Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity

Abstract

The influenza A virus (IAV) neuraminidase (NA) protein plays an essential role in the release of virus particles from cells and decoy receptors. The NA enzymatic activity presumably needs to match the activity of the IAV hemagglutinin (HA) attachment protein and the host sialic acid (SIA) receptor repertoire. We analyzed the enzymatic activities of N1 NA proteins derived from avian (H5N1) and human (H1N1) IAVs and analyzed the role of the second SIA-binding site, located adjacent to the conserved catalytic site, therein. SIA contact residues in the second SIA-binding site of NA are highly conserved in avian, but not human, IAVs. All N1 proteins preferred cleaving α2,3- over α2,6-linked SIAs even when their corresponding HA proteins displayed a strict preference for α2,6-linked SIAs, indicating that the specificity of the NA protein does not need to fully match that of the corresponding HA protein. NA activity was affected by substitutions in the second SIA-binding site that are observed in avian and human IAVs, at least when multivalent rather than monovalent substrates were used. These mutations included both SIA contact residues and residues that do not directly interact with SIA in all three loops of the second SIA-binding site. Substrate binding via the second SIA-binding site enhanced the catalytic activity of N1. Mutation of the second SIA-binding site was also shown to affect virus replication in vitro Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates.IMPORTANCE Avian and human influenza A viruses (IAVs) preferentially bind α2,3- and α2,6-linked sialic acids (SIAs), respectively. A functional balance between the hemagglutinin (HA) attachment and neuraminidase (NA) proteins is thought to be important for host tropism. What this balance entails at the molecular level is, however, not well understood. We now show that N1 proteins of both avian and human viruses prefer cleaving avian- over human-type receptors although human viruses were relatively better in cleavage of the human-type receptors. In addition, we show that substitutions at different positions in the second SIA-binding site found in NA proteins of human IAVs have a profound effect on binding and cleavage of multivalent, but not monovalent, receptors and affect virus replication. Our results indicate that the HA-NA balance can be tuned via modification of substrate binding via this site and suggest an important role of the second SIA-binding site in host tropism.

Keywords: influenza A virus; neuraminidase; second SIA-binding site; sialic acid.

FIG 1

Specific activity of H5N1 NA proteins using monovalent and multivalent substrates. (A) Specific activities of indicated H5N1 NA proteins using the substrate MUNANA are graphed normalized to the specific activity of NA HN. (B and C) Specific activities of the indicated NA proteins were determined by ELLA using different glycoprotein-lectin combinations, as indicated, and graphed normalized to the activity of NA HN. Means of three independent experiments performed in triplicate are shown. Standard deviations are indicated.

FIG 2

Specificity of H5N1 NA proteins. The specific activities of the indicated H5N1 NA proteins were determined by ELLA using different glycoprotein-lectin combinations and graphed normalized to the specific activity determined with MUNANA. Means of three independent experiments performed in triplicate are shown. Standard deviations are indicated.

FIG 3

Alignment of N1 proteins. Alignment of several N1 proteins analyzed in this study is shown. The start of the N1 protein ectodomain expressed as a recombinant soluble fusion protein is indicated. Catalytic and framework residues in the active site are shown in red and yellow, respectively (5, 6). SIA contact residues in the 2nd SIA-binding site, based on the N9 crystal structure (7), are shown in light blue. Residues introduced in HN N1 are shown in purple. N2 numbering is indicated.

FIG 4

Structure of the N9 2nd SIA-binding site. (A and B) Crystal structure of N9 from A/tern/Australia/G70C/75 in complex with SIA (N-acetylneuraminic acid) (PDB 1MWE) (7). (A) Surface representation. The NA active site and the 2nd SIA-binding site (SIA contact residues) are shown in red and light blue, respectively. The SIA moieties in these sites are shown as sticks (oxygen in red, nitrogen in blue, and carbon in gray). (B) Structure of the 2nd SIA-binding site. SIA is shown as sticks (oxygen in red, nitrogen in blue, and carbon in gray). Residues in the 2nd SIA-binding site that directly contact SIA (S367, S370, S372, N400, W403, and K432) are shown in stick representation (light blue), amino acids at positions 368 and 369 that differ between HN and WSN and CA/09 are shown in cartoon representation (yellow). Hydrogen bonds between SIA and residues in the 2nd SIA-binding site are shown as dashed red lines. Figures were made using PyMOL.

