Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase.
We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment.
Nuss
J M
JM
Department of Microbiology, University of Alabama, Birmingham 35294.
Whitaker
P B
PB
Air
G M
GM
D000595:Amino Acid Sequence; D000914:Antibodies, Viral; D000937:Antigen-Antibody Reactions / Q000235:genetics; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005814:Genes, Viral; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; D000939:Epitopes; D009439:Neuraminidase