SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.
The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.
Graduate Institute of Molecular and Cellular Biology, Tzu Chi University, Hualien, Taiwan.
Wei-Yen
WY
Wei
Hui-Chun
HC
Li
Chiung-Yao
CY
Chen
Chee-Hing
CH
Yang
Shen-Kao
SK
Lee
Chia-Wen
CW
Wang
Hsin-Chieh
HC
Ma
Yue-Li
YL
Juang
Shih-Yen
SY
Lo
D000818:Animals; D002460:Cell Line; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000472:pathogenicity*; D020798:Two-Hybrid System Techniques
D019590:Nucleocapsid Proteins; D011770:Pyruvate Kinase