Adenovirus replaces mitotic checkpoint controls

Abstract

Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant.

Importance: Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state.

FIG 1

Click-iT EdU labeling protocol and DNA profile analysis of EdU-labeled cells. (A) HeLa cells were labeled with Click-iT EdU for six 4-h periods (indicated by arrows) over the course of 24 h prior to infection. At time zero h, EdU-labeled cultures were infected with the E1B-55K/E4orf3 double mutant virus at a multiplicity of infection (MOI) of 10 and processed for immunofluorescence at 72 hpi or were collected for flow cytometric analysis. Shading indicates the phase of the cell cycle the EdU-labeled cells are expected to have reached at infection (S phase, gray; G₂/M, black; G₁, white). (B) Samples of cells labeled with Click-iT EdU were collected at the time of infection (0 h) and stained with propidium iodide for DNA content and for EdU as described in Materials and Methods. The shaded density profile represents the DNA profile for the entire population of cells. The unshaded density profile shows the DNA profile of EdU-positive cells.

FIG 2

Early G₁ cells give rise to mitotic-like nuclei after infection with the E1B-55K/E4orf3 double mutant virus. Synchronously dividing HeLa cells were obtained by mitotic shake and hydroxyurea selection as described in Materials and Methods. Synchronously dividing cells were either harvested for DNA profile analysis or infected at the indicated time after entering S phase with the E1B-55K/E4orf3 double mutant virus at an MOI of 10. (A) Cell cycle distribution by DNA profile analysis. (B) At 72 hpi, cells were stained for DNA with DAPI and evaluated by fluorescence microscopy to determine the frequency of cells with condensed DNA. The stage of the cell cycle at the time of infection is indicated below. A representative experiment is shown of three that were performed with overlapping times of infection after entering S phase.

FIG 3

HeLa cells infected with the E1B-55K/E4orf3 double mutant virus show evidence of mitotic distress. HeLa cells were mock infected or infected at an MOI of 10 with the indicated viruses and stained at 72 h postinfection with DAPI to visualize DNA (blue), antibodies to phospho-H3 as a marker of early mitosis (red), and antibodies to β-tubulin to visualize mitotic spindles (green). (A) Representative images of mock (a and b), wild-type (c), and E1B-55K/E4orf3 mutant (d and e) viral infections are shown. (B) The frequency of cells containing mitotic-like condensed DNA, staining for phospho-H3 and asymmetrically stained mitotic spindles, was quantified for at least 500 cells for each viral infection. A representative experiment of three is shown. Error bars indicate the 95% exact binomial confidence interval for the representative experiment. (C) HeLa cells were infected with the E1B 55K/E4orf3 double mutant virus at an MOI of 10. Cells were stained for DNA at the indicated times postinfection, and the frequency of mitotic-like cells was determined for approximately 500 cells at each time point. The symbols with the dashed line for times beyond 72 hpi indicate imprecision due to the probable loss of cells because of death or detachment. The experiment shown is representative of three independent experiments with similar outcomes. Error bars indicate the 95% exact binomial confidence interval for the representative experiment.

FIG 4

Colcemid traps cells infected with the E1B-55K mutant virus in a mitotic-like state. HeLa cells were infected at an MOI of 10 with the indicated viruses and treated with colcemid for 12 h at the indicated times postinfection before being fixed and stained with DAPI to visualize DNA. The frequency of mitotic-like cells was determined for approximately 500 cells for each virus at each time point.

FIG 5

The ability to inhibit entry into a mitotic-like state maps to the p53-binding ability of the E1B-55K protein. (A) HeLa cells were infected at an MOI of 10 with the indicated E1B-55K-null viruses and treated with colcemid or vehicle control 12 h prior to staining with DAPI at 72 hpi to visualize DNA. The frequency of mitotic-like cells was determined by fluorescence microscopy. A representative experiment of three independent experiments is shown. For each of the E1B-55K-null viruses, colcemid significantly increased the fraction of mitotic-like cells (P < 10⁻⁶ by Fisher's exact test). Error bars indicate the 95% exact binomial confidence interval for the representative experiment. (B) HeLa cells were infected at an MOI of 10 with the indicated viruses bearing point mutations in the E1B-55K gene (described in Table 2) and treated with colcemid 12 h prior to staining at 72 hpi with DAPI to visualize DNA. The frequency of mitotic-like cells is shown for each mutant infection. The proportion of mitotic-like nuclei was nonrandomly distributed among the 11 virus-infected samples exclusive of the E1B-55K-null virus H5pm4149 (P < 0.0001, chi-square test). The chi-square test was repeated after systematically excluding each sample. The P value was nonsignificant (P = 0.56) only when the virus H5pm4109 was excluded from the analysis. This analysis was again repeated by excluding individual samples in the collection that also excluded H5pm4109. The chi-square test reported nonsignificant P values for each subset lacking H5pm4109 and one other sample. This indicates that among the non-null viruses, only H5pm4109-infected cells exhibited a significant change in the number of condensed nuclei in the presence of colcemid. Results from a representative experiment of three independent experiments with similar outcomes are shown. (C) p53-null H1299 cells were transfected with a plasmid to express p53 24 h before being mock infected or infected at an MOI of 10 with either the E1B-55K single mutant or E1B-55K/E4orf3 double mutant virus. The cells were either left untreated or were treated with 0.2 μg/ml colcemid 12 h prior to immunostaining for p53 and visualization of DNA with DAPI staining at 72 hpi. Cells were classified as either p53 positive or negative, and the frequency of mitotic-like cells was determined. As noted in the text, condensed nuclei in mock-infected cells resembled pyknotic nuclei of apoptotic cells. The number of p53-positive virus-infected cells with condensed DNA was increased significantly over the number of p53-negative cells with condensed DNA (P = 2 × 10⁻⁶ by Fisher's exact test).

