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

Date Published: December 30, 2009

Publisher: Public Library of Science

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

Abstract: 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.

Partial Text: Influenza A viruses are enveloped viruses expressing two major transmembrane glycoproteins incorporated into the viral envelope: the hemagglutinin (HA), and the neuraminidase (NA). Based on the antigenicity of hemagglutinin and neuraminidase, influenza A viruses are subdivided into 16 HA (H1-H16) and 9 NA (N1-N9) subtypes.

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 activity-dependent. Enhancement of fusion and infectivity increased with increasing the amount of NA protein at the surface of cells and of virions, until a plateau was reached. This effect was strictly dependent of the enzymatic activity of NA, as an active-site mutant of the enzyme (Y406F) had no impact on HA-mediated fusion and infectivity. Our results also strongly suggest that the impact of NA on infectivity and fusion was mediated by desialylation of residues expressed in virus producer cells or on virions, as opposed to desialylation of residues on target cells. Indeed, the extent that NA increased infectivity of pseudoparticles was comparable whether NA was coexpressed at the surface of virions or added to the pseudoparticle suspension as a soluble bacterial sialidase. In the latter case, enhancement of infectivity was detected even after ultracentrifugation and removal of soluble NA from the inoculum, thereby excluding an effect of NA on target cell sialic acid residues. This clearly rules out a non-specific enhancement through removal of repulsive negative charges from cell surfaces, similar to that described by Sun et al. [23], [24], where propagation and syncytium formation by HIV-1, a virus that does not express NA, was enhanced by sialidase treatment of the infected cultures.



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