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The Role of NA in Cell Fusion & Virus Entry

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  • The Role of NA in Cell Fusion & Virus Entry

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

    Background
    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.

    Methodology/Principal Findings
    The 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/Significance
    The 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.

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    We then determined the pH dependence of the fusion process. Not surpringly, cell fusion was only obtained when cocultures were transiently treated with acidic buffer at pH 5.0, while no fusion was observed at neutral pH (7.0).

    Not surprisingly, the infectivity of H1-expressing pseudoparticles was not affected when the defective N1-Y406F mutant was co-expressed, regardless of the plasmid ratios tested. These results again suggest that the potentialisation of the entry process mediated by the HA envelope proteins is related to the sialidase activity of NA, but that excess NA may interfere with this enhancement effect.

    Interestingly, after normalization for HIV-1 p24 content, no difference in infectivity was seen between these two viral preparations, demonstrating that the NA-mediated infectivity increase in influenza HA pseudoparticles is the consequence of NA activity on a substrate that is expressed at the surface of virions, rather that at the surface of target cells.

    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.

    Several virological functions have been assigned to the sialidase activity of influenza NA. First, it has been known for some time that NA activity facilitates the release of progeny virions from infected cells by cleaving the interactions between HA proteins and terminal sialic acid expressed on the cell surface or on adjacent virions. Indeed, efficient propagation in cell culture of mutant viruses lacking sialidase activity requires addition of large amounts of exogenous soluble neuraminidase to the medium.

    Second, the NA protein is able to cleave sialic acid residues in mucus and cellular glycocalyx, a process that is believed to facilitate diffusion and dissemination of the virus within the respiratory tract, giving influenza virions larger access to target cells of the airway epithelium.

    Third, it was also proposed that the NA protein could restrict the rate of superinfection of influenza A target cells by removing surface sialic acid receptors, thereby limiting the emergence of reassortant virus variants. The data presented in our study now establish that neuraminidase can exert a direct enhancing effect on influenza infectivity and membrane fusion.

    PLoS http://tinyurl.com/y9zhp8s
    The salvage of human life ought to be placed above barter and exchange ~ Louis Harris, 1918

  • #2
    Re: The Role of NA in Cell Fusion & Virus Entry

    Question for other FT memebers: do these findings support a hypothesis that use of NA inhibitors post infection may pre-dispose individuals/ increase risks of co-infection (non removal of scialic acid receptors) and therefore increase risks of re-assortment? Does anyone know of any trial work looking at this issue?

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