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.

Carbohydrate modifications of the NDV fusion protein heptad repeat domains influence maturation and fusion activity.

The amino acid sequence of the fusion protein (F) of Newcastle disease virus (NDV) has six potential N-linked glycosylation addition sites, five in the ectodomain (at amino acids 85, 191, 366, 447, and 471) and one in the cytoplasmic domain at amino acid 542. Two of these sites, at positions 191 and 471, are within heptad repeat (HR) domains implicated in fusion activity of the protein. To determine glycosylation site usage as well as the function of added carbohydrate, each site was mutated by substituting alanine for the serine or threonine in the addition signal. The sizes of the resulting mutant proteins, expressed in Cos cells, showed that sites at amino acids 85, 191, 366, and 471 are used. This conclusion was verified by comparing sizes of mutant proteins missing all four used sites with that of unglycosylated F protein. The role of each added oligosaccharide in the structure and function of the F protein was determined by characterizing stability, proteolytic cleavage, surface expression, and fusion activity of the mutant proteins. Elimination of the site in F(2) at amino acid 85 had the most detrimental effect, decreasing cleavage, stability, and surface expression as well as fusion activity. The protein missing the site at 191, at the carboxyl terminus of the HR1 domain, also showed modestly reduced surface expression and negligible fusion activity. Proteins missing sites at 366 and 471 (within HR2) were expressed at nearly wild-type levels but had decreased fusion activity. These results suggest that all carbohydrate side chains, individually, influence the folding or activity of the NDV F protein. Importantly, carbohydrate modifications of the HR domains impact fusion activity of the protein.

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.

Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus.

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.

Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein.

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.

Mutations in the cytoplasmic domain of the fusion glycoprotein of Newcastle disease virus depress syncytia formation.

The role of the cytoplasmic domain of the Newcastle disease virus fusion protein in syncytia formation was explored by characterizing the intracellular processing and activities of proteins with deletions and point mutations in this region. Deletion of the entire domain (amino acids 523 to 553) resulted in a protein which was minimally proteolytically cleaved and had no syncytia forming activity. Deletion of the carboxy terminal half of the domain (amino acids 540 to 553) resulted in a protein that was normally processed but had no syncytia forming activity. Deletion of amino acids 547 to 553 resulted in a protein with approximately 30% wild-type levels of activity while deletion of amino acids 550 to 553 yielded a protein with wild-type activity. The results suggested that amino acids 540 to 550 are important for syncytia formation and this conclusion was supported by two internal deletions as well as point mutations in this region. Mutation of two cysteine residues in and adjacent to the transmembrane domain, which are potential sites for fatty acid acylation, had no effect on syncytia formation either singly or in combination.

Role of a conserved sequence in the maturation and function of the NDV HN glycoprotein.

The hemagglutinin-neuraminidase (HN) proteins of viruses in the Paramyxovirus genus have a short conserved sequence, G(A, S)EGR(I, L, V). The role of this sequence in the intracellular processing and function of the Newcastle disease virus HN protein was explored by site directed mutagenesis. Mutations in this region fall into two categories. One set of mutants (G398A, E400D, R402K, and a deletion removing amino acids 400-403) was defective in folding. These mutant proteins formed little or no mature, disulfide linked oligomer. They had few or no antigenic sites found on the mature protein and they were transported to the cell surface poorly or not at all. The second class of mutants (A399G, G401A, G401L) was minimally affected in folding and intracellular transport. When normalized to surface expression, this group of mutant proteins had wild type levels of attachment activity, neuraminidase activity, and fusion promotion activity. Thus mutations in this region directly affect intracellular processing but not the biological activities of the protein. This sequence may, therefore, be conserved in the HN proteins of Paramyxoviruses because it is critical to the folding of the molecule.

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.

Mutations in the transmembrane domain of the HN protein of Newcastle disease virus affect the structure and activity of the protein.

To explore the role of the transmembrane domain of the HN protein in the structure and function of the molecule, three conserved leucine residues in this domain which occur in a heptad-repeat motif were changed to alanine singly or in combination by site-specific mutagenesis. None of the mutant proteins were defective in translocation and intracellular transport. All mutant proteins formed disulfide-linked dimers. However, tetrameric structures of proteins with mutations in the third or most carboxy-terminal leucine could not be detected by sucrose gradient analysis, and mutant proteins with changes in both the first and second leucine formed less-stable tetramers. These results suggest that the transmembrane domain plays a role in the tetrameric structure of the HN protein. These mutations also altered the biological activities of the protein. Mutant proteins with alterations in the third leucine were very defective in attachment activity and somewhat defective for neuraminidase activity while all other mutant proteins had wild-type levels of attachment and neuraminidase activity. While all mutant proteins showed diminished fusion-promotion activity, proteins with mutations in the third leucine and proteins with changes in both the first and second leucines were very defective in fusion promotion. These results suggest that elimination or destabilization of the tetrameric structure affects attachment activity and fusion-promotion activity of the HN protein.

The role of the amino terminus of F1 of the Newcastle disease virus fusion protein in cleavage and fusion.

Phenylalanine is the amino acid at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains of Newcastle disease virus, which have a leucine residue in this position (Toyoda et al. (1989) Virology 169, 273-282). To explore the role of this phenylalanine in the fusion activity of the protein, this residue, amino acid 117 in the fusion protein sequence, was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. While both wild-type and the F117G protein were proteolytically cleaved and F1 was detected, the F117L protein was not cleaved. In the presence of the HN protein, both wild-type F and F117G proteins stimulated fusion, but the F117L protein was inactive in fusion. However, incubation in trypsin activated the fusion activity of the protein. Thus the phenylalanine at the amino terminus of the F1 component of the fusion protein is not required for the fusion activity of the protein. The presence of a leucine at this position blocks cleavage even though the cleavage site sequence is unchanged.

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.