Target cell membrane sialic acid modulates both binding and fusion activity of influenza virus

Maria C Pedroso De Lima,João Ramalho-Santos,Diana Flasher,Vladimir A Slepushkin,Shlomo Nir,Nejat Düzguneş

doi:10.1016/0005-2736(95)00067-d

Abstract

Influenza virus binds to cell surface sialic acid receptors, and following endocytosis fuses with the endosome membrane at low pH. Whether sialic acid plays a role in the virus-cell membrane fusion step is not known. We investigated the effect of the removal of cell membrane sialic acid on the fusion activity of influenza virus (A/PR/8/34 strain) toward human T lymphocytic leukemia (CEM) cells at low pH. Fusion was monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Removal of sialic acid by neuraminidase resulted in a drastic reduction in both viral binding and fusion. The association of the virus with neuraminidase-treated cells was enhanced at pH 5, compared to that at neutral pH, probably due to the unfolding of the hemagglutinin and the resulting increase in viral surface hydrophobicity, but the fusion capacity of the virus was reduced significantly. The results were analysed with a mass-action kinetic model which could explain and predict the kinetics of fusion. Our results indicate that binding of influenza virus to sialic acid residues on the cell surface leads to rapid and extensive fusion and partially inhibits the low pH-induced viral inactivation.

Highlights

Cell surface sialic acid residues are considered to be the primary receptors for influenza virus [1,2,3,4]
We investigated the role of cell surface sialic acid residues in influenza virus fusion with CEM cells using this assay
Several observations indicate that cell surface sialic acid residues modulate the fusion activity of influenza virus, as well as mediating its binding to the cell surface: (i) The fusion of virions prebound to neuraminidasetreated cells is inhibited compared to fusion with control cells (Fig. lb). Under these conditions where the unbound virions were removed, the receptor binding step is bypassed, and the fluorescence dequenching provides a measure of the fusion of virions which were already bound to the cells, possibly via non-sialic acid receptors. (ii) The fusion rate constant is decreased significantly when the sialic acid receptors are removed (Table 1). (iii) Prebinding of the virus to cells at 37° C does not enhance fusion with cells treated with neuraminidase, in contrast to control cells (Fig. 2)

Summary

Introduction

Cell surface sialic acid residues are considered to be the primary receptors for influenza virus [1,2,3,4]. Structural studies of the influenza hemagglutinin have shown sialic acid as the main component of the cellular receptor for the virus [8]. It has been suggested that the interaction of gangliosides GT1b or Grab with influenza virus hemagglutinin causes an increase in the fluidity of the viral envelope, thereby promoting fusion with target membranes [12]. We previously examined the low pH-induced fusion of influenza virus with the plasma membranes of living CEM cells as a model to study the fusion of the virus with endosome membranes [13]. We have utilized this model system to investigate the role

Methods

Results

Conclusion