Nucleocapsid Formation and/or Subsequent Conformational Change of Rabies Virus Nucleoprotein (N) Is a Prerequisite Step for Acquiring the Phosphatase-Sensitive Epitope of Monoclonal Antibody 5-2-26

• Published in: Virology (New York, N.Y.) N.Y.), 1999-10, Vol.263 (2), p.395-407
• Main Authors: Kawai, Akihiko, Toriumi, Harufusa, Tochikura, Tadafumi S., Takahashi, Takuo, Honda, Yoshikazu, Morimoto, Kinjiro
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antigens, Viral - chemistry; Antigens, Viral - genetics; Antigens, Viral - immunology; Antigens, Viral - metabolism; Blotting, Western; Cell Fractionation; Cell Line; conformational epitope; Cricetinae; encapsidation; Epitopes - immunology; Fluorescent Antibody Technique; molecular conformation; nucleocapsid; Nucleocapsid - chemistry; Nucleocapsid - genetics; Nucleocapsid - immunology; Nucleocapsid - metabolism; Nucleocapsid Proteins; nucleoprotein; Phosphates - metabolism; Phosphoproteins - metabolism; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Precipitin Tests; Protein Binding; Protein Conformation; Rabies virus; Rabies virus - genetics; Rabies virus - immunology; Rabies virus - metabolism; Solubility; Time Factors; Viral Structural Proteins - metabolism
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.1999.9962

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N–P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.