Amino acids 1 to 422 of the spike protein of SARS associated coronavirus are required for induction of cyclooxygenase-2

Abstract

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV). To evaluate the molecular mechanisms involved in the viral infection, in this study, we investigated the role of SARS-CoV Spike (S) protein in the regulation of cyclooxygenase-2 (COX-2). Expression of COX-2 stimulated by the S protein was verified by RT-PCR and western blot assay. To explore the relationship between S and COX-2, we constructed a series of plasmids containing truncated N-terminal fragments of the SARS-CoV S gene (designated from Sa to Si), which encoded truncated S proteins, and investigated whether these truncated proteins could induce effective expression of COX-2 in 293T cells. Our results showed that S(d) that encoded a truncated S protein with 422 amino acid residues (from 1 to 422 aa), a part of 672 amino-acid S1 subunit is crucial for the induction of COX-2 expression. Immunofluorescence examinations also give the evidence that these N terminal 422 amino acids of the S protein were also required for the correct localization of the protein. We also compared S protein sequences of SARS-CoV isolated during the SARS break with that from palm civets, a possible source of SARS-CoV found in humans. S protein residues (344, 360), which mutated in the epitome from palm civet to human being were characterized in 3D modeling of 252-375 amino acid fragment. Collectively, these results indicate that S protein of SARS-CoV induces the expression of COX-2 and an N-terminal fragment of the Spike protein is crucial for the induction. Our finding may provide clue for the induction of inflammation by SARS-CoV and cast insight into the severity of the SARS epidemic.

Fig. 1

Effects of SARS-CoV S protein on COX-2 promoter and its expression. (A) HEK293T, COS-7 or A549 cells were co-transfected with COX-2-wt-Luc and pCMV-S, which is the spike encoding plasmid. Luciferase activity was measured as described in “experimental procedures”. The results were expressed as the mean ± SD of three independent experiments performed in triplicate. * P < 0.05 compared with control vector. (B) To detect COX-2 expression, transiently transfecred pCMV-S into HEK293T cell, after 24 h serum starvation, cell extracts were separated by SDS-PAGE in a 12% gel and followed by being transferred to nitrocellulose membrane. The expression of COX-2 and the S protein level were shown by western blot using anti-COX-2 antibody and anti-flag protein antibody, respectively. Similar results were obtained in five independent experiments

Fig. 2

Identification of the functional regions in SARS-CoV S protein mediating COX-2 expression. (A) Structures of wild-type and deletion mutant forms of SARS-CoV S protein and their inducibility to COX-2 expression. Boxes represent the structure of different constructs of S protein with the length indicated above. (B) For COX-2 expression, transiently transfected a series of truncated S mutant encoding plasmids which can induce the activity of COX-2 promoter in HEK293T cells, the expression of COX-2 and the S mutant protein were shown by western blot using anti-COX-2 antibody and anti-flag protein antibody, respectively. (C) HEK293T cells were transiently transfected with a series of truncated mutants, after 24 h serum starvation, luciferase activity were determined. The results were expressed as the mean ± SD of three independent experiments performed in triplicate. *P < 0.05 compared with full-length of S

Fig. 3

Identification of the location of functional regions in SARS-CoV S protein mediating COX-2 expression. COS-7 cells were transfected with pCMV-S (A), pCMC-S_1–255 (B), pCMV-S_1–422 (C) and pCMV-S_255–1255 (D), respectively. 24 h after transfection, localizations were detected by anti-flag antibody

Fig. 4

The important sites of N-terminal domain of Spike protein. (A) Representation of the 3D structure of S_272–375 domain of Spike protein. Three residues of Spike protein of SARS-CoV are show in colorful balls. G311 was yellow, K344 was red and F360 was green. (B) A view identical to that in A except that the molecule has been rotated 90° about the vertical axis. (C) A view identical to that in A except that the molecule has been rotated 90° about the horizontal axis