Receptor binding specificity of recent human H3N2 influenza viruses

Abstract

Background: Human influenza viruses are known to bind to sialic acid linked alpha2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection.

Results: Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of alpha2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for alpha2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of alpha2-6 and alpha2-3 sialic acids, including human A549 and HeLa cells with high levels of alpha2-6 sialic acid, and CHO cells that have only alpha2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells.

Conclusion: Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of alpha2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of alpha2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with alpha2-6 receptor specificity.

Figure 1

Hemagglutination titers of recent viruses with chicken and human red blood cells. The viruses were grown in MDCK cells and concentrated by centrifugation to obtain a ratio of HA titer with chicken red cells compared to human red cells. For Mem/31/98, OK/323/03 and OK/369/05 there was still no agglutination of chicken red blood cells. The 2006 viruses were not concentrated. The data are average ± standard deviation of 3 experiments.

Figure 2

Binding of viruses to the Glycan Array. Glycan printed array V2 binding analyses of A/Oklahoma/323/03 (A), egg adapted vaccine reassortant A/Wyoming/03/03 X-147 (B), A/Oklahoma/369/05 (C), A/Oklahoma/1992/05 (D), and an older vaccine strain A/Phillipines/2/82 X-79 (E). The microarray slides used for these experiments had 246 glycans, shown along the X axis. The shaded regions show the α2-6-sialylsaccharides. The fluorescence is the average of 4 replicate spots ± standard error after the highest and lowest readings of six were excluded. OK/369/05 and OK/323/03, that do not bind chicken red cells, bind to the same α2-6-sialylsaccharides as do agglutinating and vaccine strain egg grown viruses.

Figure 3

Binding motifs of H3N2 viruses. The minimal motifs bound by 2003–2005 H3N2 viruses (A) and A/Philippines/2/82/X-79 (B). Recent viruses require a minimum of 3 sugars to bind while Philippines binds a disaccharide. It can accommodate an N-glycolyl in place of N-acetyl on the terminal sialic acid.

Figure 4

Hemadsorption of chicken and human red blood cells to cell surface-expressed HA. Red cells were bound to recombinant HA expressed on the surface of Sf-9 cells and to influenza HA expressed on MDCK cells. Bound red cells were lysed with water and released hemoglobin was measured at 540 nm. The results shown are an average of 4 experiments ± standard deviation.

Figure 5

Binding of biotinylated α2-3- and α2-6- linked sialic acid PAA-compounds to Memphis/31/98 (HG). Oligosaccharide bound to virus immobilized in wells of a microtiter plate was detected by AP-streptavidin as described in the Methods. The data points are mean of triplicates ± standard deviation. The line is the fit of the data to the standard binding equation generated using Kaleidagraph software. A similar experiment that shows the binding specificity of NWS-Mem/98 has been published [11].

Figure 6

Binding, internalization and synthesis of viral proteins in different cell lines. Surface binding of virus (4°C, 1hr), internalization (37°C,1hr), replication to amplify NP (37°C, 6 hr), and cell surface expression of HA seen by hemadsorption at 18 hr (rbc binding). Immuno staining and red cell binding was done as described in Methods.