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Journal of Virology, August 1999, p. 6743-6751, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

Darwyn Kobasa,1,dagger Shantha Kodihalli,1 Ming Luo,2 Maria R. Castrucci,3 Isabella Donatelli,3 Yasuo Suzuki,4 Takashi Suzuki,4 and Yoshihiro Kawaoka1,5,*

Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 381011; Center for Macromolecular Crystallography, Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 352942; Department of Virology, Istituto Superiore di Sanita, Rome, Italy3; Department of Biochemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka 422, Japan4; and Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 537065

Received 29 January 1999/Accepted 4 May 1999

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-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 substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha 2-3Gal and NeuAcalpha 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 similar specificities for NeuAcalpha 2-3Gal, while the activities for NeuAcalpha 2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha 2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha 2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower 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 by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail: kawaokay{at}svm.vetmed.wisc.edu.

dagger Present address: DVP/OVRR/CBER/FDA, 29 Lincoln Drive, Bethesda, MD 20892.

Journal of Virology, August 1999, p. 6743-6751, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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