Glycoproteins of Sendai virus (HVJ) have a critical ratio for fusion between virus envelopes and cell membranes

The biological activity of two glycoproteins, hemagglutinin and neuraminidase (HN) and fusion (F) proteins, of Sendai virus (HVJ) were studied using purified proteins. The proteins were purified by chromatography on DEAE and CM cellulose in the presence of Nonidet P-40 (NP40). The glycoproteins were reconstituted at various ratios of F to HN into lipid vesicles containing fragment A of diphtheria toxin. The association of HN and F proteins with the vesicles was confirmed by electron microscopy and sucrose density gradient centrifugation. The cytotoxic activity of vesicles containing fragment A on fusion with L cells was determined by measuring colony formation of the cells. It was found that for maximum cytotoxic activity of the vesicles, there was an optimal ratio of F to HN of two. This suggests that HN is not merely the initial binding site to the cell surface, and that interactions between HN and F proteins on the virus surface may be important for the biological activities of these proteins on the cells.     

Monoclonal antibodies to hemagglutinin-neuraminidase and fusion glycoproteins of Newcastle disease virus: relationship between glycosylation and reactivity.

Eighteen hybridoma lines obtained by immunization of mice with Newcastle disease virus (NDV) lentogenic strain La Sota or velogenic strain Italien produced hemagglutinating monoclonal antibodies. The 18 monoclones were divided into four groups according to their reactivity toward native hemagglutinin neuraminidase protein (HN), nonglycosylated HN precursor, and heat-denatured HN blotted on nitrocellulose membranes. Only group II reagents were reactive toward their targets in all conditions tested. They were considered sequence-specific antibodies. Group I antibodies did not require glycosylation but lacked reactivity towards the denatured glycosylated antigen. Monoclonal antibodies from group III recognized only the native HN. Group IV was made up of a single monoclone that lacked reactivity with NDV Italien but recognized the La Sota strain in hemagglutination inhibition and enzyme-linked immunosorbent assays. Five hybridoma lines produced monoclonal antibodies which neutralized viral infectivity but failed to inhibit hemagglutination. One monoclonal antibody obtained after immunization of mice with NDV La Sota showed a low neutralization index versus NDV Italien. Four monoclonal antibodies derived from mice immunized with NDV Italien showed higher neutralization indices towards this strain. Neither the denatured F protein nor its nonglycosylated precursor was reacted against by the five monoclonal antibodies.     

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.     

HN glycoprotein of HVJ (Sendai virus) enhances the selective cytotoxicity of diphtheria toxin fragment A-containing liposomes on subacute sclerosing panencephalitis virus-infected cells

Fragment A of diphtheria toxin-containing liposomes (naked liposomes) selectively kill subacute sclerosing panencephalitis virus-infected cells (SSPE cells) (Exp cell res 132 (1981) 259) [10]. Fragment A-containing liposomes associated with either hemagglutinating and neuraminidase (HN) or fusion (F) glycoprotein of HVJ (Sendai virus) were prepared. These liposomes did not kill normal cultured cells. Fragment A-containing liposomes associated with HN protein were much more cytotoxic than naked liposomes containing fragment A to SSPE cells. Their cytotoxicity to the SSPE cells was influenced by the duration of incubation and the amount of HN protein. Fragment A-containing liposomes associated with F protein had about the same cytotoxicity on SSPE cells as had naked liposomes containing fragment A. Fragment A-containing liposomes associated with wheat germ agglutinin (WGA) were also prepared, but these also had the same toxicity as naked liposomes containing fragment A. The effects of monoclonal antibodies against HN protein on the cytotoxicity on SSPE cells of fragment A-containing liposomes associated with HN were studied. The significance of these results with regard to the actions of HN protein and possible reasons for the selective killing of SSPE cells are discussed.     

Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion

Fluorescence photobleaching recovery has been employed to study the lateral mobility of the Sendai virus envelope glycoproteins (HN, neuraminidase/hemagglutinin protein (HN) fusion protein (F) on the surface of human erythrocytes. Our results indicate that the two viral glycoproteins are laterally immobile on the cell surface prior to fusion, and become mobile during the fusion process. The two fused glycoproteins are mobilized to the same extent (diffusion coefficients of 3.1-3.3 X 10(-10) cm2/sec with mobile fractions of 0.53-0.57 for both HN and F). Their mobilization is blocked under conditions that allow virus adsorption and hemagglutination, but not virus-cell or cell-cell fusion. These findings suggest a possible role for the lateral diffusion of the viral glycoproteins in the mechanism of cell-cell fusion, enabling them to perturb the membranes of adjacent cells and lead to cell-cell fusion.     

Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.     

