Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.
An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.
Mateu
M G
MG
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid, Spain.
Escarmís
C
C
Domingo
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D005537:Aphthovirus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002213:Capsid / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D011498:Protein Precursors