Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway

Abstract

Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these "accessory" viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.

FIG. 1.

Expression of the viral proteins in 293T cells and the effects on apoptosis. (A) The CaspACE fluorometric assay system from Promega Corporation was used to measure the activation of caspase-3 protease activity, which is a hallmark of apoptosis, in cells that were transfected with a positive control (HA-BAX; column 1), a negative control (HA-GST; column 2), and the different SARS-CoV proteins (columns 3 to 7). All experiments were performed in duplicate; the average values with standard deviations are plotted. For cell viability assays, experiments were performed in triplicate, and the average percentages (± standard deviations) of live cells, compared to that for HA-GST, which is normalized to 100%, are shown in parentheses above each column. (B) Western blot analysis to determine the cleavage of endogenous full-length PARP, which is a substrate of activated caspase-3, from 116 to 83 kDa (upper panel). Expression levels of the HA-tagged proteins were determined with anti-HA antibody (middle panel), and the amounts of total cell lysates loaded were verified by measuring the level of endogenous actin (lower panel). (C) The CaspACE fluorometric assay system from Promega Corporation was used to measure the activation of caspase-3 in cells that were transfected with 7a-HA, 7a, or HA-BAX in the presence of a pan-caspase inhibitor, zVAD-fmk (columns 2 to 4) or an irrelevant peptide, zFA-fmk (columns 5 to 7). (D) Western blot analysis were performed to determine the cleavage of endogenous PARP (upper panel), expression levels of HA-GST, 7a-HA, 7a, and HA-BAX (anti-7a or anti-HA; middle panels), and endogenous actin as a loading control (antiactin; lower panel). Since the anti-7a antibody was obtained by using a GST-fusion protein, it recognizes both the 7a and GST proteins.

FIG. 2.

Induction of apoptosis by 7a in cell lines derived from different organs. The cell lines used were HeLa (cervical carcinoma) (lanes 1 to 3), HepG2 (liver carcinoma) (lanes 4 to 6), A549 (lung carcinoma) (lanes 7 to 9), and COS-7 and Vero E6 (kidney) (lanes 10 to 12 and 13 to 15, respectively). All cell lines were purchased from the American Type Culture Collection (Manassas, Va.). The CaspACE fluorometric assay system from Promega Corporation was used to measure the activation of caspase-3 protease in different cell lines that were transfected with HA-GST, 7a-HA, and HA-BAX (first panel). Western blot analysis were performed to determine the cleavage of endogenous PARP (second panel), expression levels of HA-GST, 7a-HA, and HA-BAX (anti-HA) (third panel), and endogenous actin as a loading control (antiactin) (fourth panel).

FIG. 3.

Induction of apoptosis by overexpression of wild-type 7a and 7a mutants (7a-L and mat7a) in A549 and Vero E6 cells and by SARS-CoV infection of Vero E6 cells. (A) The CaspACE fluorometric assay system from Promega Corporation was used to measure the activation of caspase-3 protease in different cell lines that were transfected with HA-GST (negative control) (lanes 1 and 5), wild-type 7a (lanes 2 and 6), mutant 7a-L (lanes 3 and 7), and mutant mat7a (lanes 4 and 8) (first panel). 7a-L contains mutations at the signal peptide cleavage site and is cleaved less efficiently than the wild type, and mat7a does not contain the signal peptide (see reference 11). Western blot analyses were performed to determine the expression levels of the GST and 7a proteins (anti-7a; second panel) and endogenous actin as a loading control (antiactin; third panel). (B) Caspase-3 protease activities in Vero E6 cells transfected with 1.0 μg (lane 1) or 2.0 μg (lane 2) of 7a plasmid or mock-infected cells (lane 3) or SARS-CoV-infected cells (lane 4) were determined (first panel). Western blot analyses were performed to determine the expression levels of 7a (anti-7a; second panel), SARS-CoV N (anti-N [12a]; third panel), and endogenous tubulin as a loading control (monoclonal antitubulin [Sigma]; fourth panel) in 20 μg of cell lysates.