infected in G1 phase (4 h after replating) (C) or early S phase (15 h after replating) (D) as indicated. MCMVUV, MCMV irradiated with 1,000 J/m2 UV-C light. Cell cycle progression was monitored at constant intervals by flow cytometry analysis of cellular DNA content as indicated.
FIG. 7.
FIG. 7.
MCMV ie3 has cell cycle arrest activity. HA-tagged versions of the indicated CMV genes or an empty vector control were transfected together with the KK surface marker (Miltenyi Biotec) into proliferating NIH 3T3 cells. (A) Cells were controlled for expression of the transfected genes by immunoblot analysis. An anti-HA antibody was used for detection of the viral proteins and an anti-GAPDH antibody to control for equal loading. (B) The cells were further analyzed for cell cycle distribution of the KK positive fraction by flow cytometry. To assay for G1 arrest activity, cycling cells were blocked in prometaphase by nocodazole treatment (+ noco, lower panel). Cell cycle distributions are given as percent G1/percent S/percent G2 + M.
FIG. 8.
FIG. 8.
ie3 is essential for the MCMV-mediated cell cycle arrest. NIH 3T3 cells were released from density arrest and infected in G1 phase (4 h after replating) (A) or early to mid-S phase (16 h after replating) (B) with the indicated MCMV recombinants (MCMV, parental virus; MCMVdie1, ie1 deletion mutant; MCMVdie3, ie3 deletion mutant; MCMVrev, ie3 revertant virus). Cell cycle progression was monitored at constant intervals by flow cytometry of cellular DNA content as indicated.
FIG. 9.
FIG. 9.
Comparative model of CMV-mediated cell cycle arrest functions. This model summarizes our current knowledge of the interdependencies between CMV immediate-early gene expression and the cell cycle position of the host cell. Both HCMV and MCMV can infect cells throughout the cell cycle. The expression of the cell cycle regulator IE2 is restricted to G0/G1 cells during HCMV infection, which causes these cells to run into the well-described cell cycle block at the G1/S transition. In contrast, the cell cycle-independent expression of ie3 can result in early (G1/S) and late (G2) cell cycle blocks. The exact position of the early ie3-mediated cell cycle block is unknown, but in analogy to IE2 it appears likely that it occurs at a similar position as in HCMV-infected cells. This model implies that HCMV might also be able to block cells at a later time point of the cell cycle if immediate-early genes were expressed in those cell cycle phases.
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