Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: insights on viral regulation of apoptosis and proliferation

Abstract

We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.

Fig. 1

Time course study of S2-induced apoptosis in Vero E6 cells. (a) Morphology of Vero E6 cells under light microscope (40×) after rAd-Ctrl or rAd-S2 transductions from 0 to 96 h p.t. in 12- to 24-h intervals. Representative figures of three independent experiments are shown. (b) Cell viability along the time course is estimated quantitatively with Trypan blue exclusion assay. (c) The percentage of rAd-Ctrl or rAd-S2-transduced cells showing chromatin condensation was counted under fluorescence microscope after Hoechst 33342 staining. For both panels b and c, average of three independent experiments is shown with standard error of the mean (SEM). (d) Agarose gel electrophoresis showing the characteristic DNA laddering pattern resulting from internucleosomal DNA cleavage in rAd-S2-transduced cells in four selected time points. Three micrograms of low molecular weight DNA was loaded into each well. The ladders with 200 bp increments are indicated with arrowheads. The result is the representative of three independent experiments. ^⁎p < 0.01 when compared with cells transduced by rAd-Ctrl under the same condition.

Fig. 2

Functional classification of differentially expressed probe sets. Probe sets that were differentially expressed by more than two-folds were classified into 11 groups manually, with 9 groups of known functions and two groups representing hypothetical/miscellaneous proteins and unknown proteins. The number of probe sets with known functions showing up- and down-regulations is shown with empty and filled bars respectively. The first group represents the probe sets related to apoptosis while probe sets regulating cell cycle and proliferation are grouped into group 7.

Fig. 3

Bcl-xL blocks S2-induced apoptosis in Vero E6 cells. Different dosage of rAd-Bcl-xL was co-infected with 50 MOI of rAd-S, -S1, -S2 or rAd-Ctrl and the effect on cell death and apoptosis induced by the rAds were assayed through (a) Trypan blue exclusion assay and (b) Hoechst 33342 staining at day 5 and day 3 p.t. respectively. Both figures indicated the average of three independent experiments with SEM.

Fig. 4

S2-mediated inhibition of cell proliferation in Vero E6 cells. Vero E6 cells were transduced with the indicated rAds and their proliferations were estimated with MTT cell proliferation assay and are expressed as percentage of proliferation of mock-infected cells. Time- and dose-dependent anti-proliferation effect of S2 was shown in panels a and b, respectively. The time-dependent effect was performed with rAd of 25 MOI and cells in the dose-dependent experiment were collected at 3 days p.t. Results shown are the average of three independent experiments with SEM. ^⁎p < 0.01 when compared with cells transduced by rAd-Ctrl under the same condition.