Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Abstract

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in HA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in HA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

FIG. 1.

Recent H3N2 influenza viruses show reduced binding to synthetic α-2,3- and α-2,6-linked SA. K_ass for H3N2 influenza viruses (1968 to 2003) binding to 3′SL-PAA and 6′SLN-PAA were determined as a function of the gradient of the slope defined in Scatchard plots. Binding assays were run on different days, and an overall trend was observed. Representative data sets are shown.

FIG. 2.

H3N2 influenza viruses isolated between 1969 and 1996 sensitize target cells for NK lysis more efficiently than recent virus isolates (1999 to 2000). Representative results from ⁵¹Cr release assays comparing the percentage of specific lysis of uninfected 143BTK⁻ cells (dashed line) to that of cells infected with different influenza virus isolates by unseparated PBMC effectors (A) or isolated CD56⁺ CD3⁻ NK cells (B) at a range of E:T ratios. Solid lines and filled symbols are used to denote target cells infected with older virus isolates (1969 to 1996), and dotted lines and open symbols denote target cells infected with more recent viruses.

FIG. 3.

Summary of NK lysis of target cells infected with H3N2 influenza viruses isolated between 1969 and 1996 and recent virus isolates (1999 to 2003). Each panel shows the increase in the percentage of specific lysis at different E:T ratios induced by infection of target cells with older (1969 to 1996; squares) and more recent (1999 to 2003; triangles) virus isolates. The mean increase in the percentage of specific lysis induced by each group of viruses is indicated by the horizontal lines. Statistical analysis of the pooled data from all E:T ratios indicated that the difference in the increase in the percentage of specific lysis induced by the two groups of viruses was significant (P = 0.008).

FIG. 4.

NA treatment of NK cells reduces lysis of influenza virus-infected target cells. PBMCs were treated with NAs or were left untreated. The cells then were stained with DIG-labeled MAA or SNA followed by a fluorescein isothiocyanate-conjugated anti-DIG antibody to determine the expression of SA-α-2,3-Gal or SA-α-2,6-Gal, respectively. NK cells were identified by costaining with antibodies against CD3 and CD56. (A) The upper left panel shows the forward scatter (FSC) versus side scatter (SSC) profile of total PBMCs and the R1 gate set to identify the lymphocyte population. The upper right panel shows CD3 versus CD56 staining of the R1 population and the R2 gate set to identify the CD3⁻ CD56⁺ NK cell population. In the lower two panels, the gray-shaded histograms represent staining of gated NK cells without lectins, the solid lines represent NK cells without NA treatment, and the dotted lines represent NK cells with NA treatment. The results shown are representative of findings from three independent experiments using PBMCs from different donors. ⁵¹Cr release assays also were performed to investigate the percentage of specific lysis of uninfected 143BTK⁻ cells (B), 143BTK⁻ cells infected with A/England/878/69 (C), or 143BTK⁻ cells infected with A/England/24/00 (D) by control and NA-treated PBMCs at a range of E:T ratios. In each graph, the solid line represents the lysis mediated by control PBMCs, and the dotted line represents the lysis mediated by PBMCs treated with NAs. The data are means of triplicate values, and the results shown are representative of findings from three different experiments.

FIG. 5.

Manipulation of potential glycosylation site motifs in the HA of a recent virus. (A) Sequence analysis of HA from A/England/26/99 determined that it contained two new potential glycosylation site motifs at positions 122 to 124 and 133 to 135 that were not found in strains from the previous influenza season or from older viruses such as A/Victoria/3/75. Using site-directed mutagenesis, the new potential glycosylation site motif at 122 to 124 was destroyed by mutating the motif from NES to TEG to create G1Δ. Similarly, the new potential glycosylation site motif at 133 to 135 was destroyed by mutating the motif from NGT to NGG to create G2Δ. (B) Western blot analysis of HA proteins from purified virions obtained following infection of MDCK cells with recombinant influenza viruses bearing HA and NA genes of A/England/26/99 or HA modified as illustrated in panel A to alter the glycosylation motifs at amino acids 122 to 124 (G1Δ) or 133 to 135 (G2Δ).

FIG. 6.

Glycosylation on HA reduces the efficiency of lysis of influenza virus-infected cells. (A) Cell surface expression of modified HA proteins following infection of 143BTK⁻ cells with recombinant influenza viruses. 143BTK⁻ cells were infected with the viruses indicated and were incubated overnight, and then surface HA expression was detected using a polyclonal anti-H3-specific rabbit serum and fluorescent anti-rabbit secondary (2ry) antibody. The mean fluorescence intensity (MFI) of cells stained with the secondary antibody only (white bars) or with both the anti-HA antiserum and secondary antibody (black bars) is shown. (B) Data from a ⁵¹Cr release assay comparing the percent specific lysis of uninfected 143BTK⁻ cells (dashed line, filled squares) to those of cells infected with the A/Victoria/3/75 influenza virus isolate (solid line, filled squares) or recombinant influenza viruses 26/99 HA/NA (dashed line, filled triangles), G1Δ (solid line, open circles), or G2Δ (solid line, open squares) by PBMC effectors at a range of E:T ratios. These results are representative of findings from two independent experiments. Ab, antibody.