Characterization of monoclonal antibodies raised against recombinant respiratory syncytial virus nucleocapsid (N) protein: identification of a region in the carboxy terminus of N involved in the interaction with P protein.
To investigate structure and biological properties of the nucleocapsid (N) protein of respiratory syncytial virus (RSV), we have generated a panel of 16 monoclonal antibodies, raised against recombinant N protein, and epitope mapped seven of these to three antigenic sites (Site I aa 16-30; Site II aa 341-350; Site III aa 351-365). Characterization by immunofluorescence and by immunoprecipitation assay demonstrated that a monoclonal antibody to antigenic site I can detect N protein complexed with phospho (P) protein. Antibodies to antigenic sites II and III, which are adjacent to each other near the carboxyl terminus of the N protein, have distinct properties. A site III monoclonal antibody detected N protein in cytoplasmic inclusion bodies and in the cytosol, but not when N was complexed to P protein, while the site II antibody reacted with N protein in the nucleocapsid fraction but did not detect cytosolic N protein. Further investigation into the reactivities of the antibodies after binding of P to N in vitro demonstrated that antigenic sites II and III were blocked by the interaction, indicating an involvement for the carboxy domain of N in the N-P interaction. This was confirmed by the ability of peptides from the carboxy terminus of N to inhibit the N-P interaction in vitro.
Murray
J
J
MRC Virology Unit, Church Street, Glasgow, G11 5JR, Scotland, United Kingdom.
Loney
C
C
Murphy
L B
LB
Graham
S
S
Yeo
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000914:Antibodies, Viral / Q000096:biosynthesis; D001665:Binding Sites; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology*; D010750:Phosphoproteins / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins