Effects of site-specific mutation on structure and activity of influenza virus B/Lee/40 neuraminidase.
A cDNA copy of the neuraminidase (NA) gene of a high-growing reassortant influenza virus which has the hemagglutinin of B/Hong Kong/8/73 and the NA of B/Lee/40 was cloned into plasmid pUC 9 and subcloned into a late-replacement SV40 vector so that the NA gene could be expressed in eukaryotic cells. The expressed protein was antigenically and enzymatically active. To study structure-function relationships in the B/Lee NA, particularly in comparison to the known structure of influenza A (N2) NA, specific mutations were introduced using synthetic oligonucleotides. Mutation of an apparently unpaired cysteine residue to serine at position 251 had no effect on protein transport or folding as judged by cell-surface reactivity with monoclonal antibodies, and the NA retained enzyme activity, confirming that this Cys is not essential for correct folding of the polypeptide. Mutation of Trp 364 to Leu abolished detectable enzyme activity, while mutation of Thr 368 to Val reduced enzyme activity to less than 25% of wild-type levels. Neither mutation affected antigenic properties. Therefore it is likely that both these side chains extend into the NA active site pocket. The results of these experiments are in accord with similarities in the structure of the B/Lee NA compared with that of influenza A (N2) NA. Although there is about 70% amino acid sequence difference between influenza A and B NAs, residues in the active site are highly conserved. Our in vitro mutagenesis experiments help to confirm the tentative alignment of sequences and have identified conserved amino acid side chains involved in enzyme activity.
D000595:Amino Acid Sequence; D001483:Base Sequence; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D009981:Influenza B virus / Q000201:enzymology / Q000235:genetics*; D008970:Molecular Weight; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D009439:Neuraminidase