Antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies

Abstract

A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274-283 and 373-382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286-295, 316-325 and 361-367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316-325 and 361-367 on one nucleoprotein molecule to amino acid 286-295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.

Fig. 1

Western blot and cross-reactivity analysis. Lysates of coronavirus-infected cells were separated by SDS-PAGE and reactivity of proteins with antibody 8G5 A1A was determined by Western blotting. Lane 1: N-MBP fusion protein (89 kDa) and protein degradation products; lane 2: SARS CoV-infected Vero76 cells; lane 3: uninfected Vero76 cells; lane 4: CoV-229E-infected MRC-5 cells; lane 5: uninfected MRC-5 cells; lane 6: CoV-NL63-infected LLC-MCK2 cells; lane 7: uninfected LLC-MCK2 cells; lane 8: CoV-OC43-infected MDCK cells; lane 9: uninfected MDCK cells; lane 10: SARS CoV-infected Vero76 cells; lane 11: uninfected Vero76 cells; lane 12: TGEV-infected ST cells; lane 13: uninfected ST cells; lane 14: MHV A59-infected 17-C11 cells; lane 15: uninfected 17-C11 cells. Similar results were obtained with the other monoclonal antibodies.

Fig. 2

Intracellular distribution of the SARS CoV nucleocapsid protein. Confocal laserscan microscopy of SARS CoV-infected Vero76 cells. Superimposition of 6 consecutive sections of 0.2 μm. (A) Staining with the mAb 4C11A3 and a secondary FITC-conjugated rabbit anti-mouse antiserum; (B) propidium iodide staining of the nuclei; (C) overlay of A and B shows granular distribution of the SARS N protein concentrated around the nucleus.

Fig. 3

Overlapping SARS nucleoprotein peptides on a cellulose membrane recognised by anti-SARS N monoclonal antibodies: (A) clone 1G7D1; (B) clone 2G8D4; (C) clone 4E10A3A1; (D) clone 4F3C4; (E) clone 6D11C1; (F) clone 8G7B2C; (G) antibody epitopes are indicated on the amino acid sequence of the C-terminal portion of the nucleoprotein.