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.
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.
Kawai
A
A
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Sakyo-ku, 606-8501, Japan. akawai@pharm.kyoto-u.ac.jp
Toriumi
H
H
Tochikura
T S
TS
Takahashi
T
T
Honda
Y
Y
Morimoto
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002458:Cell Fractionation; D002460:Cell Line; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D018832:Molecular Chaperones; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011820:Rabies virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012995:Solubility; D013997:Time Factors; D015678:Viral Structural Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D018832:Molecular Chaperones; D019590:Nucleocapsid Proteins; C103961:P phosphoprotein, Rabies virus; D010710:Phosphates; D010750:Phosphoproteins; D015678:Viral Structural Proteins; C099601:nucleocapsid protein, Rabies virus; D010744:Phosphoric Monoester Hydrolases