A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (CoV) spread from China to more than 30 countries, causing severe outbreaks of atypical pneumonia and over 800 deaths worldwide. CoV primarily infects the upper respiratory and gastrointestinal tract; however, SARS-CoV has a unique pathogenesis because it infects both the upper and lower respiratory tracts and leads to human respiratory diseases. SARS-CoV genome has shown containing 14 open reading frames (ORFs) and 8 of them encode novel proteins. Previous reports show that overexpression of ORF-3a, ORF-3b and ORF-7a induce apoptosis. In this report, we demonstrate that overexpression of ORF-6 also induces apoptosis and that Caspase-3 inhibitor and JNK inhibitor block ORF-6 induced apoptosis. Importantly, the protein level of ER chaperon protein, GRP94, was up-regulated when ORF-6 was overexpressed. All these data suggest that ORF-6 induces apoptosis via Caspase-3 mediated, ER stress and JNK-dependent pathways.

Fig. 1

Overexpression of ORF-6-induced apoptosis in different cell lines. (A), COS-7 and (B) Vero E6 cells were transiently transfected with GFP and HA-Bax, GFP-N1, GFP-ORF-6 and GFP-ORF-7a for 24 h. The nuclei of the cells were stained by Hoechst for 15 min. The number of healthy cells was counted under fluorescence microscopy with no DNA condensation and fragmentation. The percentage of apoptotic cells was calculated by the number of healthy cells over the total number of transfected cells. Experiments were repeated three times. Standard deviations are shown.

Fig. 2

Overexpression of ORF-6 induced apoptosis is Caspase-3 and JNK-dependent. (A) Caspase-3 inhibitor (z-DEVD) blocks ORF-6-induced apoptosis. Vero E6 cells were incubated with either Dimethyl Sulfoxide (DMSO) or 50 μM z-DEVD-fmk for 30 min before they were transiently transfected with 3 μg of GFP-Cb5, GFP-ORF-6 and GFP-ORF-7a for 24 h. The nuclei of the cells were stained by Hoechst for 15 min. The number of healthy cells was counted under fluorescence microscopy with no DNA condensation and fragmentation. The percentage of apoptotic cells was calculated by the number of healthy cells over the total number of transfected cells. Experiments were repeated three times and the standard deviations are shown. (B) Overexpression of ORF-6- and ORF-7a induces Caspase-3 activation. Vero E6 cells were transiently transfected with GFP-ORF-6 and GFP-ORF-7a for 24 h. Cell lysates were normalized to 2 μg/ μl by lysis buffer and subjected to Western Blot with α-GFP, α-Caspase-3 and α-Actin. For positive controls, cells were treated with 1 μM of Staurosporine (STS) for 8 h. Cells without any treatment were served as negative control. (C) JNK inhibitor blocked ORF-6 induced apoptosis. Vero E6 cells were incubated with either Dimethyl Sulfoxide (DMSO) or 40 μM JNK inhibitor for 30 min before they were transiently transfected with 3 μg of GFP-Cb5, GFP-ORF-6 and GFP-ORF-7a for 24 h. Cell counts were done as mentioned in B.

Fig. 3

ORF-6 and ORF-7a induces ER stress. (A) Overexpression of ORF-6 and ORF-7a increase endogenous GRP94 protein level. Vero E6 cells were transiently transfected with 6 μg of GFP-ORF-6 and GFP-ORF-7a for 24 h. Cell lysates were normalized to 1 μg/μl by lysis buffer and subjected to Western blot with α-GFP, α-GRP94 and α-Actin. For positive controls, cells were treated with 1 μM of Thapsigargin for 24 h. Cells without treatment served as negative control. (B, C), ORF-6 and ORF-7a increase endogenous GRP94 protein level in a dose dependent manner. Vero E6 cells were transiently transfected with 2 μg, 6 μg and 12 μg of GFP-ORF-6 or GFP-ORF-7a for 24 h. Cell lysates were normalized to 1 μg/μl by lysis buffer and subjected to Western blot with α-GFP, α-GRP94 and α-Actin. For positive controls, cells were treated with 1 μM of Thapsigargin for 24 h. Cells without treatment served as negative control.