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.

Mutational analysis of the membrane proximal heptad repeat of the newcastle disease virus fusion protein.

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309; Zhao et al. 2000, Proc. Natl. Acad. Sci. USA 97, 14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease virus fusion protein in residues that are likely to form contacts with the HR1 core trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as fusion activity as measured by syncytia formation, content mixing, and lipid mixing. While all mutant proteins were transport competent and proteolytically cleaved, these mutations did variously affect fusion activity of the protein. Nonconservative mutations in the "g" position had no effect on fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in fusion, a role related to content mixing and syncytia formation.

Mutations in the fusion peptide and adjacent heptad repeat inhibit folding or activity of the Newcastle disease virus fusion protein.

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172-14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the "a" and "d" positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the "a" position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven "d" position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these "d" mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most "d" position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the "d" position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.

A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion.

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

Role of carbohydrate processing and calnexin binding in the folding and activity of the HN protein of Newcastle disease virus.

The role of carbohydrate processing and calnexin binding in the folding pathway and activity of the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) was explored in infected cells using the inhibitor castanospermine (CST). Calnexin-HN protein complexes were demonstrated by coimmunoprecipitation using antibody specific for calnexin or HN protein. As in other systems, this complex was not detected in CST treated cells. In cells incubated in CST, the synthesis and stability of the HN protein was unaffected. However, as monitored by the appearance of conformationally sensitive antigenic sites, the folding of the HN protein in CST treated cells was approximately twice as slow than in untreated cells. This folding was ultimately efficient since there was no evidence for significant amounts of irreversibly aggregated forms which never acquired a mature conformation. Most significantly, the folding sequence as measured by the order of appearance of conformationally sensitive antigenic sites (McGinnes and Morrison, Virology 199, 255) was unaffected by CST. Thus while calnexin functions to speed the folding of the HN protein, it is not required for the folding of this protein. In addition, the protein synthesized in the presence of CST had significant levels of neuraminidase and hemagglutination activity suggesting that processing of the carbohydrate has a minimal role in the activity of the protein.

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.

Role of cotranslational disulfide bond formation in the folding of the hemagglutinin-neuraminidase protein of Newcastle disease virus.

The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakman et al. (EMBO J. 11, 1717-1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, the t1/2 for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.

The role of individual oligosaccharide chains in the activities of the HN glycoprotein of Newcastle disease virus.

To explore the role of N-linked carbohydrate in the activities of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), the six glycosylation addition sites (G1-G6) in the HN sequence of the AV strain of NDV were mutated. Migration of mutant protein on polyacrylamide gels as well as endoglycosidase H digestion of mutant protein showed that four of the addition sites (G1, G2, G3, and G4 at amino acids 119, 341, 433, and 481, respectively) are used while two (G5 and G6 at amino acids 508 and 538, respectively) are not used. Proteins expressed from single and all possible combinations of double and triple mutant DNAs as well as the unglycosylated molecule were characterized for the presence of specific antigenic sites, formation of disulfide-linked dimers, stability, transport to the cell surface, and biological activity. Results showed that glycosylation at positions G1 and G2 play little detectable role in the folding, stability, or transport of the molecule either singly or in combination with other mutations. Mutation of these sequons, however, significantly increased the cell attachment and fusion promotion activities of the protein, particularly in combination. Mutation of the glycosylation site at G4 either singly or in combination with other site-eliminating mutants inhibited the formation of the mature protein, while a mutation eliminating the addition site at G3 had a slight effect on the efficiency of folding, particularly in combination with mutation of the site G4. When normalized to surface expression, elimination of carbohydrate addition sites at G3 and G4 singly or in combination with other mutations depressed in particular the neuraminidase activity of the protein but not the fusion promotion activity. Thus two oligosaccharides do not have a detectable role in maturation but do modulate the biological activities of the protein. The other two oligosaccharides influence both folding and activity of the protein.

Modulation of the activities of HN protein of Newcastle disease virus by nonconserved cysteine residues.

