The nucleocapsid protein of SARS-CoV induces transcription of hfgl2 prothrombinase gene dependent on C/EBP alpha.
Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and beta-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.
Han
Meifang
M
Department of Infectious Disease; Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Yan
Weiming
W
Huang
Yuancheng
Y
Yao
Huaning
H
Wang
Zhanhui
Z
Xi
Dong
D
Li
Weina
W
Zhou
Yaoyong
Y
Hou
Jinlin
J
Luo
Xiaoping
X
Ning
Qin
Q
D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; D006224:Cricetinae; D003412:Cricetulus; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D064370:Spike Glycoprotein, Coronavirus; D015533:Transcriptional Activation; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha; D000086862:Coronavirus M Proteins; D000086462:Coronavirus Nucleocapsid Proteins; C095202:FGL2 protein, human; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D005340:Fibrinogen