Enhancement of the Influenza A Hemagglutinin (HA)-Mediated Cell-Cell Fusion and Virus Entry by the Viral Neuraminidase (NA)

Bin Su,Marie-Anne Rameix-Welti,Sylvie van der Werf,Béatrice Labrosse,Sébastien Wurtzer,Dominic Dwyer,François Clavel,Nadia Naffakh,Robert J. Geraghty

doi:10.1371/journal.pone.0008495

Abstract

BackgroundThe major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity.Methodology/Principal FindingsThe role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogeneous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA.Conclusion/SignificanceThe NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.

Highlights

Influenza A viruses are enveloped viruses expressing two major transmembrane glycoproteins incorporated into the viral envelope: the hemagglutinin (HA), and the neuraminidase (NA)
When the precursor carries a monobasic cleavage site, which is the case in most influenza A strains, HA0 is cleaved on the cell surface during natural infection by extracellular trypsin-like proteinase [20]
Using a cell-cell fusion assay and an HIV-based pseudotype infectivity assay, we demonstrate here that NA expression can directly enhance HA-dependent influenza virus fusion and infectivity in a manner that is both dose-dependent and activitydependent

Summary

Introduction

Influenza A viruses are enveloped viruses expressing two major transmembrane glycoproteins incorporated into the viral envelope: the hemagglutinin (HA), and the neuraminidase (NA). The HA glycoproteins are expressed as trimers at the surface of virions [1,2] and mediate viral entry into target cells through recognition and binding to terminal sialic acid groups on membrane-bound proteins and lipids of the host cell. The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity

Methods

Results

Conclusion