Comparisons of the sequences of the hemagglutinin-neuraminidase (HN) protein from thirteen different strains of Newcastle disease virus (NDV) show that while 12 cysteine residues are conserved in all strains, two cysteine residues are variably present (Sakaguchi et al. (1989) Virology 169, 260-272). One of these residues, at amino acid 6, is in the cytoplasmic domain. The other cysteine is at amino acid 123 in the ectodomain and is responsible for disulfide-linked HN dimers detected in some NDV strains (McGinnes and Morrison (1994) Virology 200, 470-483). To explore the role of these nonconserved residues in the structure and function of the protein, cysteine residues at amino acid 6 and 123 in the HN protein of the AV strain of NDV were mutated individually and in combination by site specific mutagenesis to serine and tryptophan, respectively. Proteins with mutations in either residue (C6S or C123W) or in both residues (C6S,123W) were transported to the cell surface. However, all three mutants had reduced attachment, neuraminidase, and fusion promotion activities. All three mutant proteins also showed an alteration in an antigenic site specific for oligomers of HN protein while all other antigenic sites were present at wild type levels. These results suggest that the nonconserved cysteine residues in the HN sequence may modulate the biological activities of the protein by affecting the oligomeric structure of the protein.

The role of the individual cysteine residues in the formation of the mature, antigenic HN protein of Newcastle disease virus.

The amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) has 14 cysteine residues, two of which are variably present in the sequences of the HN proteins of different strains of NDV while the rest are absolutely conserved. The role of each residue in the formation of the mature, oligomeric structure of the HN protein was assessed by characterizing proteins with mutations in each of the cysteine residues by Western analysis, immunoprecipitation with conformationally sensitive antibodies, immunofluorescence, and sedimentation on sucrose gradients. Proteins with mutations in the first cysteine (amino acid 6) or the second cysteine (amino acid 123), the nonconserved cysteine residues, formed antigenically mature oligomers which were transported to the cell surface like wild type. Protein with a mutation at cysteine 2 did not, however, form covalently linked oligomers demonstrating that it is this residue that is responsible for intermolecular disulfide bonds in the mature oligomer. Proteins with mutations in cysteine 3 (amino acid 172) or cysteine 5 (amino acid 196) formed proteins with all antigenic sites except one, site 23. These mutant proteins formed disulfide-linked dimers and were efficiently transported to the cell surface. They did not, however, sediment on gradients like the wild-type protein. They were also defective in the biological activities associated with the wild-type protein. Proteins with mutations in cysteines 4 (amino acid 186), 6 (amino acid 238), 7 (amino acid 247), 8 (amino acid 251), 13 (amino acid 531), or 14 (amino acid 542) contained no mature antigenic sites but formed noncovalently linked oligomers. Proteins with mutations in cysteines 9 (amino acid 344), 10 (amino acid 455), 11 (amino acid 461), or 12 (amino acid 465) formed proteins with antigenic site 4 but no other mature antigenic sites. These mutant proteins also formed noncovalently linked oligomers. These results suggest that mutations in different cysteine residues block the maturation of the HN protein at different stages.

Conformationally sensitive antigenic determinants on the HN glycoprotein of Newcastle disease virus form with different kinetics.

The mature Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein is a type 2, oligomeric glycoprotein which has seven antigenic sites, sites 1, 2, 3, 4, 23, 12, and 14 (lorio et al., 1989, Virus Res. 13, 245). The folding of the HN protein was explored by characterizing the formation of representative epitopes specific for each of the six of antigenic sites in infected cells. None of these epitopes was present on the nascent, 5-min pulse-labeled HN protein, while all epitopes appeared after a 1- to 2-hr chase. All epitopes formed in the presence of monensin or during a chase at 15 degrees, suggesting that all these determinants appear while the molecule is in the rough endoplasmic reticulum. However, none of the epitopes appeared during chases in the presence of carbonyl cyanide m-chlorophenylhydrazone, suggesting that the formation of all determinants requires ATP. The kinetics of formation of each of the determinants was quantitated in both NDV-infected Cos cells and chick embryo cells. In both cell types, antigenic determinants formed with different kinetics. The epitope specific for site 4 appeared first, followed by the simultaneous appearance of epitopes in sites 1 and 3. The epitope in site 2 appeared next and that in site 23 last. The kinetics of appearance of the epitope in site 14 relative to those in other sites varied with cell type. In chick cells, this epitope appeared with the site 2 epitope, while in Cos cells this epitope appeared with or just after sites 1 and 3 epitopes. Nonradioactive chases at 15 degrees slowed the formation of the antigenic determinants. The disulfide-linked dimer form of the HN protein appeared concomitant with epitopes in sites 1 and 3.

The fusion promotion activity of the NDV HN protein does not correlate with neuraminidase activity.

