G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells

Abstract

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), cause of the life-threatening atypical pneumonia, infects many organs, such as lung, liver and immune organ, and induces parenchyma cells apoptosis and necrosis. The genome of SARS-CoV, not closely related to any of the previously characterized coronavirus, encodes replicase and four major structural proteins and a number of non-structural proteins. Published studies suggest that some non-structural proteins may play important roles in the replication, virulence and pathogenesis of viruses. Among the potential SARS-CoV non-structural proteins, 3b protein (ORF4) is predicted encoding 154 amino acids, lacking significant similarities to any known proteins. Till now, there is no report about the function of 3b protein. In this study, 3b gene was linked with the EGFP tag at the C- terminus. Through cell cycle analysis, it was found that over-expression of 3b-EGFP protein in Vero, 293 and COS-7 cells could induce cell cycle arrest at G0/G1 phase, and that especially in COS-7 cells, expression of 3b-EGFP was able to induce the increase of sub-G1 phase from 24 h after transfection, which was most obvious at 48 h. The apoptosis induction of 3b fusion protein in COS-7 cells was further confirmed by double cell labeling with 7-AAD and Annexin V, the function of 3b protein inducing cell G0/G1 arrest and apoptosis may provide a new insight for further study on the mechanism of SARS pathogenesis.

Figure 1

Induction of cell cycle arrest and cell apoptosis by 3b protein expression. p3b/EGFP-N1 plasmid was transfected into COS-7 cells, and the DNA contents of cells were measured by flow cytometry. EGFP expression positive and negative cells were gated with forward scatter (on the left row). The middle and right rows were the assay of cell cycle in p3b/EGFP-N1 negative and positive cells. In p3b/EGFP-N1 positive cells, sub-G1 phase was changed from 11.80% to 53.50%, 48.36% at 24 h or 36 h, 48 h separately. The proportion was decreased to 23.34 and 24.85% at 60 and 72 h respectively. However, in EGFP negative cells, the changes of sub-G1 phase were not obvious.

Figure 2

Histogram of cell cycle arrest and cell apoptosis. Histogram showing the percentages of cells at various phases of cell cycle. p3b/EGFP-N1 positive cells were showed with grey columns, and p3b/EGFP-N1 negative cells were showed with crisscross. Data were means of three independent experiment ± s.d. (bars).

Figure 3

Apoptosis assay of COS-7 cells transfected with p3b/EGFP-N1. COS-7 cells were transfected with pEGFP-N1 and p3b/EGFP-N1 respectively. At 48 h after transfection, cells were collected and resuspended in binding buffer containing Annexin V-PE and 7-AAD, and then processed for flow cytometry analysis. On the left row, EGFP positive cells were gated. The middle and right rows were the results of EGFP negative and positive cells analyzed with Annexin V-PE and 7-AAD staining. In each box, the upper left corner included damaged cells, the lower left corner included viable cells, which were negative for 7-AAD and Annexin V-PE binding, the upper right corner included necrotic or late apoptotic cells, which were positive for Annexin V-PE staining and for 7-AAD uptake, while the lower right corner included apoptotic cells, which were Annexin V-PE positive but impermeable to 7-AAD. In p3b/EGFP-N1 transfected cells (a), the percentage of apoptosis cells in EGFP positive cells increased significantly, compared with negative ones, while there were no changes between positive and negative cells transfected with pEGFP-N1 (b). One of three experiments with similar results was shown.