FIG 5

Substitution N369H affects H5N1 NA enzymatic activity. Specific activities of the indicated (mutant) H5N1 NA proteins determined by MUNANA assay (A) or by ELLA using fetuin (B) or transferrin (C) in combination with lectin ECA were graphed normalized to the activity of N1 HN. Means of two independent experiments performed in triplicate are shown. Standard deviations are indicated.

FIG 6

Substrate binding of NA proteins via the 2nd SIA-binding site. (A). HN and HB NA proteins and N9 proteins with different binding properties (10) were complexed to lumazine synthase nanoparticles displaying domain B of protein A using anti-Strep-tag monoclonal antibodies. Limiting dilutions of these complexes were incubated with red blood cells in the presence of OsC, and hemagglutination titers were determined. Standard deviations are indicated. (B and C) Membrane vesicles containing full-length HN, HN N369H, or HB protein were analyzed for their ability to bind 3′-SLNLNLN or 6′-SLNLNLN in the presence of OsC using biolayer interferometry. Representative experiments are shown.

FIG 7

Sequence logos of the 2nd SIA-binding site of avian and human N1 proteins. Sequence logos were generated for the three loops that constitute the 2nd SIA-binding site using the WebLogo website (http://weblogo.berkeley.edu/) (40) using all sequences available of avian N1 viruses, human N1 viruses prior to 2009 (seasonal human N1), and human H1N1pdm09 viruses from 2009 to 2014 (pandemic human N1) via the Influenza Research Database (https://www.fludb.org/). Asterisks indicate SIA contact residues according to the N9 crystal structure (7).

FIG 8

Enzymatic activity of HN, WSN, and CA/09 NA proteins using monovalent and multivalent substrates. Specific activities of indicated N1 proteins determined by MUNANA assay (A) or by ELLA using fetuin (B) and transferrin (C) in combination with lectin ECA were graphed normalized to the activity of N1 HN. (D) Substrate specificities of the indicated N1 proteins were determined by ELLA using different glycoprotein-lectin combinations and graphed normalized to the specific activity determined with MUNANA. Means of 2 to 3 independent experiments performed in triplicate are shown for the MUNANA and ELLA assays. Standard deviations are indicated.

FIG 9

Effect of substitutions in the 2nd SIA-binding site on NA enzymatic activity. Specific activities of wild-type and mutant HN determined by MUNANA assay (A) or by ELLA using fetuin (B) and transferrin (C) in combination with lectin ECA were graphed normalized to that of wild-type HN. Means of 2 to 3 independent experiments performed in triplicate are shown. Standard deviations are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 10

Substrate specificity of mutant HN NA proteins. Substrate specificities of the indicated mutant HN proteins (same as those shown in Fig. 5) were determined by ELLA using different glycoprotein-lectin combinations and graphed normalized to the specific activity determined with MUNANA. Means of 2 to 3 independent experiments performed in triplicate are shown. Standard deviations are indicated.

FIG 11

Effect of substitutions outside the 2nd SIA-binding site on NA enzymatic activity. Specific activities of wild-type and mutant HN determined by MUNANA assay (A) or by ELLA using fetuin (B) or transferrin (C) in combination with lectin ECA were graphed normalized to the activity of wild-type HN. Means of at least two independent experiments performed in triplicate are shown. Standard deviations are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 12

Replication of viruses with and without a 2nd SIA-binding site. MDCK-II cells were infected a multiplicity of infection of 0.001 with PR8 (7+1) viruses carrying wild-type (K432) or mutant (E432) HN NA proteins. Virus in the supernatant at the indicated time points postinfection was titrated. Means of three independent experiments are graphed. Standard deviations are indicated. Significant differences were analyzed using a Student t test, and the P values are shown. TCID₅₀, 50% tissue culture infective dose.