Three activities, attachment, neuraminidase, and fusion promotion, have been associated with the hemagglutinin-neuraminidase (HN) protein encoded by paramyxoviruses such as Newcastle disease virus. The fusion promotion activity of the HN protein can be separated from its attachment activity by mutation (Sergel et al., 1993, Virology 193, 717-726). To determine if neuraminidase activity of the HN protein has any role in fusion promotion, two sets of mutants were characterized. First, a change of amino acid 193 from a serine to a proline and a change of amino acid 175 from isoleucine to a methionine diminished neuraminidase activity as previously reported. However, these mutant proteins retained fusion promotion activity. In addition, mutation of amino acid 200 from a histidine to a proline resulted in nearly twice the neuraminidase activity of wild-type as previously reported. This mutant also had wild-type levels of fusion promotion activity. Second, substitution of three leucine residues at amino acids 94, 96, and 97 with three alanines resulted in a mutant protein with full neuraminidase as well as full attachment activity but no fusion promotion activity. Thus, two sets of HN protein mutants demonstrate that the fusion promotion activity does not correlate with the level of neuraminidase activity.

The attachment function of the Newcastle disease virus hemagglutinin-neuraminidase protein can be separated from fusion promotion by mutation.

Using Cos-7 cells and an SV40-based vector, quantitative assays for fusion directed by the Newcastle disease virus hemagglutinin-neuraminidase (HN) and fusion glycoproteins were developed. Data from these assays showed that after cotransfection of the HN protein and the fusion protein DNAs, there was a progressive increase in syncytia size over background levels while transfection with HN protein or F protein DNA alone resulted in no changes over background. Using immunofluorescence, the efficiency of syncytia formation directed by the glycoproteins was assessed. Virtually all cells expressing the fusion protein alone or the HN protein alone existed as single nucleated cells. However, all cells coexpressing the two genes existed in syncytia with 4 to 50 nuclei. Using these assays, the fusion promotion activity of two deletion mutants of the HN protein was quantitated. One mutation deleted amino acids 4 through 26, effectively removing the cytoplasmic domain of the protein. This mutant protein was found at the cell surface, although in very reduced amounts. The mutant protein could bind red blood cells and could promote fusion, although the syncytia formed were smaller (4 to 9 nuclei) than those promoted by the wild-type protein. A second mutation deleted nine amino acids (91-99) located 37 amino acids from the transmembrane region in the ectodomain. This mutant was detected at the cell surface at 33% the level of wild type and it retained near normal ability to bind red blood cells. However, this mutation completely eliminated the fusion promotion activity of the HN protein. This mutation effectively separates the attachment function of the HN protein from fusion promotion.

Mature, cell-associated HN protein of Newcastle disease virus exists in two forms differentiated by posttranslational modifications.

Characterization of the posttranslational modifications of the mature, cell-associated hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) revealed that the HN protein exists in two forms differentiated by disulfide bonds and glycosylation. One form, HNa, contains intermolecular disulfide bonds and is endoglycosidase H partially resistant. The other form, HNb, is not linked by disulfide bonds and is endoglycosidase H sensitive. Both forms of the protein are modified with fucose indicating transport to the Golgi membranes. Both forms are detected at the cell surface by monoclonal antibody. Furthermore, both forms are transported to the cell surface with identical kinetics. HNa is incorporated into virions. HNb is not incorporated into virions and is presumably degraded. The cDNA derived from the HN gene was expressed from a retrovirus vector. The majority of the protein expressed was in the nonvirion-associated form b. Evidence is presented that the level of gene expression determines the ratio of the two forms of HN protein. At high levels of expression, the virion-associated form is favored while at low levels of expression the nonvirion-associated form is favored. The results presented have implications for persistent infections as well as expression of viral genes from different vectors.

Avian cells expressing the Newcastle disease virus hemagglutinin-neuraminidase protein are resistant to Newcastle disease virus infection.

The cDNA derived from the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene was inserted into a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. Chick embryo cells transfected with this vector expressed HN-sized protein which could be precipitated with anti-HN antibody. These cells adsorbed avian red blood cells and the cell surfaces exhibited neuraminidase activity while cells transfected with an antisense version of the gene were negative for hemadsorption and neuraminidase. The cells transfected with the retroviral vector containing the HN gene were resistant to infection by NDV and influenza virus, viruses which bind to sialic acid containing receptors, but sensitive to vesicular stomatitis virus (VSV). Cells transfected with the antisense version of the HN gene were sensitive to NDV, influenza virus, and VSV infection. Thus the HN protein-expressing cells are likely resistant to NDV and influenza virus due to the destruction of the cellular receptors by the neuraminidase of the HN protein. The expression of the influenza virus HA protein using the same retrovirus vector has been reported previously (L. A. Hunt, D. W. Brown, H. L. Robinson, C. W. Naeve, and R. G. Webster, 1988, J. Virol. 62, 3014-3019). Cells infected with this vector were sensitive to infection with influenza virus, NDV, and VSV. Thus expression of a viral surface protein does not necessarily confer resistance of the cell to the homologous virus.

