Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses

Abstract

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Fig 1. HA sequences and designations of 2:6 recombinant IAVs used in this study.

(a) Amino acid differences between HAs of two 1968 pandemic virus lineages, their putative avian ancestor and 2:6 recombinant PR8-based viruses. Dots depict sequence identity with the HA of A/Hong Kong/1/1968. Numbering of amino acid positions starts from the N-terminus of the mature protein. Green background marks asparagine residues of glycosylation sites 63–65 and 81–83. (b) Location of amino acid substitutions shown as yellow space-filling models on the X-ray structure of the H3 HA complex with human receptor analogue LSTc (2YPG.pdb) [23]. Two HA monomers are colored gray, and the third monomer is colored green (HA1) and blue (HA2). LSTc is shown as red stick model, N-linked glycans are shown as dotted space-filling models. Cyan spheres show location of N₆₃ present in the HA of A/Memphis/1/1968 lineage. The model was generated using PyMOL 2.0.6 (Schrödinger, LLC).

Fig 2. Conformational stability and membrane fusion properties of HK, R5, R2 and R7.

(a) pH of acid-induced conformational transition of HA. Solid-phase adsorbed viruses were incubated in acidic buffers and treated with proteinase K. Viral binding of fet-HRP was assayed, and pH values that corresponded to 50% reduction of HA binding activity (pH₅₀) were determined from binding-versus-pH curves. (b) pH threshold of polykaryon formation. Inoculated MDCK cells were cultured for 16 h, treated with trypsin and exposed to different pH buffers. After returning to neutral medium and incubation for 3 h, the cells were fixed, stained and analysed under the microscope. The data show highest pH values at which polykaryon formation was detected. (c) Inhibition of viral infection by ammonium chloride. MDCK cells were inoculated in the presence of various concentrations of NH₄Cl, incubated overnight, fixed, and immunostained for NP. Concentrations of NH₄Cl that reduced numbers of infected cells by 50% (IC₅₀) were determined from dose-response curves. (d) HA stability at elevated temperature. Solid-phase adsorbed viruses were incubated in PBS at 65°C for different time periods and assayed for their binding to fet-HRP to determine incubation time required for 50% reduction of the binding activity (t₅₀). (e) HA stability in chaotropic buffer. Solid-phase adsorbed viruses were incubated in buffers containing GnHCl for 60 min at 4°C washed with PBS and assayed for binding to fet-HRP. Data show concentrations of GnHCl that reduced viral binding activity by 50%. (f) Reduction of infectivity after incubation of the viruses for 2 h at 45°C determined by focus assay in MDCK cells. All panels show data points, mean values and SDs from 1 to 4 independent experiments performed with 2 to 7 replicates. P values for the differences between the viruses were determined with Tukey’s multiple comparison procedure.

Fig 3. Binding of IAVs to biotinylated SGPs.

The solid-phase adsorbed viruses were allowed to bind biotinylated SGPs from solution, and association constants of viral complexes with SGPs (K_ass) were determined as described in Materials and Methods. Higher values of K_ass reflect stronger binding. (a) Binding to six low molecular mass Neu5Acα2-3Gal-containing SGPs differing by structure of penultimate sugar moieties. Wild type IAVs A/mallard/Alberta/279/1998 (H3N8) and A/ruddy turnstone/ Delaware/2378/1988 (H7N7) were tested in parallel with R7 and R2. Two to 4 experiments were performed on different days with one replicate for each virus-SGP pair per experiment. (b) Binding to high molecular mass SGPs 3’SLN and 6’SLN. Two experiments were performed on different days with two replicates per each virus-SGP pair per experiment. All panels show the individual values adjusted for day as described in Materials and Methods with geometric mean (bars) and SDs. P values for the differences between Kass presented in the panel 3a are shown in S3 Table.

Fig 4. Receptor-binding properties of HA point mutants of HK and R5.

(a-c) Association constants of viral complexes with biotinylated SGPs 6’SLN (20 kDa), 3’SLN and SLe^c (both 1 MDa) were determined as described in Materials and Methods. Data represent combined results from 4 to 11 experiments performed on different days with 1 replicate for each virus-SGP pair per experiment. (d) Inhibition of viral cell entry by Vibrio cholerae sialidase. MDCK cells were incubated with solutions of gradually diluted sialidase for 30 min, inoculated with 200 FFU of the viruses without removing sialidase, fixed after one cycle of replication and immunostained for viral NP. The figure shows concentrations of sialidase that reduced numbers of infected cells by 50% (IC₅₀). From 2 to 9 experiments were performed on different days using 3 to 4 replicates per virus. All panels show the individual values adjusted for day as described in Materials and Methods with geometric mean (bars) and SDs. Vertical dotted line separates point mutants of HK and point mutants of R5. Asterisks depict P values for the differences between single-point mutants and the corresponding parental virus, either HK or R5 (dark gray bars). Asterisks over horizontal lines depict differences between HK and R5 and between HK-81 and HK-81-63.

Fig 5. Attachment of HK and R5 to cells in HTBE cultures.

