Individual N-glycans added at intervals along the stalk of the Nipah virus G protein prevent fusion but do not block the interaction with the homologous F protein.

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F.

Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding.

Role of the two sialic acid binding sites on the newcastle disease virus HN protein in triggering the interaction with the F protein required for the promotion of fusion.

Newcastle disease virus (NDV)-induced membrane fusion requires an interaction between the hemagglutinin-neuraminidase (HN) attachment and the fusion (F) proteins, triggered by HN's binding to receptors. NDV HN has two sialic acid binding sites: site I, which also mediates neuraminidase activity, and site II, which straddles the membrane-distal end of the dimer interface. By characterizing the effect on receptor binding avidity and F-interactive capability of HN dimer interface mutations, we present evidence consistent with (i) receptor engagement by site I triggering the interaction with F and (ii) site II functioning to maintain high-avidity receptor binding during the fusion process.

Paramyxoviruses: different receptors - different mechanisms of fusion.

Paramyxovirus-mediated membrane fusion usually requires an interaction between the viral-attachment and -fusion proteins. The mechanism by which this interaction regulates fusion differs between paramyxoviruses that bind to sialic acid-containing receptors and those that recognize specific proteins. The recently solved structure of the globular head of the measles virus hemagglutinin suggests that this difference might be related to the location of the receptor-binding sites on the attachment proteins of the two classes of paramyxoviruses.

Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion.

The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37 degrees C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.

An oligosaccharide at the C-terminus of the F-specific domain in the stalk of the human parainfluenza virus 3 hemagglutinin-neuraminidase modulates fusion.

The promotion of membrane fusion by the fusion (F) protein of human parainfluenza virus 3 (hPIV3) is dependent on a virus-specific contribution from the hemagglutinin-neuraminidase (HN) protein. By evaluation of chimeric hPIV3-Newcastle disease virus (NDV) HN proteins, we have previously shown that hPIV3-F-specificity is determined by a domain that extends from the middle of the membrane anchor to the 82nd residue in the ectodomain [Virology 209, (1995) 457; Arch. Virol. 13 (1997) 115]. If the corresponding NDV-derived residues replace the two C-terminal residues in this domain, no fusion is detected. However, these substitutions restore a glycosylation site present in NDV HN, but not in hPIV3 HN. Deletion of this site from a nested set of chimeras with hPIV3-derived N-terminal portions of decreasing length partially restores fusion, suggesting that an oligosaccharide near the top of hPIV3 HN stalk modulates fusion. In addition, further mutational analyses show that a chimera with only 125 N-terminal hPIV3-derived residues (72 in the stalk) actually promotes fusion more efficiently than the wt protein. These findings localize the C-terminus of the F-specific domain in hPIV3 HN a full 10 residues closer to the membrane than previously shown.

Glycoprotein interactions in paramyxovirus fusion.

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus.