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

Abstract

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 (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) 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 NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), 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 NeuAcalpha2-6Gal 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.

FIG. 1

NeuAcα2-6Gal substrate specificities of avian, swine, and human virus N2 NAs. Virus strains were compared for their ability to release sialic acid from sialyllactoses containing either the NeuAcα2-3Gal or the NeuAcα2-6Gal linkage. (A) Ratio of sialic acid released from a 0.1 mM concentration (50 μl) of each substrate (NeuAcα2-6Gal and NeuAcα2-3Gal) by 10.0 mU of viral NA per ml. (B) Absolute amount of sialic acid released from a 0.1 mM concentration (50 μl) of NeuAcα2-6Gal-containing sialyllactose by 100.0 mU of NA per ml in 30 min at 37°C. The amount of released sialic acid was determined by the periodate-thiobarbituric acid assay (22) and reported as the absorbance of the colored chromophore derivative at 549 nm.

FIG. 2

Determination of amino acid residues involved in NeuAcα2-6Gal specificity by use of A/Singapore/1/57-A/England/12/62 chimeric constructs. (A) In these constructs, the sequences of A/England/12/62 N2 NA (□; pCAT3DKENG62NASAP) were replaced with the sequences of A/Singapore/1/57 N2 NA (■; pCAT3DKSING57NASAP) by use of shared restriction sites to generate chimeras 1 to 5. Amino acid mutations K258→E and I275→V in A/Singapore/1/57 N2 NA were generated in constructs pCAT3DKSING-258E (258E) and pCAT3DKSING-275V (275V) by substitution of the NheI-BlpI and BlpI-EcoRV fragments of pCAT3DKENG62NASAP into the equivalent regions of pCAT3DKSING57NASAP, respectively. (B and C) 293T cells were transfected with 2 μg of each plasmid expression vector containing parental or chimeric NA per well (six-well plate). Cells expressing parental NAs and chimeric constructs 1 to 5 (B) or mutant constructs pCAT3DKSING-258E and pCAT3DKSING-275V (C) were harvested at 40 h posttransfection. Cell-expressed NA (0.5 mU) was assayed for the amount of sialic acid released from 0.1 mM sialyllactose containing the NeuAcα2-6Gal linkage in 30 min at 37°C. Released sialic acid was determined as described in the legend to Fig. 1.

FIG. 2

Determination of amino acid residues involved in NeuAcα2-6Gal specificity by use of A/Singapore/1/57-A/England/12/62 chimeric constructs. (A) In these constructs, the sequences of A/England/12/62 N2 NA (□; pCAT3DKENG62NASAP) were replaced with the sequences of A/Singapore/1/57 N2 NA (■; pCAT3DKSING57NASAP) by use of shared restriction sites to generate chimeras 1 to 5. Amino acid mutations K258→E and I275→V in A/Singapore/1/57 N2 NA were generated in constructs pCAT3DKSING-258E (258E) and pCAT3DKSING-275V (275V) by substitution of the NheI-BlpI and BlpI-EcoRV fragments of pCAT3DKENG62NASAP into the equivalent regions of pCAT3DKSING57NASAP, respectively. (B and C) 293T cells were transfected with 2 μg of each plasmid expression vector containing parental or chimeric NA per well (six-well plate). Cells expressing parental NAs and chimeric constructs 1 to 5 (B) or mutant constructs pCAT3DKSING-258E and pCAT3DKSING-275V (C) were harvested at 40 h posttransfection. Cell-expressed NA (0.5 mU) was assayed for the amount of sialic acid released from 0.1 mM sialyllactose containing the NeuAcα2-6Gal linkage in 30 min at 37°C. Released sialic acid was determined as described in the legend to Fig. 1.

