Recent evidence suggests that the attachment (HN) and fusion (F) glycoproteins of Newcastle disease virus interact at the cell surface in a virus-specific manner to promote syncytium formation. Consistent with the existence of such an interaction, we have shown that it is possible to coimmunoprecipitate (co-IP) the two proteins from the surface of transiently expressing cells using a monoclonal antibody to either protein. Further, we show that a point mutation in the globular domain of HN that abolishes its receptor recognition and neuraminidase (NA) and fusion activities also abolishes its ability to interact with F in the co-IP assay. The mechanism by which this mutation might interfere with the interaction between the two proteins is discussed in terms of the postulate that recognition by HN of cellular receptors triggers its interaction with F and the apparently conflicting evidence for an interaction between the two proteins in the endoplasmic reticulum. Also, characterization of a set of chimeric HN proteins, having short overlapping sequences from a heterologous HN protein in the F-specific domain in the protein stalk, reveals that a weakened interaction between HN and F is still sufficient to trigger 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.

The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.The paramyxoviruses are enveloped, negative-stranded RNA viruses, including human parainfluenza viruses types 1 to 4, mumps virus, and the animal pathogens Newcastle disease virus (NDV), Sendai virus, and simian virus 5. The hemagglutinin-neuraminidase (HN) glycoprotein spike not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, sialic acid (35). The presence of both receptor recognition and NA activities on the same protein is in contrast to influenza virus, in which the two activities reside on independent spike structures.The HN spike is a type II homotetramer. The ectodomain consists of a long stalk, supporting a terminal globular head, in which reside the receptor recognition, NA, and antigenic sites (26, 42). All HN tetramers are pairs of dimers. In the case of the Australia-Victoria isolate of NDV (NDV-AV), the monomers in each dimer are disulfide linked via a cysteine at position 123 in the stalk region (26). NDV HN utilizes four of its six potential glycosylation sites. Elimination of two of them, G1 and/or G2, results in an increase in hemadsorption (HAd) activity (24).Based on the conservation in HN of amino acids in the active site of the influenza NA protein, the globular head of the HN spike has been predicted to have a six ␤-sheet propeller structures, similar to the influenza virus protein; putative NA activesite residues are contributed by five of the six ␤-sheets (5, 21). This prediction is consistent with the antibody escape mutant mapping of discontinuous epitopes on the NDV (17, 19), human parainfluenza virus type 3 (hPIV3) (4), and simian virus 5 (1) HN proteins. In the influenza virus NA protein, residue D151 is thought to be important to catalysis by virtue of its position within hydrogen-bonding distance of the glycosidic oxygen of the substrate (2, 44). Several different substitutions for the ...

Paramyxovirus-mediated membrane fusion usually requires an interaction between the viralattachment 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. Paramyxoviruses use different receptorsThe Paramyxoviruses are enveloped, negative-stranded RNA viruses, each of which contains two surface glycoproteins, an attachment protein and a fusion (F) protein [1]. The most common type of paramyxovirus attachment protein, called the hemagglutinin-neuraminidase (HN), found on viruses, such as Newcastle disease virus (NDV), human parainfluenza virus 3 (hPIV3) and parainfluenza virus 5 (PIV5) (known formerly as simian virus 5), recognizes the ubiquitous sugar, sialic acid, on cell-surface moieties and has the ability to cleave the same moiety by virtue of its neuraminidase (NA) activity [1]. The structures of the globular heads of all of the above HN proteins [2-4] reveal a conserved β-sheet propeller motif, as identified originally in the influenza virus NA protein [5], but with a sialic acid-binding site on each monomer of the homotetramer that can mediate both receptor binding and NA (hereafter referred to as the NA site). A second binding site was identified subsequently in NDV HN [6] and has been postulated in hPIV3 HN [7]. Unlike HN, the hemagglutinin (H) of measles virus (MV) lacks NA and recognizes specific proteins. Although both wild-type and vaccine strains recognize signal lymphocyte-activating molecule (SLAM), vaccine strains also use CD46 as a receptor [8][9][10][11]. The recent publication by two groups of the structure of the globular domain of the MV H protein [12,13] provides our first look at a paramyxovirus attachment protein that binds to a specific protein receptor, revealing differences from the HN structures that might be related to the differences in the mechanisms by which these viruses mediate fusion. Location of receptor-binding sites on paramyxovirus attachment proteinsThe NA site in the various HN proteins is highly conserved and located in a pocket a short distance from the dimer interface (Figure 1a). The second sialic acid-binding site in NDV HN spans the membrane-distal end of the dimer interface and is composed of residues from both

The fusion (F) proteins of Newcastle disease virus (NDV) and