Influenza Hemagglutinin and Neuraminidase: Yin⁻Yang Proteins Coevolving to Thwart Immunity

Abstract

Influenza A virions possess two surface glycoproteins-the hemagglutinin (HA) and neuraminidase (NA)-which exert opposite functions. HA attaches virions to cells by binding to terminal sialic acid residues on glycoproteins/glycolipids to initiate the infectious cycle, while NA cleaves terminal sialic acids, releasing virions to complete the infectious cycle. Antibodies specific for HA or NA can protect experimental animals from IAV pathogenesis and drive antigenic variation in their target epitopes that impairs vaccine effectiveness in humans. Here, we review progress in understanding HA/NA co-evolution as each acquires epistatic mutations to restore viral fitness to mutants selected in the other protein by host innate or adaptive immune pressure. We also discuss recent exciting findings that antibodies to HA can function in vivo by blocking NA enzyme activity to prevent nascent virion release and enhance Fc receptor-based activation of innate immune cells.

Keywords: Influenza A virus; antigenic drift; hemagglutinin; neuraminidase; viral evolution.

Figure 1

Naturally observed mechanisms to optimize HA–NA stoichiometry. (A) The HA H1 (pdb entry 3lzg) is colored pink with wt-RBS in black and mutated RBS with decreased binding in red. The NA N2 is colored turquoise (the model was created by superimposition of pdb entries 2hty and 6crd-containing tetrabrachion stalk) with the receptor destroying/catalytic site in blue, receptor binding site in magenta, and combined receptor binding/destroying site in yellow. (B) After the HA (pink) acquires an avidity decreasing mutation (blue), the NA (green) follows with a mutation (bright green) that impairs NA intracellular trafficking and incorporation into virions. (C) During host adaptation an NP mutation (red-orange) can decrease NA gene segment incorporation leading to diminished amounts of virion NA to rebalance HA–NA levels. UCSF Chimera 1.13 was used to render the 3-D structures.

Figure 2

NA structural conservation. We aligned roughly 400 NA amino acid sequences from IAV strains spanning the last 100 years (200 hundred H1N1, 50 H5N1, and 150 H3N2) using MUSCLE software at the fludb website (www.fludb.org). We projected residue conservation onto the N2 NA A/Perth/16/2009 H3N2 (pdb entry 6br5) structure using UCSF Chimera 1.13 software. Blue represents residues with maximal variability while red represents minimal variability. (A) Side view showing surface rendering of half the structure with the other half showing ribbon rendering, demonstrating the variability of NA surface residues vs. the conservation of internal residues. (B) Top view. The black squares show the catalytic site, which is magnified on the right. The residues forming the catalytic site are surface-rendered; note the high conservation except at the base, which is variable and consistent with its lack of substrate interaction.

Figure 3

Naturally observed mechanisms of NA–HA epistasis. (A) Some oseltamivir-resistant IAV clinical isolates completely lack the NA gene segment and adapt by acquiring mutations that reduce HA receptor avidity. HA is colored pink, NA green, NA inhibitor yellow, HA mutation blue. (B) NA inhibitors can select escape mutants with changes exclusively in HA that alter HA receptor binding properties. The H1 HA (pdb entry 3lzg) colored pink with wt-RBS black and mutated RBS red, rendered as side view. The N1 NA colored turquoise (pdb entry 3ti6) with or without oseltamivir (yellow-green) bound to receptor destroying site (blue) rendered as top view. UCSF Chimera 1.13 software was used to visualize the molecules.

Figure 4

Biogenesis of HA antigenic sites. The HA head antigenic site Sb and HA stem protective epitopes are formed on HA monomers during biogenesis, while for other sites on the HA head, full maturation requires HA trimerization. Contrary to HA stem epitopes, which are destroyed by conformational changes triggered by low pH, HA head antigenic sites are remarkably resistant and remain intact even after proteolytic liberation of the monomeric head domain. The pdb entries 1htm, 1ibn, and 2vir (A/Aichi/2/1968 H3N2) were used to visualize various conformational stages during the fusion process. The HA1 is colored bright blue, HA2 orange, and fusion peptide red. Approximate localization of the canonical antigenic sites and the HA stem epitopes are highlighted by translucent purple with dashed edges. UCSF Chimera 1.13 software was used to visualize the molecules.

Figure 5

The mechanisms of humoral response modulation towards HA stem domain. If regular HA (top left) is used as an immunogen, the dominant antibody response is directed towards the head domain (dark red). Immunization with hyperglycosylated HA head (complex glycans colored bright green) or sequential immunization with dissimilar head (olive green, dark red, bright blue), identical stem (dim gray) constructs (top middle), or physically separated stem (top right) all result in anti-stem dominated Ab responses.