Influence of the human parainfluenza virus 3 attachment protein's neuraminidase activity on its capacity to activate the fusion protein

In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. F activation was assessed by quantitating the irreversible binding of target erythrocytes (RBC) to HN/F-coexpressing cells in the presence of 4-GU-DANA (zanamivir) to release target cells bound only by HN-receptor interactions; the remaining, irreversibly bound target cells are retained via the fusion protein. In cells coexpressing wild-type (wt) or variant HNs with wt F, the fusion promotion capacity of HN was distinguished from target cell binding by measuring changes with time in the amounts of target RBC that were (i) reversibly bound by HN-receptor interaction (released only upon the addition of 4-GU-DANA), (ii) released by HN's neuraminidase, and (iii) irreversibly bound by F-insertion or fusion (F triggered). For wt HN, lowering the pH (to approach the optimum for HPIV3 neuraminidase) decreased F triggering via release of HN from its receptor. An HN variant with increased receptor binding avidity had F-triggering efficiency like that of wt HN at pH 8.0, but this efficiency was not decreased by lowering the pH to 5.7, which suggested that the variant HN's higher receptor binding activity counterbalanced the receptor dissociation promoted by increased neuraminidase activity. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37 degrees C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HN's competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).     

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.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

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.     

Functional modification of Sendai virus by siRNA

Sendai virus (hemagglutinating virus of Japan; HVJ) is a negative-strand RNA virus with robust fusion activity, and has been utilized for gene transfer and drug delivery. Hemagglutinin-neuraminidase (HN) protein on the viral membrane is important for cell fusion, but causes agglutination of red blood cells. HN-depleted HVJ has been desired for in vivo transfection in order to improve safety. Here, we succeeded in producing HN-depleted HVJ using HN-specific short interfering RNA (siRNA). Viral production was not affected by the siRNA. HN protein was markedly decreased in the new HVJ, while other viral proteins were retained. Consequently, the hemagglutinating activity was substantially reduced and infection activity was suppressed. When the HN-depleted HVJ was mixed with cultured cells and the mixture was centrifuged for 10min at 2000xg, the modified HVJ recovered its infectivity to approximately 80% of wild HVJ. However, infectivity was abolished in the presence of anti-F antibody. Moreover, transfection of FITC-labeled oligodeoxynucleotides using the modified HVJ was also recovered by centrifugation. Thus, the HN-depleted HVJ produced using siRNA technology will be applicable to a delivery vector.     

Binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of F-glycoprotein of HVJ (Sendai virus): possible implication in the fusion reaction

Specificity of the binding of sterols and related compounds with purified F-protein (fusion protein) of the HVJ (Sendai virus) was studied by binding competition with [3H]cholesterol. Requirement for cholesterol or the A/B ring trans structure and nonrequirement for the 3-hydroxyl group were found in this binding. Binding of 125I-labeled Z-Phe-Tyr, an inhibitory peptide of viral membrane-cell membrane fusion, was studied by using purified proteins and virions. F-Protein and virions showed a specific binding with the peptide, whereas the result was negative with hemagglutinin and neuraminidase protein. Thermolysin-truncated F-protein (an F-protein derivative deprived of a 2.5-kDa fragment from the N-terminal of the F1 subunit and without fusogenic activity) exhibited a considerably diminished binding ability both to cholesterol and to inhibitory peptides. Therefore, the N-terminal hydrophobic sequence that was previously assigned as fusogenic seems to be the binding site of these molecules. In support of this, the binding of cholesterol with F-protein was inhibited by Z-Phe-Tyr and other fusion inhibitory peptides, whereas it was not affected with non-fusion-inhibitory Z-Gly-Phe. These results are discussed in relation to the notion that the binding of the N-terminal portion of the F1 subunit of F-protein with cholesterol in the target cell membranes facilitates the fusion reaction.     

Delineation of four antigenic sites on a paramyxovirus glycoprotein via which monoclonal antibodies mediate distinct antiviral activities

Six hybridoma antibodies specific for the hemagglutinin-neuraminidase (HN) molecule of the parainfluenza type 1 virus strain 6/94 were used to demonstrate the existence of four distinct antigenic sites on the HN molecule. Three of the sites (A, B-C, D) are topologically nonoverlapping, because antibodies to these sites bind noncompetitively to the HN molecule. Two sites (B, C) are operationally nonoverlapping, because mutations in site B do not detectably modify the antigenic site C. Although antibodies to each site had similar potencies (activity per microgram of antibody) in hemagglutination inhibition tests, antibodies to sites A and C or D differed approximately 100-fold in their potency to neutralize virus. Also, the antibody to site A strongly inhibited viral neuraminidase activity, whereas antibodies to sites C and D (and to a lesser extent to site B) enhanced the neuraminidase activity. Lastly, only antibodies to sites C and D formed precipitates in Ouchterlony double diffusion against detergent-disrupted virus. Because all six anti-HN antibodies are of IgG isotype and exhibited similar avidity for HN, these findings suggest that the ability of anti-HN antibodies to interact with the viral protein and to alter viral functions is largely dependent on their fine specificity.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

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.     

Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation

To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.     

Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.     

Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes

Receptor binding of paramyxovirus attachment proteins and the interactions between attachment and fusion (F) proteins are thought to be central to activation of the F protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F protein interactions and HN protein attachment to sialic acid receptors, HN and F protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN protein receptor binding, cells transfected with HN and F protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F protein-expressing cells resulted in a twofold decrease in amounts of HN and F protein complexes. In contrast, high levels of complexes containing HN protein and an uncleaved F protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F protein cleavage are necessary for disassociation of the HN and F protein-containing complexes.     

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.     

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.