Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: relationship to growth in the duck intestine

Abstract

The 1957 human pandemic strain of influenza A virus contained an avian virus hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for the human receptor, N-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an alpha2-6 bond (NeuAcalpha2-6Gal). Although the NA retained considerable specificity for NeuAcalpha2-3Gal, its original substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift in substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient viral growth in the intestines of orally inoculated ducks. The growth-supporting capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, we localized the determinants of high specific NA activity to a region containing six amino acid substitutions in A/England/12/62: Ser331-->Arg, Asp339-->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and Pro431-->Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs for their cellular receptor.

FIG. 1

Relative specific enzymatic activities of avian and human virus NAs. Enzymatic activity was determined with the MU-NANA substrate, as described in Materials and Methods. Relative specific activity was calculated by normalizing NA activity to the level of NA protein in virions, as determined by nonreducing polyacrylamide gel electrophoresis of ³⁵S-labeled virions, followed by autoradiography and densitometric analysis of the NA bands. The results are shown in relation to the activity of the avian NA of Dk78/75N2, set at 100%. The error bars indicate standard deviations.

FIG. 2

Identification of the NA region associated with high specific activity. (A) The sequence coding for the FLAG epitope tag (DYKDDDDK) was inserted, in frame, into the NA genes of A/Singapore/1/57 (Sing/57) and A/England/12/62 (Eng/62), and chimeras 1 to 5 were generated by using shared restriction sites. All genes were subcloned into the plasmid expression vector pCAGGS/MCS. (B) Two 6-cm² wells of 293T cells were transfected with 2 μg of each NA-expressing plasmid/well. 48 h later, the cell-associated NA activity was determined by using equal numbers of cells transfected with each NA and 0.1 mM NeuAcα2-3Gal sialyllactose substrate. NA protein expression levels were determined by isolation of cell membranes and measurement of NA by chemiluminescent Western blot analysis with the anti-FLAG antibody. Enzymatic activities were normalized to protein expression for each NA and are reported relative to that of A/England/12/62. The error bars indicate standard deviations.

FIG. 3

Location of amino acid differences between A/Singapore/1/57 and A/England/12/62 in the region of NA exchanged in chimera 5. The positions of the amino acid residues (cyan) and the enzymatic active site, indicated by the position of the bound sialic acid (purple), are shown on the NA structure of A/Tokyo/3/67 (H3N2) (23). The fourfold-symmetry axis (▪) that generates the tetrameric head of NA is perpendicular to the plane of the figure.

FIG. 4

Determination of the contributions of individual amino acid residues to high specific activity. (A) Mutants were generated in which amino acid residues in the A/England/12/62 (Eng/62) NA were replaced (X) individually and in groups with the corresponding A/Singapore/1/57 (Sing/57) residues. (B) The relative specific activities of the mutants were determined as described in the legend to Fig. 2. The error bars indicate standard deviations.