Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation

Abstract

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.Influenza virus hemagglutinin (HA) mediates virus entry into a host cell through two functions, receptor binding and membrane fusion. HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem (26). The receptor binding site is located in the head region, while the fusion site is carried by the stem region. Proteolytic cleavage of HA into the HA1 and HA2 subunits is a prerequisite for fusion activity, and cleaved HA mediates membrane fusion under acidic conditions (11,22,25).The HA1 and HA2 subunits are functionally specialized; HA1 binds to cellular receptors, and HA2 mediates membrane fusion. It is generally accepted that HA1 and HA2 are functionally independent of each other; for example, H7/Rostock HA-expressing cells show high cell fusion activity even when receptor binding activity is not detectable (16). Epand et al. (8) and Leikina et al. (12) showed that, in the absence of HA1, the ectodomain of HA2 promotes lipid mixing between liposomes and cell-cell hemifusion (fusion of outer leaflets of lipid bilayer membranes), respectively.On the other hand, there are several reports indicating that HA1 is important for the function of HA2. The fusion-active form of HA2 is believed to be maintained by a structural constraint of the HA1 region (3, 4, 5). Ramalho-Santos and Pedriso de Lima (21) proposed that receptor binding activity plays an essential role...

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