Glycosylation within an antigenic site on the HN glycoprotein of Newcastle disease virus interferes with its role in the promotion of membrane fusion.

The binding of monoclonal antibodies to antigenic site 3 on the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus neutralizes viral infectivity and prevents syncytium formation by a mechanism other than the prevention of viral attachment. The virus can escape neutralization by these antibodies by the addition of an N-glycan at a site introduced by a D287N mutation in HN. The variant has significantly reduced ability to induce fusion from within, the mode of fusion promoted by the viral glycoproteins deposited on the cell surface late in infection. Conversely, and unlike the parent virus, the variant has acquired the ability to promote fusion from without, the mode of fusion directly mediated by input virions at high multiplicity. This finding is consistent with different roles for the HN protein in virion-cell and cell-cell fusion. D287N-mutated HN with its additional N-glycan shows a markedly reduced ability, compared to wild-type HN, to complement the viral fusion protein in the promotion of fusion in a BHK cell transient expression system. This confirms that the addition of an N-glycan in HN antigenic site 3 and the deficiency in syncytium formation are causally related. Moreover, no alteration in cell surface expression, hemadsorption, or neuraminidase activity was detected in the mutated protein. This monoclonal antibody-selected mutation suggests that a fusion-related function, secondary to receptor recognition, may be defined by the globular head of the HN spike. However, D287C or D287S-mutated HN is as effective as the wild-type protein in the promotion of fusion in the coexpression system. This suggests that the diminished fusogenicity of the D287N-mutated protein is probably due to a more global effect of glycosylation in site 3 rather than an alteration at the amino acid level.