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 nucleoca psid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (res idues 112 to 123) was shown to be essential for binding of N-specific confo rmation-dependent monoclonal antibodies (MAbs), In the present study, the i mportance of individual residues within this C-terminal domain for antigeni city was investigated using eight different mutant constructs of N expresse d 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 th e 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 posi tions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Seconda ry-structure predictions indicate that amino acids 111 to 117 form a beta-s trand. In view of the fact that replacement of beta-strand-forming amino ac ids with proline elicited the greatest effect on MAb binding, it appears th at secondary structure in the C terminus of the N protein is an important d eterminant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studie s broaden our understanding of the secondary structures that make up the PR RSV N protein and shed some light on how they may relate to function.