Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein.
Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.
Wootton
S
S
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Koljesar
G
G
Yang
L
L
Yoon
K J
KJ
Yoo
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D016056:Immunodominant Epitopes; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000235:genetics / Q000276:immunology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology*; D013552:Swine
D000956:Antigens, Viral; D016056:Immunodominant Epitopes