Inhibition of human cytomegalovirus immediate-early gene expression by cyclin A2-dependent kinase activity

Abstract

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

Fig 1

Cyclin A2 overexpression is sufficient to block the onset of IE gene expression in HCMV-infected cells. (A) Schematic overview of lentivirus-expressed cyclin A2 mutants and the endogenous wild-type form. The D box of cyclin A2 was destroyed by the double point mutation R47A/L50V (ΔD). An arginine residue essential for CDK binding was eliminated by the R211A mutation. HA, hemagglutinin tag. (B to G) HEK293 (B to D) and U373 (E to G) cells were transduced with expression vectors for the indicated cyclin A2 mutants or with an empty expression vector (vector control) or were left untransduced. (B and E) The efficiency of transduction was controlled by flow cytometry of the vector-encoded enhanced green fluorescent protein (EGFP) marker. (C and F) The relative levels of exogenous and endogenous cyclin A2 protein expression were determined by immunoblot analysis. (D and G) Cells were infected with HCMV and analyzed at 5 h postinfection by flow cytometry for IE1/IE2 expression and DNA content. Shown are dot plots (n = haploid number of chromosomes) in which cells were divided into four subpopulations: IE1/2-negative G₀/G₁ cells (lower left quadrant), IE1/2-positive G₀/G₁ cells (upper left quadrant), IE1/2-positive S/G₂/M cells (upper right quadrant), and IE1/2-negative S/G₂/M cells (lower right quadrant). The proportion of each subpopulation is indicated as a percentage of the total cells.

Fig 2

Nuclear entry of viral genomes is unaffected by cyclin A2. Cyclin A2-overexpressing cells and the indicated control U373 cells were grown to confluence and infected with BrdU-labeled HCMV. (A) At 3 h postinfection, cells were fixed and analyzed for IE1/IE2 protein expression and subcellular localization of viral genomes by confocal immunofluorescence microscopy. DAPI was used as a nuclear counterstain. (B and C) In a parallel approach, cells were harvested at 3 h postinfection and fractionated into nuclei (nu) and cytoplasm (cy) by hypotonic lysis. (B) The purity of fractions was confirmed and compared to that of whole-cell lysates (wh) by immunoblot analysis of β-tubulin (cytoplasmic marker) and lamin A/C (nuclear marker). In addition, the nucleocytoplasmic distribution of exogenous cyclin A2 was controlled using HA and cyclin A2 antibodies. (C) Total DNA isolated from whole cells and from purified nuclei was analyzed for the relative amount of viral DNA by quantitative real-time PCR using UL112/113-specific primers. The ratios of nuclear and whole-cell viral DNA contents are given as mean values of two independent experiments.

Fig 3

Cyclin B1 overexpression does not interfere with IE1/IE2 gene expression. U373 cells were stably transduced with lentiviral expression vectors for HA-tagged wt forms of cyclin A2 and cyclin B1, as indicated. (A) After puromycin selection, the expression of cotransduced EGFP was controlled by flow cytometry and compared to that of nontransduced cells. (B) Protein levels of exogenous HA-tagged cyclins and the corresponding endogenous cyclins were compared by immunoblot analysis. (C) Five hours after HCMV infection, cells were analyzed for IE1/IE2 protein expression and DNA content as described in the legends to Fig. 1D and G.

Fig 4

MCMV is insensitive to cyclin A2 overexpression and induces cyclin A2-associated kinase activity after infection of quiescent cells. (A) Cyclin A2ΔD and cyclin A2ΔD(R211A)-expressing U373 derivatives (see the legend to Fig. 1), as well as control cells, were infected with MCMV. Five hours postinfection, the cells were harvested and analyzed for IE1 protein expression and DNA content by flow cytometry. (B, C, and D) Mouse 3T3 fibroblasts were lentivirus transduced with expression vectors for human and mouse cyclin A2. (B) Flow cytometric analysis of EGFP marker expression was performed to document homogeneously transduced cell pools. (C) The expression of exogenous cyclin A2 protein was checked by HA immunoblot analysis. A nonspecific band is indicated by an asterisk. (D) Cyclin A2-overexpressing and control 3T3 fibroblasts were infected with MCMV and analyzed at 5 h postinfection for IE1 protein expression and cellular DNA content by flow cytometry. (E) Fibroblasts of human (HEL) and mouse (3T3) origin were made quiescent by 48 h of serum starvation. Then (at 0 h), the cells were either restimulated by 10% fetal calf serum containing medium (+ serum), CMV infected in the presence of medium without serum (+ HCMV/MCMV), or both restimulated and infected by adding virus and serum containing fresh medium simultaneously (+ HCMV/MCMV, + serum). Cells were harvested at regular intervals and prepared for analysis of their endogenous cyclin A2-, B1-, and E1-associated kinase activities. Shorter intervals were chosen for the murine system, because both the 3T3 cell division and the MCMV replication cycle proceeded faster than for their human counterparts. Cyclin-CDK complexes were isolated from cell extracts by immunoprecipitation (IP) using antibodies that are proven to specifically bind the indicated cyclins without destroying the associated kinase activity. To measure this kinase activity, in vitro kinase assays were performed using histone H1 as a substrate. Shown are autoradiographic images of histone H1 phosphorylation. The results are representative of more than three independent experiments.

Fig 5

Cyclin A2-CDK activity has a long-lasting negative effect on the early and late phases of HCMV infection. U373 cells were lentivirus transduced as indicated and enriched in G₁ phase by density arrest before HCMV infection. (A) The cell cycle distributions at the time of infection and at the indicated time points postinfection were analyzed by flow cytometry. Shown are DNA histograms of propidium iodide-stained cells (n is the haploid number of chromosomes). (B) After infection, cells were harvested at regular intervals and analyzed for expression of immediate-early (IE1/2), early (gB), and late (pp28) proteins by flow cytometry. (C) In parallel, virus growth curves were obtained by determining the number of infectious particles in the cell culture supernatants. (B and C) Means and standard deviations of three independent experiments (until 96 h p.i.), all of them using an MOI of approximately 10. From 120 to 240 h p.i., the values represent averages from two of these experiments. (D) The experiment was repeated using an MOI of approximately 0.1. Shown are growth kinetics, where the data points show the mean values of biological triplicates and the error bars indicate standard deviations.

Fig 6

Cyclin A2 has a stronger effect on IE2 than on IE1 expression. The indicated U373 derivatives were grown to confluence and infected with HCMV using an MOI of approximately 10. (A) The expression of selected viral gene products and of exogenous, HA-tagged cyclin A2 was monitored by immunoblot analysis from 5 to 96 h p.i. (B) IE1 and IE2 protein expression was analyzed in more detail by using exon 4 (IE1 [top])-, exon 5 (IE2 [second panel from top])-, and exon 3 (IE1 and IE2 [third panel from top])-specific antibodies. The relative molecular masses of the different splice variants are indicated. As the 55-kDa form of IE2 lacks the exon 3-encoded N terminus of IE1 and IE2 full-length proteins, it appears only in the exon 5-specific blot. (A and B) Equal protein content of samples was controlled by Coomassie staining (bottom gels). Shown are representative results from three independent experiments.

Fig 7

Sustained inhibition of IE2 mRNA accumulation by cyclin A2-CDK. U373 parental cells and the indicated lentivirus-transduced derivatives were density arrested and HCMV infected. At the indicated times postinfection, total RNA was prepared from infected cells and analyzed for IE1, IE2, and UL112/113 mRNA expression by quantitative real-time PCR. The results are shown as fold change of mRNA expression compared to the values of nontransduced cells at 5 h p.i. Presented are the means and standard deviations of technical triplicates. Where no standard deviation is indicated, the means of technical duplicates are shown.

Fig 8

Reduced IE2 mRNA levels in cyclin A2-overexpressing cells are not due to decreased IE2 mRNA stability. The indicated stably transduced U373 derivatives were first infected with HCMV and then, at 72 h p.i., treated with 5 μg/ml actinomycin D or dimethyl sulfoxide (DMSO) as a solvent control. At the beginning of actinomycin D treatment (0 h) and at the indicated intervals thereafter, cells were harvested and analyzed for IE1 and IE2 mRNA content by quantitative real-time PCR. (A) Shown are the relative IE1 (left) and IE2 (right) mRNA expression levels at 0 h posttreatment (72 h p.i.). The values of empty-vector-transduced cells (vector control) were used as a reference and set to 1. (B) Profiles of IE1 (left) and IE2 (right) mRNA expression over the actinomycin D/DMSO treatment period of 24 h. The results are shown as fold change of the corresponding initial values of each cell line at 0 h (72 h p.i.). The means and standard deviations of technical triplicates are presented. Where no standard deviation is indicated, only means of technical duplicates are shown. The data are representative of two independent experiments.

Fig 9

Suppression of UL37 splicing by cyclin A2-CDK. Samples already tested for IE1/IE2 mRNA content (see the legend to Fig. 5) were analyzed for relative mRNA expression levels of TRS1; unspliced UL37x1; spliced UL37 (using an exon 1- and 2-specific primer pair); and unspliced, single-spliced (SS), and double-spliced (DS) US3 and UL111A, as indicated. The results are depicted as fold change of mRNA expression compared to the values of nontransduced cells at 5 h p.i. Shown are the means and standard deviations of technical triplicates. Where no standard deviation is indicated, means of technical duplicates are shown. The data are representative of two independent experiments.