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.     

Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease virus hemagglutinin-neuraminidase is due to a temperature-dependent defect in receptor binding

The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37 degrees C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.     

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.     

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.     

Biological significance of the second receptor binding site of Newcastle disease virus hemagglutinin-neuraminidase protein

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-alpha(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.     

The second receptor binding site of the globular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.     

Second sialic acid binding site in Newcastle disease virus hemagglutinin-neuraminidase: implications for fusion

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.     

Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.     

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN₀) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase β-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN₀ and associated reduced virulence.     

Interacting domains of the HN and F proteins of newcastle disease virus

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.     

Mutations in the stalk of the measles virus hemagglutinin protein decrease fusion but do not interfere with virus-specific interaction with the homologous fusion protein

The hemagglutinin (H) protein of measles virus (MV) mediates attachment to cellular receptors. The ectodomain of the H spike is thought to consist of a membrane-proximal stalk and terminal globular head, in which resides the receptor-binding activity. Like other paramyxovirus attachment proteins, MV H also plays a role in fusion promotion, which is mediated through an interaction with the viral fusion (F) protein. The stalk of the hemagglutinin-neuraminidase (HN) protein of several paramyxoviruses determines specificity for the homologous F protein. In addition, mutations in a conserved domain in the Newcastle disease virus (NDV) HN stalk result in a sharp decrease in fusion and an impaired ability to interact with NDV F in a cell surface coimmunoprecipitation (co-IP) assay. The region of MV H that determines specificity for the F protein has not been identified. Here, we have adapted the co-IP assay to detect the MV H-F complex at the surface of transfected HeLa cells. We have also identified mutations in a domain in the MV H stalk, similar to the one in the NDV HN stalk, that also drastically reduce fusion yet do not block complex formation with MV F. These results indicate that this domain in the MV H stalk is required for fusion but suggest either that mutation of it indirectly affects the H-dependent activation of F or that the MV H-F interaction is mediated by more than one domain in H. This points to an apparent difference in the way the MV and NDV glycoproteins interact to regulate fusion.     

Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity--5'5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody--inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.     

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.     

A second receptor binding site on human parainfluenza virus type 3 hemagglutinin-neuraminidase contributes to activation of the fusion mechanism

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Structural and functional relationship between the receptor recognition and neuraminidase activities of the Newcastle disease virus hemagglutinin-neuraminidase protein: receptor recognition is dependent on neuraminidase activity

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.     

Decreased dependence on receptor recognition for the fusion promotion activity of L289A-mutated newcastle disease virus fusion protein correlates with a monoclonal antibody-detected conformational change

It has been shown that the L289A-mutated Newcastle disease virus (NDV) fusion (F) protein gains the ability to promote fusion of Cos-7 cells independent of the viral hemagglutinin-neuraminidase (HN) protein and exhibits a 50% enhancement in HN-dependent fusion over wild-type (wt) F protein. Here, we show that HN-independent fusion by L289A-F is not exhibited in BHK cells or in several other cell lines. However, similar to the results in Cos-7 cells, the mutated protein plus HN does promote 50 to 70% more fusion above wt levels in all of the cell lines tested. L289A-F protein exhibits the same specificity as the wt F protein for the homologous HN protein, as well as NDV-human parainfluenza virus 3 HN chimeras. The mutated F protein promotes fusion more effectively than the wt when it is coexpressed with either the chimeras or HN proteins deficient in receptor recognition activity. In addition, its fusogenic activity is significantly more resistant to removal of sialic acid on target cells. These findings are consistent with the demonstration that L289A-F interacts more efficiently with wt and mutated HN proteins than does wt F by a cell surface coimmunoprecipitation assay. Taken together, these findings indicate that L289A-F promotes fusion by a mechanism analogous to that of the wt protein with respect to the HN-F interaction but is less dependent on the attachment activity of HN. The phenotype of the mutated F protein correlates with a conformational change in the protein detectable by two different monoclonal antibodies. This conformational change may reflect a destabilization of F structure induced by the L289A substitution, which may in turn indicate a lower energy requirement for fusion activation.     

Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process

Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.     

Role of the hemagglutinin-neuraminidase protein in the mechanism of paramyxovirus-cell membrane fusion

Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth beta sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.