gms | German Medical Science

Background. Apoptosis represents an important antiviral defence mechanism, and viruses have evolved strategies to counteract and regulate apoptosis in order to maximize the production of virus progeny and its spread to neighboring cells. Replication of SARS-associated coronavirus (SARS-CoV) in vitro causes extensive cytopathic effect (c.p.e.) and rapid destruction of cell monolayer; in addition, apoptotic cell death has been hypothesized to play a pathogenetic role in the disease. In this study, the role of apoptosis in the appearance of c.p.e. during SARS-CoV infection was analyzed, by using as host cell system Vero cells either wild type or transfected with the antiapoptotic proto-oncogene bcl-2.

Methods. Vero cells, transfected with either a bcl-2 or control plasmid, were infected at either high (10) or low (0.01) m.o.i., to run the analysis under either single or multiple replication cycle conditions, respectively. Infection parameters were determined at various time points by c.p.e. inspection, viral RNA measurement, back titration of both cell-associated and shed viral infectivity. Viable cells were counted by dye exclusion; apoptotic cell death was assessed by measuring cellular DNA content. Transmission electron microscopy (TEM) was used to monitor viral particle formation and ultrastructural cell modifications.

Results. At high m.o.i., virus replication cycle was completed within 8 hours from infection. Most of progeny virus, measured as both infectivity and viral RNA, remained cell associated. Virus yield remained at plateau for the subseguent 48 hours p.i., and after this time the proportion of cell-associated and released virus was reversed, coincident with massive cell death. Surprisingly, replication kinetics, and virus yield were similar in bcl-2-transfected as compared to control Vero cells. Extensive cytopathic effect was observed in control plasmid-transfected cells, starting from 24 hours p.i., with both cell necrosis and apoptosis contributing to its appearance. A slight delay of c.p.e. appearance was observed in bcl-2-expressing cells, along with a significant reduction of both apoptosis and cell necrosis. Ultrastructural analysis revealed that SARS-CoV replication is associated to a range of cytoplasmic organelle membrane modifications, and confirmed that c.p.e. resulted from both apoptotic and necrotic cell death. Expression of bcl-2 did not alter the pattern of ultrastructural changes, but resulted in a delay of its appearance.

The addition of the caspase inhibitor z-VAD-FMK showed only a partial inhibition of cell death, and did not affect virus replication kinetics.

Discussion. These results show that replication cycle of SARS-CoV requires about 8 hours to be completed, and that both apoptosis and necrosis may contribute to the appearance of c.p.e. and of the death of infected cells in vitro. However, bcl-2 expression, although leading to significant protection from both apoptosis and necrosis, did not alter the overall kinetics of virus replication, and only delayed the appearance of ultrastructural modifications associated with SARS-CoV-induced c.p.e.