Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus

• Published in: Journal of general virology 2000-01, Vol.81 (1), p.119-127
• Main Authors: Goto, Hideo, Minamoto, Nobuyuki, Ito, Hiroshi, Ito, Naoto, Sugiyama, Makoto, Kinjo, Toshio, Kawai, Akihiko
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; antigenic sites; Antigens, Viral - genetics; Antigens, Viral - immunology; Antigens, Viral - metabolism; Blotting, Western; Cell Line; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Fluorescent Antibody Technique, Indirect; Molecular Sequence Data; Nucleocapsid Proteins - chemistry; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - immunology; Nucleocapsid Proteins - metabolism; Nucleoproteins - chemistry; Nucleoproteins - genetics; Nucleoproteins - immunology; Nucleoproteins - metabolism; Peptides - chemical synthesis; Peptides - immunology; Plasmids - genetics; Rabies virus; Rabies virus - genetics; Rabies virus - immunology; Rabies virus - physiology; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Sequence Deletion
• ISSN: 0022-1317; 1350-0872; 1465-2099; 1465-2080
• DOI: 10.1099/0022-1317-81-1-119

Department of Veterinary Public Health, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 1 Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku 606, Kyoto, Japan 2 Author for correspondence: Nobuyuki Minamoto. Fax [phone]. e-mail minamoto{at}cc.gifu-u.ac.jp Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358–367, and that the other site IV epitope of MAb 7-12 mapped to aa 375–383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.