Nucleotide sequence of the gene encoding the Newcastle disease virus hemagglutinin-neuraminidase protein and comparisons of paramyxovirus hemagglutinin-neuraminidase protein sequences.

The nucleotide sequence of cloned cDNA copies of the mRNA encoding the Newcastle disease virus (NDV), strain A-V, hemagglutinin-neuraminidase (HN) protein was determined. A single open reading frame in the sequence encodes a protein of 570 amino acids with a calculated molecular weight of 62,280. The predicted protein sequence contains only one obvious potential membrane spanning region, located 27 amino acids from the amino terminus of the sequence. The predicted sequence contains 6 glycosylation sites and 14 cysteine residues. Comparison of the NDV HN protein sequence with three other paramyxovirus HN protein sequences reveals two regions that have homologies in all four sequences. The conserved cysteine residues are clustered in these two regions. One conserved region is located near the middle of the predicted sequence while the second region is in the carboxy terminal third of the molecule. The presence of conserved regions suggests the importance of these areas of the molecule in the structure or function of the protein.

Conformational change in a viral glycoprotein during maturation due to disulfide bond disruption.

The fusion glycoprotein of Newcastle disease virus is synthesized as an inactive precursor, F0. During intracellular transport and maturation, F0 undergoes a conformational change resulting from the loss of intramolecular disulfide bonds. F0 is also cleaved to yield F1, F2, the active, membrane-fusing form of the protein. Two monoclonal antibodies were used to explore this conformational change and its relationship to cleavage. These antibodies failed to precipitate the pulse-labeled fusion protein but did precipitate the F0 and the F1, F2 forms of the "chase" fusion protein. Use of the inhibitors carbonylcyanide m-chlorophenylhydrazone and monensin showed that the fusion protein acquired the ability to react with the monoclonal antibodies after it left the rough endoplasmic reticulum but before it left the medial Golgi membranes and before it was cleaved. The acquisition of antigenicity correlates with the disruption of intramolecular disulfide bonds during transit through the cell. This correlation was directly confirmed. The pulse-labeled fusion protein could be recognized by both monoclonal antibodies if the protein was first reduced. The formation and disruption of intramolecular disulfide bonds as a posttranslational modification of glycoproteins is discussed.

The nucleotide sequence of the gene encoding the Newcastle disease virus membrane protein and comparisons of membrane protein sequences.

The nucleotide sequence of a cloned cDNA copy of the mRNA encoding the Newcastle disease virus (NDV) membrane (M) protein was determined. A single open reading frame in the sequence encodes a protein of 364 amino acids with a calculated mol wt of 39,742. The predicted protein sequence does not contain extensive hydrophobic regions. The sequence does contain eight pairs of basic amino acid residues, five of which are located in the carboxyl-terminal half of the sequence. Comparisons of the NDV M protein sequence with other paramyxovirus M protein sequences reveals little homology common to all sequences. There is only 17% homology with Sendai virus M protein. A short region of homology with the VSV M protein sequence, was, however, found.

Nucleotide sequence of the gene encoding the Newcastle disease virus fusion protein and comparisons of paramyxovirus fusion protein sequences.

The nucleotide sequence of cloned cDNA copies of the mRNA encoding the Newcastle disease virus fusion protein was determined. A single open reading frame in the sequence encodes a hydrophobic protein of 553 amino acids with a calculated molecular weight of 58 978. The previously determined protein sequence of the amino terminus of the F1 (Richardson, G.D. et al. (1980) Virology 105, 205-222) was located within the predicted protein sequence. The predicted protein sequence contains a hydrophobic stretch of 29 amino acids near the carboxy terminal end and likely represents the membrane spanning region of the protein. The F2 portion of the sequence contains one glycosylation site while F1 contains four which are potentially used. The predicted sequence contains 13 cysteine residues. Comparison of the NDV fusion protein sequence with three other paramyxovirus fusion protein sequences reveals little homology common to all four viruses except for the amino terminus of the F1 proteins. However, the positions of the cysteine residues within the sequence are conserved, particularly among the members of the paramyxovirus subgroup, suggesting the importance of disulfide bond formation in the conformation of paramyxovirus fusion proteins.

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.