SARS-CoV nucleocapsid protein induced apoptosis of COS-1 mediated by the mitochondrial pathway.
To investigate the apoptosis effect of SARS coronavirus nucleocapsid protein on cultured cell lines and to explore the possible pathway of apoptosis. pCDNA3.1(-)/his-myc vector containing the SARS coronavirus nucleocapsid gene (N), matric gene (M), spike gene (S) were transfected into COS-1, Huh-7 and HepG2 cells. Apoptosis induced by SARS coronavirus N protein under starvation of serum of COS-1 cells was monitored by Annexin V and electron microscopy assays. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim) were determined by flow cytometric assay. Cytochrome C, cleaved caspase (cysteine aspartic acid protease)-3, 9, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. After removal of serum in COS-1 cells, we observed the loss of DeltaPsim, the increase of ROS and cytochrome C release into cytosol and subsequent activation of caspase-3 and PARP cleavage. The pan-caspase inhibitor z-VAD-fmk can block the activation of caspase 3, 9 and PARP cleavage. In conclusion, SARS coronavirus N protein can induce apoptosis of COS-1 cells by activating mitochondrial pathway. SARS coronavirus M, S protein can not induce apoptosis in COS-1, HepG2 and Huh-7 and SARS coronavirus N protein can not induce apoptosis in HepG2 and Huh-7 by methods used in this study.
Zhang
Lu
L
Hepatology Institute, Peking University, People's Hospital, Beijing, People's Republic of China.
Wei
Lai
L
Jiang
Dong
D
Wang
Jianghua
J
Cong
Xu
X
Fei
Ran
R
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D000086462:Coronavirus Nucleocapsid Proteins; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D008854:Microscopy, Electron; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure / Q000821:virology*; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D064370:Spike Glycoprotein, Coronavirus; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D000086462:Coronavirus Nucleocapsid Proteins; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D045304:Cytochromes c; D053453:Caspase 9