FIG. 2

Determination of amino acid residues involved in NeuAcα2-6Gal specificity by use of A/Singapore/1/57-A/England/12/62 chimeric constructs. (A) In these constructs, the sequences of A/England/12/62 N2 NA (□; pCAT3DKENG62NASAP) were replaced with the sequences of A/Singapore/1/57 N2 NA (■; pCAT3DKSING57NASAP) by use of shared restriction sites to generate chimeras 1 to 5. Amino acid mutations K258→E and I275→V in A/Singapore/1/57 N2 NA were generated in constructs pCAT3DKSING-258E (258E) and pCAT3DKSING-275V (275V) by substitution of the NheI-BlpI and BlpI-EcoRV fragments of pCAT3DKENG62NASAP into the equivalent regions of pCAT3DKSING57NASAP, respectively. (B and C) 293T cells were transfected with 2 μg of each plasmid expression vector containing parental or chimeric NA per well (six-well plate). Cells expressing parental NAs and chimeric constructs 1 to 5 (B) or mutant constructs pCAT3DKSING-258E and pCAT3DKSING-275V (C) were harvested at 40 h posttransfection. Cell-expressed NA (0.5 mU) was assayed for the amount of sialic acid released from 0.1 mM sialyllactose containing the NeuAcα2-6Gal linkage in 30 min at 37°C. Released sialic acid was determined as described in the legend to Fig. 1.

FIG. 3

NeuGc substrate specificities of avian, swine, and human virus N2 NAs. The abilities of avian, swine, and human virus N2 NAs (A) and NAs from additional human viruses isolated in 1962 and 1967 to 1969 and swine viruses (B) to release sialic acid from GM3 ganglioside substrates containing NeuAcα2-3Gal or NeuGcα2-3Gal were compared. Viral NA (10 mU/ml) was incubated with 0.1 mM substrate containing 0.1% SDC (50 μl) for 30 min at 37°C, and the amount of liberated sialic acid was determined as described in the legend to Fig. 1. The ratio of sialic acid released from NeuGcα2-3Gal versus that released from NeuAcα2-3Gal is reported.

FIG. 4

Determination of amino acid residues contributing to NeuGc specificity. (A) Chimeras 6 to 10, in which the sequences of A/England/12/62 N2 NA (□; pCAT3DKENG62NASAP) were replaced with the equivalent sequences of A/Tokyo/3/67 N2 NA (■; pCAT3TOKYO67NASAP), were generated as described in the legend to Fig. 2 for chimeras 1 to 5. Parental NAs and chimeras 6 to 10 (B) or A/England/12/62 mutant constructs containing amino acid substitutions N401→D and W403→R (pCAT3DKENG-401D,403R) and Q431→K (pCAT3DKENG-431K) (C) were expressed as described in the legend to Fig. 2. Cell-expressed NA (0.5 mU) was incubated with 0.1 mM GM3 ganglioside substrate containing NeuAc or NeuGc in the presence of 0.1% SDC for 30 min at 37°C. The amount of sialic acid released from NeuGcα2-3Gal was determined as described in the legend to Fig. 1.

FIG. 4

Determination of amino acid residues contributing to NeuGc specificity. (A) Chimeras 6 to 10, in which the sequences of A/England/12/62 N2 NA (□; pCAT3DKENG62NASAP) were replaced with the equivalent sequences of A/Tokyo/3/67 N2 NA (■; pCAT3TOKYO67NASAP), were generated as described in the legend to Fig. 2 for chimeras 1 to 5. Parental NAs and chimeras 6 to 10 (B) or A/England/12/62 mutant constructs containing amino acid substitutions N401→D and W403→R (pCAT3DKENG-401D,403R) and Q431→K (pCAT3DKENG-431K) (C) were expressed as described in the legend to Fig. 2. Cell-expressed NA (0.5 mU) was incubated with 0.1 mM GM3 ganglioside substrate containing NeuAc or NeuGc in the presence of 0.1% SDC for 30 min at 37°C. The amount of sialic acid released from NeuGcα2-3Gal was determined as described in the legend to Fig. 1.

FIG. 5

Active-site residues of N2 influenza virus NA and bound NeuAc presented as a ball-and-stick model. Valine at residue 275 (Val275) is located below the center of the active site, while lysine at residue 431 (Lys431) lies above the center of the active site (at twice the distance of Val275).