Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses.
Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.
Tang
Tswen-Kei
TK
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Wu
Mark P-J
MP
Chen
Shui-Tsung
ST
Hou
Ming-Hon
MH
Hong
Ming-Hsiang
MH
Pan
Fu-Ming
FM
Yu
Hui-Ming
HM
Chen
Jenn-Han
JH
Yao
Chen-Wen
CW
Wang
Andrew H-J
AH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D000956:Antigens, Viral / Q000737:chemistry; D001665:Binding Sites; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D028941:Coronavirus 229E, Human / Q000378:metabolism*; D000086462:Coronavirus Nucleocapsid Proteins; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry; D019590:Nucleocapsid Proteins / Q000737:chemistry*; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D011817:Rabbits; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis; D014709:Vero Cells
D000596:Amino Acids; D000914:Antibodies, Viral; D000941:Antigens; D000956:Antigens, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D000939:Epitopes; C000706247:N protein, SARS-CoV; D019590:Nucleocapsid Proteins; D010455:Peptides; D012313:RNA; D004247:DNA