Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.     

Disulfide bond formation is a determinant of glycosylation site usage in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

Determinants of glycosylation site usage were explored by using the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus Newcastle disease virus. The amino acid sequence of the HN protein, a type II glycoprotein, has six N-linked glycosylation addition sites, G1 to G6, two of which, G5 and G6, are not used for the addition of carbohydrate (L. McGinnes and T. Morrison, Virology 212:398-410, 1995). The sequence of this protein also has 13 cysteine residues in the ectodomain (C2 to C14). Mutation of either cysteine 13 or cysteine 14 resulted in the addition of another oligosaccharide chain to the protein. These cysteine residues flank the normally unused G6 glycosylation addition site, and mutation of the G6 site eliminated the extra glycosylation found in the cysteine mutants. These results suggested that failure to form an intramolecular disulfide bond resulted in the usage of a normally unused glycosylation site. This conclusion was confirmed by preventing cotranslational disulfide bond formation in cells by using dithiothreitol. Under these conditions, the wild-type protein acquired extra glycosylation, which was eliminated by mutation of the G6 site. These results suggest that localized folding events on the nascent chain, such as disulfide bond formation, which block access to the oligosaccharyl transferase are a determinant of glycosylation site usage.     

Proteins of Newcastle disease virus envelope: interaction between the outer hemagglutinin-neuraminidase glycoprotein and the inner non-glycosylated matrix protein

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.     

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.     

Neutralization map of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition.

Monoclonal antibodies (MAbs) to the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus delineate seven overlapping antigenic sites which form a continuum on the surface of the molecule. Antibodies to five of these sites neutralize viral infectivity principally by preventing attachment of the virion to cellular receptors. Through the identification of single amino acid substitutions in variants which escape neutralization by MAbs to these five antigenic sites, a neutralization map of HN was constructed, identifying several residues that contribute to the epitopes recognized by MAbs which block the attachment function of the molecule. These epitopes are defined, at least in part, by three domains on HN: residues 193 to 201; 345 to 353 (which include the only linear epitope we have identified in HN); and a C-terminal domain composed of residues 494, 513 to 521, and 569. To identify HN residues directly involved in receptor recognition, each of the variants was tested for its ability to agglutinate periodate-modified chicken erythrocytes. One variant with a single amino acid substitution at residue 193 was 2.5- to 3-fold more resistant to periodate treatment of erythrocytes than the wild-type virus, suggesting that this residue influences the binding of virus to a sialic acid-containing receptor(s) on the cell surface.     

Fusion mutants of Newcastle disease virus selected with monoclonal antibodies to the hemagglutinin-neuraminidase.

The Australia-Victoria (AV) isolate of Newcastle disease virus (NDV) induces fusion from within but not fusion from without. L1, a neuraminidase (NA)-deficient virus derived from AV, has the opposite fusion phenotype from the wild-type virus. It fails to induce the former mode of fusion, but has gained a limited ability to promote the latter. Monoclonal antibodies to antigenic site 23 on the hemagglutinin-neuraminidase (HN) glycoprotein have previously been shown to select variants of the AV isolate that have altered NA activity or receptor-binding affinity. By using an antibody to this site, variants of L1 have been selected. Three of the variants have gained an increased affinity for sialic acid-containing receptors, as evidenced by the resistance of their hemagglutinating activity to the presence of reduced amounts of sialic acid on the surface of chicken erythrocytes. All four variants still have very low levels of NA activity, comparable to that of the parent virus, L1. The alteration in receptor-binding affinity results in a decreased potential for elution from cellular receptors and correlates with an increased ability to promote both modes of fusion. A single amino acid substitution in the HN protein of each variant, responsible for its escape from neutralization, has been identified. These studies identify two HN residues, 193 and 203, at which monoclonal antibody-selected substitution influences the receptor recognition properties of NDV and may influence its ability to promote syncytium formation.     

Intracellular processing of the Newcastle disease virus fusion glycoprotein.

The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site (Nagai et al., Virology 69:523-538, 1976) into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with [3H] fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.     

Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors

The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage.     

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.     

Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus

Viruses of the Paramyxoviridae family are the leading cause of respiratory disease in children. The human parainfluenza viruses (hPIV) are members of the Paramyxovirinae subfamily, which also includes mumps virus, Newcastle disease virus (NDV), Sendai virus (SV) and simian type 5 virus (SV5). On the surface of these viruses is the glycoprotein hemagglutinin-neuraminidase (HN), which is responsible for cell attachment, promotion of fusion and release of progeny virions. This multifunctional nature of HN makes it an attractive target for the development of inhibitors as a treatment for childhood respiratory diseases. Here we report the crystal structure of NDV HN in complex with a derivative of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, that has a functional group designed to occupy a large conserved binding pocket around the active site. The purpose of this study was to examine the effect of a bulky hydrophobic group at the O4 position of Neu5Ac2en, given the hydrophobic nature of the binding pocket. This derivative, with a benzyl group added to the O4 position of Neu5Ac2en, has an IC₅₀ of ∼10 μM in a neuraminidase assay against hPIV3 HN. The IC₅₀ value of the parent compound, Neu5Ac2en, in the same assay is ∼25 μM. These results highlight the striking difference between the influenza neuraminidase and paramyxovirus HN active sites, and provide a platform for the development of improved HN inhibitors.     

Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is an important determinant of its virulence. We investigated the role of each of the four functional N-linked glycosylation sites (G1 to G4) of the HN glycoprotein of NDV on its pathogenicity. The N-linked glycosylation sites G1 to G4 at residues 119, 341, 433, and 481, respectively, of a moderately pathogenic NDV strain Beaudette C (BC) were eliminated individually by site-directed mutagenesis on a full-length cDNA clone of BC. A double mutant (G12) was also created by eliminating the first and second glycosylation sites at residues 119 and 341, respectively. Infectious virus was recovered from each of the cDNA clones of the HN glycoprotein mutants, employing a reverse genetics technique. There was a greater delay in the replication of G4 and G12 mutant viruses than in the parental virus. Loss of glycosylation does not affect the receptor recognition by HN glycoprotein of NDV. The neuraminidase activity of G4 and G12 mutant viruses and the fusogenicity of the G4 mutant virus were significantly lower than those of the parental virus. The fusogenicity of the double mutant virus (G12) was significantly higher than that of the parental virus. Cell surface expression of the G4 virus HN was significantly lower than that of the parental virus. The antigenic reactivities of the mutants to a panel of monoclonal antibodies against the HN protein indicated that removal of glycosylation from the HN protein increased (G1, G3, and G12) or decreased (G2 and G4) the formation of antigenic sites, depending on their location. In standard tests to assess virulence in chickens, all of the glycosylation mutants were less virulent than the parental BC virus, but the G4 and G12 mutants were the least virulent.     

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.     

Expression of a cloned hemagglutinin-neuraminidase gene from Newcastle disease virus in murine myeloma cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin-neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoid fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.     

Conformational changes in Newcastle disease virus fusion glycoprotein during intracellular transport.

The migration on polyacrylamide gels of nascent (pulse-labeled) and more processed (pulse-labeled and then chased) forms of nonreduced Newcastle disease virus fusion glycoprotein were compared. Results are presented which demonstrate that pulse-labeled fusion protein, which has an apparent molecular weight of 66,000 under reducing conditions (Collins et al., J. Virol. 28: 324-336), migrated with an apparent molecular weight of 57,000 under nonreducing conditions. This form of the Newcastle disease virus fusion protein has not been previously detected. This result suggests that the nascent fusion protein has extensive intramolecular disulfide bonds which, if intact, significantly alter the migration of the protein on gels. Furthermore, upon a nonradioactive chase, the migration of the fusion protein in polyacrylamide gels changed from the 57,000-molecular-weight species to the previously characterized nonreduced form of the fusion protein (molecular weight, 64,000). Evidence is presented that this change in migration on polyacrylamide gels is due to a conformational change in the molecule which is likely due to the disruption of some intramolecular disulfide bonds: Cleveland peptide analysis of the pulse-labeled nonreduced fusion protein (molecular weight, 57,000) yielded a pattern of polypeptides quite different from that obtained from the more processed form of the fusion protein (molecular weight, 64,000). However, the pattern of polypeptides obtained from the nonreduced 64,000-molecular-weight species was quite similar to that obtained from the fully reduced nascent protein (molecular weight, 66,000). This conformational change occurred before cleavage of the molecule. To determine the cell compartment in which the conformational change occurs, use was made of inhibitors which block glycoprotein migration at specific points. Monensin allowed the appearance of the 64,000-molecular-weight form of the fusion protein, whereas carboxyl cyanide m-chlorophenylhydrazine blocked the appearance of the 64,000-molecular-weight form of the fusion protein. Thus, the fusion protein undergoes a conformational change as it moves between the rough endoplasmic reticulum and the medial Golgi membranes.     

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.     

Structure of the major oligosaccharides in the fusion glycoprotein of Newcastle disease virus

The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.     

Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences

Highly virulent Newcastle disease virus (NDV) isolates are List A pathogens for commercial poultry, and reports of their isolation among member nations must be made to the Office of International Epizootes (OIE). The virus is classified as a member of the order Mononegavirales in the family Paramyxoviridae of the subfamily Paramyxovirinae. Two interactive surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, play essential roles in NDV attachment and fusion of cells during infection. Antibodies to the F or HN proteins are capable of virus neutralization; however, no full-length sequences are available for these genes from recently obtained virulent isolates. Therefore, nucleotide and predicted amino acid sequences of the F and HN protein genes from 16 NDV isolates representing highly virulent viruses from worldwide sources were obtained for comparison to older virulent isolates and vaccine strains. The F protein amino acid sequence was relatively conserved among isolates maintaining potential glycosylation sites and C residues for disulfide bonds. A dibasic amino acid motif was present at the cleavage site among more virulent isolates, while the low virulence viruses did not have this sequence. However, a Eurasian collared dove virus had a K114Q substitution at the F cleavage site unique among NDV isolates. The HN protein among NDV isolates maintained predicted catalytic and active site residues necessary for neuraminidase activity and hemagglutination. Length of the HN for the Eurasian collared dove isolate and a previously reported heat resistant virulent isolate were longer relative to other more recent virulent isolates. Phylogenetically NDV isolates separated into four groups with more recent virulent isolates forming a diverse branch, while all the avian paramyxoviruses formed their own clade distinct from other members of the Paramyxoviridae.     

Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein

The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.     

新城疫病毒囊膜糖蛋白七肽重复区的融合作用

在新城疫病毒（Newcastle diseasevirus,NDV）膜融合的过程中融合糖蛋白（Fusion protein,F）的两段七肽重复区（Heptad repeat,HR）发挥着重要作用。这两段七肽重复区能够形成反相平行的六螺旋束结构,这被认为是融合蛋白融合后构象的核心结构。对融合作用的深入系统研究将有助于膜融合病毒的防控。