Changes to the dynamic nature of hemagglutinin and the emergence of the 2009 pandemic H1N1 influenza virus

Abstract

The virologic factors that limit the transmission of swine influenza viruses between humans are unresolved. While it has been shown that acquisition of the neuraminidase (NA) and matrix (M) gene segments from a Eurasian-lineage swine virus was required for airborne transmission of the 2009 pandemic H1N1 virus (H1N1pdm09), we show here that an arginine to lysine change in the hemagglutinin (HA) was also necessary. This change at position 149 was distal to the receptor binding site but affected virus-receptor affinity and HA dynamics, allowing the virus to replicate more efficiently in nasal turbinate epithelium and subsequently transmit between ferrets. Receptor affinity should be considered as a factor limiting swine virus spread in humans.

Figure 1. Transmissibility of the recombinant TRsw viruses in ferrets.

Ferrets were inoculated intranasally with 10⁶ EID₅₀/ml of NC/02 (a) NC/02:TN/09^NA,M (b) NC/02^HA149 (c) NC/02^HA149:TN/09^NA,M (d) TN/09 (e) NC/02^HA149:TN/09^M (f) and NC/02^HA149:TN/09^NA (g) influenza viruses. The transmissibility percentage was based on the TCID₅₀ assay using nasal wash samples in each group.

Figure 2. Receptor binding avidity of NC/02, NC/02^HA149, and TN/09 influenza viruses.

Each virus binding to sialylglycopolymers containing either α2,3- or α2,6-sialylated was measured by solid-phase direct binding assays (a) and dose-dependent direct glycan binding assays (b). Avidity assays were confirmed by performing a standard hemagglutinin assay with the resialylated cRBCs; untreated cRBCs (upper), VCNA-treated cRBCs (middle), or α2,6-resialylated cRBCs (bottom) (c). The images are representatives from four independent experiments. In each assay described NC/02^HA149 bound more strongly to the α2,6 receptor.

Figure 3. Growth of influenza viruses in vitro.

Replication of recombinant viruses in MDCK (a) MDCK-SIAT1 (b) and dNHBE cells (c,d). Cells were infected with viruses at 0.01 MOI, and infected cultures were collected at the indicated time points. The detection limit was one log₁₀TCID₅₀/ml. Data are shown as means ± SD from three or four independent experiments. *p < 0.01 compared with the value for NC/02 virus.

Figure 4. Position 149 is located in a deep and highly-conserved cleft.

ConSurf analysis of the HA of A/California/04/2009 (PDB ID: 3UBE, chain E); the structure is colored by the evolutionary conservation grades using the color-coding bar, with variable-through-conserved corresponding to cyan-through-maroon. Position 149 (which is mildly conserved) is in orange. The analogue of the α2,6 host cell receptor (LSTc) is shown in black bond-sticks representation. The figure was made using PyMOL (The PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC).

Figure 5. Position HA149 is a central hinge in the RBD, displaying correlated dynamic fluctuations with antigenic sites and receptor binding site residues.

An overview of the HA trimer from A/California/04/2009 structure [Protein Data Bank (PDB) accession number 3UBE] in cartoon representation with each subunit in a different color. The RBDs, mediating the binding to the host cell, are at the top and the sialic acid receptors are displayed using stick model representation. Normal mode analysis revealed spring motion along the arrow, considered to be important here (a). Correlation between the dynamic fluctuations of position 149 and the other RBD residues, color-coded with red-through-blue corresponding to the most positively-through-most negatively cooperative motions (b). Amino acids of known antigenic sites are presented as spheres, and the known sialic acid-binding residues are marked with sticks; binding site residues that are also a part of an antigenic site are presented as spheres. The analogue of the α2,6 host cell receptor is shown in black bond-sticks representation. The figure was made using PyMOL (The PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC).

Figure 6. The motion in position 149 correlates with the motion of key regions of the two other monomers in the trimer.

A top-down view of the HA trimer from the human A/California/04/2009 H1N1 strain (PDB ID: 3UBE) in cartoon representation. The correlation between the fluctuations of position 149 (red bond-sticks model representation) of the bottom monomer (grey) and the two top monomers, color-coded with red-through-blue corresponding to the most positively-through-most negatively cooperative residue motions. Correlated fluctuations were observed with amino acids in the B-region of HA2, the sialic acid binding site (220-loop), and parts of the Ca and Sa antigenic sites. The analogue of the α2,6 host cell receptor is shown in black bond-sticks representation. The figure was made using PyMOL (The PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC).