Influenza neuraminidase

Abstract

Influenza neuraminidase is the target of two licensed antivirals that have been very successful, with several more in development. However, neuraminidase has been largely ignored as a vaccine target despite evidence that inclusion of neuraminidase in the subunit vaccine gives increased protection. This article describes current knowledge on the structure, enzyme activity, and antigenic significance of neuraminidase.

Figure 1

The NA active site is highly conserved. An NA tetramer (PDB 1NNB) showing 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid (DANA) in space‐filling atoms (gray, C; red, O; blue, N) bound to each of the four subunits. (B) Superposition of the conserved amino acids that make contact with DANA in influenza A (PDB 1NNB) and influenza B (PDB 1NSD). The amino acids are identified in panel C. Hydrogen bonds are shown as gold dotted lines. Panels A and B were made using the PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC and panel C was made using SwissPDB Viewer (http://www.expasy.org/spdbv/). Reproduced from Ref. 6 with permission.

Figure 2

Group 1 NAs such as N1 have an additional cavity in the active site that is not present in Group 2 NAs such as N9. (A, B) Oseltamivir bound to N1 (A, green) and N9 (B, yellow) NAs shown in surface representation with the protein main chain shown in “worm” representation. (C) Superposition of the active sites of apo‐N1 (green) and N1 complexed with oseltamivir (blue). Electron density from a difference Fourier is shown in blue to indicate the position of the 150‐cavity. Reproduced from Russell et al. ⁷ with permission.

Figure 3

The second sialic acid binding site in N9 and N6 NA. (A) The catalytic site (Sia1) and second sialic acid binding site (Sia2) in N9 NA (PDB 1MWE). (B) Sialic acid bound in the catalytic site of N6 NA is in the boat configuration but in the second site it is in the chair conformation (PDB 1W1X), as in hemagglutinin (1RVX). The figures were made using Pymol.

Figure 4

Neutralizing epitopes on NA. (A) Escape mutations in NAs surround the active site pocket. All known escape mutations (Table 1) are shown in the green and cyan subunits. The NA tetramer is tilted so that you are looking straight into the active site of the green subunit, while the cyan subunit shows that escape mutations occur on loops on the top surface. (B) A single subunit of N9 (gray) and N2 (pink) showing the amino acids that make up the epitopes of antibodies NC41 (green) and Mem5 (blue). The orientation is similar to the green subunit in A. An epitope is defined as those amino acids of NA that make direct contact with the antibody heavy and light chains. Each antibody covers about one‐third of the top surface of the NA subunit. Images were made using Pymol.

Figure 5

The Mem5 epitope has undergone antigenic drift. (A) Mutations in the Mem5 epitope since 1998. All 11 NA amino acid side chain contacts are shown. Another 6 amino acids make contacts from main chain atoms and these would not be expected to change if the side chain is substituted. (B) The interface between the NA (brown) and antibody heavy (blue) and light (cyan) chains. Amino acids shown in space‐filling representation have changed in human isolates since 1998. Adapted from Venkatramani et al. ⁹⁸ with permission.

Figure 6

Accumulation of changes in HA1 and NA of representative H3N2 human isolates. The y‐axis is overall numbers of residues changed from Aichi/1/68, so reversions and multiple substitutions are not counted. The NA changes are given for the whole NA and for the head region only. Since an antibody footprint is fairly constant, the results are given as total numbers of changes rather than percentages.