Phosphorylation of p38 MAPK and its downstream targets in SARS coronavirus-infected cells

Abstract

Severe acute respiratory syndrome (SARS) has become a global public health emergency. Understanding the molecular mechanisms of SARS-induced cytopathic effects (CPEs) is a rational approach for the prevention of SARS, and an understanding of the cellular stress responses induced by viral infection is important for understanding the CPEs. Polyclonal antibodies, which recognized nucleocapsid (N) and membrane (M) proteins, detected viral N and M proteins in virus-infected Vero E6 cells at least 6 and 12 h post-infection (h.p.i.), respectively. Furthermore, detection of DNA ladder and cleaved caspase-3 in the virus-infected cells at 24h.p.i. indicated that SARS-CoV infection induced apoptotic cell death. Phosphorylation of p38 MAPK was significantly up-regulated at 18 h.p.i. in SARS-CoV-infected cells. The downstream targets of p38 MAPK, MAPKAPK-2, HSP-27, CREB, and eIF4E were phosphorylated in virus-infected cells. The p38 MAPK inhibitor, SB203580, inhibited effectively phosphorylation of HSP-27, CREB, and eIF4E in SARS-CoV-infected cells. However, viral protein synthesis was not affected by treatment of SB203580.

Fig. 1

Detection of SARS-CoV-specific proteins and activation of caspase-3 in virus-infected cells. Vero E6 cells were infected with SARS-CoV at an m.o.i. of 5, and Western blotting was then performed using proteins obtained at 6, 12, 18, and 24 h.p.i. Polyclonal antibodies against synthetic peptides based on membrane (M) and nucleocapsid (N) proteins were used.

Fig. 2

Induction of apoptosis by SARS-CoV-infection. (A) Vero E6 cells were infected with SARS-CoV at an m.o.i. of 5 and stained with 0.05% crystal violet at 24 h.p.i. (B) DNA ladder fragmentation was detected at 24 h.p.i. (C) Western blotting analysis was performed using anti-cleaved caspase-3 antibody from 6, 12, 24, and 30 h.p.i. (C).

Fig. 3

Phosphorylation of p38 MAPK in SARS-CoV-infected Vero E6 cells. Phosphorylated and unphosphorylated p38 MAPK were detected by Western blotting analysis using proteins isolated from SARS-CoV-infected Vero E6 cells at 6, 12, 18, and 24 h.p.i.

Fig. 4

Phosphorylation of MAPKAPK-2 and HSP-27 in virus-infected cells. (A) Phosphorylated and unphosphorylated MAPKAPK-2 were detected by Western blotting analysis. (B) Phosphorylated and unphosphorylated HSP-27 were detected by Western blotting analysis.

Fig. 5

Phosphorylation of CREB and ATF-1 in virus-infected cells. Phosphorylated and unphosphorylated CREB were detected by Western blotting analysis. Phosphorylated ATF-1 was also detected using anti-phospho CREB antibody.

Fig. 6

Phosphorylation of eIF4E in virus-infected cells. Phosphorylated and unphosphorylated eIF4E were detected by Western blotting analysis.

Fig. 7

Effective doses of p38 MAPK inhibitor. SARS-CoV-infected Vero E6 cells were treated with SB203580 (p38 MAPK-specific inhibitor), PD98059 (ERK1/2-specific inhibitor), and SP600125 (JNK-specific inhibitor) for 18 h. Western blot analysis was performed using anti-phosphorylated HSP-27, CREB eIF4E antibodies. SARS-CoV N protein was also detected.

Fig. 8

Kinetics of N and M proteins synthesis in the presence of SB203580. After viral adsorption for 1 h, 20 and 30 μM of SB203580 was added to the cells. (A) Western blot analysis was performed at 6, 9, and 12 h.p.i. for detection of SARS-CoV N and M proteins. (B) Vero E6 cells were stained with 0.05% crystal violet at 41 h.p.i.