(a) The apical sides of live HTBE cultures were washed with PBS+ to remove accumulated mucins and inoculated with 0.2 ml of DMEM-BSA containing 1.3x10⁶ FFU of HK and R5. Control cultures were inoculated with DMEM-BSA. After 1-h incubation at 4°C the cultures were washed, fixed and immune-stained using anti-HK primary antibodies and HRP-labelled secondary antibodies. The mean absorbance in the control cultures was subtracted, and the results were expressed as the relative absorbance at 450 nm (A₄₅₀) in R5-treated and HK-treated cultures with respect to the mean absorbance in the latter. Open circles, closed circles and crosses depict individual data points from three experiments performed on different days. Mean, SD and P values were calculated using within-day averages. (b) Control of the concentrations of physical viral particles in suspensions of HK and R5 used for the HTBE attachment experiments. Suspensions were serially diluted in PBS and adsorbed in the wells of ELISA microplates. The wells were washed, fixed and immuno-stained as described above. Shown are the results of one experiment with 5 replicates per condition. The absorbance (A₄₅₀) reflects non-specific binding of HK and R5 to the plastic. Overlap of the A₄₅₀ vs dilution curves indicate that suspensions used in the Fig 5a contained equal amounts of viral particles.

Fig 6. Diameter of plaques formed by viruses in MDCK cells.

Cells in six-well plates were inoculated, incubated under semi-solid overlay medium for 48 h at 37°C, fixed and immunostained. Two panels represent two groups of viruses tested separately. Data were analysed after log-transformation using a general mixed model including a random intercept term accounting for day-to-day variation between experiments as described in Materials and Methods. Each panel shows diameters of individual plaques adjusted for day, geometric mean (bars) and geometric SDs from 1 to 4 experiments performed on different days. Asterisks depict P values for the differences between the mutants and the corresponding parental virus (HK in the left panel and R5 in the right panel).

Fig 7. Infectivity and multicycle replication of HK and R5 in HTBE cultures.

(a) The apical sides of HTBE cultures were washed with PBS+ to remove mucins and inoculated with 2x10⁴ FFU of HK (closed circles) and R5 (open circles) with and without addition of mucins using 6 replicate cultures per condition. The inoculum was removed after 1 h. The cultures were incubated for 7 h under ALI conditions, fixed, immuno-stained for viral NP, and numbers of infected cells were counted. (b) The apical sides of washed HTBE cultures were inoculated with 7x10⁴ FFU of the viruses without addition of mucins and processed as described above. Six cultures per virus were fixed 8 h post infection for immuno-staining and counting of infected cells. Viral progeny was periodically harvested by washing the apical sides of the remaining cultures, and the harvests were titrated simultaneously at the end of the experiment. P values were determined using Student’s t-test.

Fig 8. Competitive replication of HK and its 6 single-point HA mutants in HTBE cultures.

HTBE cultures were inoculated with the mixtures of HK and its 6 HA mutants containing 5 PFU (L, 12 replicate cultures), 20 PFU (M, 12 replicates) and 320 PFU (H, 6 replicates) of each virus. After 1-h incubation, the inoculum was removed, the cultures were incubated under ALI conditions, and the apical material was harvested at 72 h post-inoculation. Small filled circles show proportions of each HA genotype determined by next generation sequencing in the inoculated mixture (In) and in each replicate harvest in the L, M and H groups. Empty squares and error bars represent the mean values and confidence intervals of proportions. Asterisks depict statistical significance of the differences between proportion of the corresponding genotype in the harvest and in the inoculum. The proportions of the parent HK virus (shown on gray background) were inferred by subtracting proportions of the six HA variants from a theoretical value of 1. Because of the high intrinsic errors of this approach the data for HK were not analysed for statistical significance.

Fig 9. Comparison of airborne transmission of HK and R5 in ferrets.

Groups of two ferrets were inoculated intranasally with 10⁶ TCID₅₀ of recombinant viruses HK (top panels) and R5 (bottom panels) containing all eight gene segments of A/Hong Kong/1/1968. One naïve ferret was co-housed with each inoculated ferret in a separate transmission cage starting from one day after inoculation. Data show results of two replicate experiments performed on different days. Lines depict viral titers in nasal swabs (empty circles) and throat swabs (closed circles) collected from inoculated ferrets. White and black bars depict viral titers in nasal and throat swabs, respectively, of the indirect contact ferrets. Numbers show titers of hemagglutination inhibiting antibodies in the blood collected from the indirect contact animals 2 weeks post exposure.

Fig 10. Host-specific lineages of IAVs with H3 HA and variation of amino acids at selected HA positions.

(a) Phylogenetic tree for the H3 HA nucleotide sequences of representative sequences of human and swine viruses and all unique sequences of other mammalian and avian viruses available from GISAID EpiFlu database. The numbers of analysed sequences are shown in panel 10b below the lineage name. S5 Fig shows the same tree with strain names, accession numbers and amino acids at 9 HA positions under study. (b) Protein logos for indicated HA positions of the viral lineages shown in panel 10a. The overall height of each stack of letters depicts sequence conservation measured in bits. The height of each letter is proportional to the frequency of the corresponding amino acid in the alignment, the letters are ordered from most to least frequent. Only one sequence was available for the Eq/Jilin/1989 lineage.