FIG 6

Cyclin B1 levels are elevated during adenoviral infections. HeLa cells were mock infected or infected with the indicated viruses at an MOI of 10. Cellular lysates were collected in the presence of protease and phosphatase inhibitors at 72 hpi. Material from identical numbers of infected cells was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for cyclin B1 (A) and β-actin (B). An overexposed β-actin blot is presented here to permit visualization of the weaker signals. Nonsaturated exposures were used for quantitative analyses. The position of a cyclin B1-related product of 35 kDa is indicated by the arrowhead. (C) The optical density of the signal for the intact cyclin B1 products was quantified, normalized to β-actin, and then normalized to the value measured from mock-infected cells in three independent experiments. The mean and standard deviation are plotted on a log scale. Application of the t test to log-transformed values shows that levels of cyclin B1 in were significantly greater than in mock-infected cells in dl309- and dl1520-infected cells (P < 0.005). Differences were not significant in dl341- or 3112-infected cells (P = 0.97 and 0.18, respectively).

FIG 7

E4orf3 alters the distribution of cyclin B1 during adenoviral infections. HeLa cells were mock infected or infected with the indicated viruses at an MOI of 10 and stained for DNA (blue) and cyclin B1 (green) at 72 hpi. (A) Representative fields show asynchronously dividing mock-infected cells, with white arrowheads indicating a G₂-phase cell (a), open arrowheads indicate an early mitotic cell (b), and double arrowheads indicating a late mitotic cell. The white bar in panel c indicates 20 nm. (B) Representative cells show patterns of cyclin B1 staining for HeLa cells infected with wild-type (a and b), E1B-55K mutant (c and d), E4orf3 mutant (e and f), and E1B-55K/E4orf3 double mutant (g and h) viruses. (C) The relative frequencies of the three patterns (aggregates, diffuse or speckles, and absent) of cyclin B1 localization are tabulated for a representative experiment. Similar results were obtained in three independent experiments in which approximately 500 cells were evaluated for each virus.

FIG 8

E4orf3 overcomes the sustained chromatin condensation elicited by expression of the degradation-resistant R42A cyclin B1 variant. (A) The wild-type cyclin B1 fused to the yellow fluorescent protein Venus (CycB1 wt) or the degradation-resistant form of cyclin B1 fused to Venus (CycB1 R42A) was expressed by transfection in HeLa cells together with E4orf3, E4orf6/7, or an empty vector control. Cells were processed 48 h after transfection and stained for the E4 protein. The cyclin B1 protein was visualized by Venus fluorescence. DNA was visualized by DAPI staining. Chromatin was scored as condensed (mitotic like) or diffuse (interphase like) in approximately 50 cells expressing each combination of the cyclin B1 and E4 constructs in each of three independent experiments. Data are reported as the mean and standard deviation. The effect of the E4 construct (or absence of the construct) on the fraction of cells containing condensed DNA was evaluated with a pairwise t test allowing for different variances among the three groups. The P value was adjusted for multiple comparisons by the Holm-Bonferroni method. Adjusted P values below 0.05 were considered significant. (B) Three representative cells showing that the E4orf6/7 protein does not affect chromatin condensation promoted by CycB1 R42A. The scale bar in panel a indicates 10 nm. (C) Chromatin remains diffuse in three representative cells expressing the R42A cyclin B1 variant and E4orf3. The cyclin B1 fusion protein was excluded from the nucleus in most (>80%) cells expressing both fusion protein and E4orf3, as indicated in panels a and b.