Amino acid residues contributing to the substrate specificity of the influenza A virus neuraminidase

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sia lic acid, and neuraminidase (NA), which removes terminal sialic acid from o ligosaccharides. Hence, the interplay between these receptor-binding and re ceptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA s ubstrate specificity in viral replication among different animal species. W e therefore investigated the substrate specificities of NA for linkages bet ween N-acetyl sialic acid and galactose (NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had si milar specificities for NeuAc alpha 2-3Gal, while the activities for NeuAc alpha 2-6Gal ranged from marginal, as represented by avian and early N2 hum an viruses, to high (although only one-third the activity for NeuAc alpha 2 -3Gal), as represented by swine and more recent N2 human viruses. Using sit e-specific mutagenesis, we identified in the earliest human virus with a de tectable increase in NeuAc alpha 2-6Gal specificity a change at position 27 5 (from isoleucine to valine) that enhanced the specificity for this substr ate. Valine at position 275 was maintained in all later human viruses as we ll as swine viruses. A similar examination of N-glycolylneuraminic acid (Ne uGc) specificity showed that avian viruses and most human viruses had low t o moderate activity for this substrate, with the exception of most human vi ruses isolated between 1967 and 1969, whose NeuGc specificity was as high a s that of swine viruses. The amino acid at position 431 was found to determ ine the level of NeuGc specificity of NA: lysine conferred high NeuGc speci ficity, while proline, glutamine, and glutamic acid were associated with lo wer NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs b y a mechanism of amino acid substitutions that subtly alter the conformatio n of NA in and around the active site to facilitate the binding of differen t species of sialic acid.