Nucleocapsid Protein Of SARS-associated Coronavirus Upregulates The Transcription Of Hfgl2 Prothrombinase/ Fibroleukin Gene

Objective To investigate the responsible structural protein of SARS-associated coronavirus(SARS-CoV) in the activation of hfgl2 gene and to characterize the region in the hfgl2 promotor which is responsive to protein of SARS-CoV.Methods SARS-CoV was isolated from patient with SARS and gene fragments encoding nucleocapsid protein, membrane protein and spike protein were obtained by RT-PCR. and they were sub-cloned into eukaryotic expression vector( pcDNA3.1).The orientation and the sequence were ensured by restriction endonucleases and sequencing assays. The recombinated plasmid was then transfected into Chinese hamster ovary (CHO) cells, and immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV. To investigate the the responsible structural protein of SARS-CoV, Expression vectors of recombinant structural proteins N ,M and S2 of SARS-CoV were cotransfected respectively with hfgl2 promoter/luciferase reporter gene in CHO cells, and luciferase activity was detected for the assessment of promoter function.Constructs containing progressive deletions of the–1334 base pair fragment were cotransfected with N gene constrcts in CHO cells. Relative luciferase activity is expressed in fold increase compared with CHO cells cotransfected with the hfgl2 promoter constructs with PGL2-basic vector.Results Expression vectors of recombinant structural proteins of SARS-CoV were successfully constructed. Immunohistochemistry showed that structural protein of SARS-CoV was expressed only in those cells transfected with the recombinated plasmid. Cotransfection of N gene expression construct with a reporter construct containing hfgl2 promoter in CHO cells showed a remarkable increasing in luciferase activity compared with nontransfected CHO cells. However, CHO cells cotransfected with pcDNA3.1-M and pcDNA3.1-S2 with hfgl2 promoter/luciferase reporter gene did not show significant difference in luciferase activity when compared with negative controls. Preliminary mapping of the hfgl2 promoter has defined a region from–816 to–468 to be responsive to induction of N protein.Conclusions Nucleocapsid protein of SARS-CoV upregulates the transcription of hfgl2 prothrombinase/fibroleukin gene. The regulatory elements in the (–816 to–468) hfgl2 promoter account for the activation of hfgl2 gene in response to N protein of SARS-CoV.