Fusion Promotion Activity Research Articles

A Conserved Region in the F 2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F(1) and F(2). Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F(1) (CBF(1)) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F(2) subunit (CBF(2)). To analyze the functions of CBF(2), alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF(2) mutations resulted in folding and expression defects. However, the CBF(2) mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF(2) Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF(2) I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF(2) in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

Mutation at Residue 523 Creates a Second Receptor Binding Site on Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Protein

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein mediating hemagglutination (HA), neuraminidase (NA), and fusion promotion activities. It has been a matter of debate whether HN contains combined or separate sites for HA and NA activities. To clear the issue, we determined the presence of the second binding site on human parainfluenza virus (hPIV) type 1, 2, and 3 and Sendai virus (SeV) HN proteins. Results of virus elution from erythrocytes at an elevated temperature and HA inhibition by NA inhibitor BCX-2798 suggest that all hPIVs bind to the receptor only through the NA catalytic site, while SeV HN has an additional receptor binding site. Comparison of SeV and hPIV1 HN sequences revealed two amino acid differences at residues 521 and 523 in the region close to the second binding site identified in Newcastle disease virus HN. We mutated hPIV1 HN at position 523 from Asn to the residue of SeV HN, Asp, and rescued a recombinant SeV that carries the mutated hPIV1 HN by a reverse genetics system. The hPIV1 HN with Asp at position 523 hemagglutinated in the presence of BCX-2798, suggesting that the amino acid difference at position 523 is critical for the formation of a second binding site. Creation of the second binding site on hPIV1 HN, however, did not significantly affect the growth or fusion activity of the recombinant virus. Our study indicates that the presence and requirement of a second binding site vary among paramyxoviruses.

Role of sialic acid-containing molecules in paramyxovirus entry into the host cell: A minireview

Sialic acid-containing compounds play a key role in the initial steps of the paramyxovirus life cycle. As enveloped viruses, their entry into the host cell consists of two main events: binding to the host cell and membrane fusion. Virus adsorption occurs at the surface of the host cell with the recognition of specific receptor molecules located at the cell membrane by specific viral attachment proteins. The viral attachment protein present in some paramyxoviruses (Respirovirus, Rubulavirus and Avulavirus) is the HN glycoprotein, which binds to cellular sialic acid-containing molecules and exhibits sialidase and fusion promotion activities. Gangliosides of the gangliotetraose series bearing the sialic acid N-acetylneuraminic (Neu5Ac) on the terminal galactose attached in alpha2-3 linkage, such as GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are the major receptors for Sendai virus. Much less is known about the receptors for other paramyxoviruses than for Sendai virus. Human parainfluenza viruses 1 and 3 preferentially recognize oligosaccharides containing N-acetyllactosaminoglycan branches with terminal Neu5Acalpha2-3Gal. In the case of Newcastle disease virus, has been reported the absence of a specific pattern of the gangliosides that interact with the virus. Additionally, several works have described the use of sialylated glycoproteins as paramyxovirus receptors. Accordingly, the design of specific sialic acid analogs to inhibit the sialidase and/or receptor binding activity of viral attachment proteins is an important antiviral strategy. In spite of all these data, the exact nature of paramyxovirus receptors, apart from their sialylated nature, and the mechanism(s) of viral attachment to the cell surface are poorly understood.

Decreased Dependence on Receptor Recognition for the Fusion Promotion Activity of L289A-Mutated Newcastle Disease Virus Fusion Protein Correlates with a Monoclonal Antibody-Detected Conformational Change

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

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.

High cell surface expression of Newcastle disease virus proteins via replicon vectors demonstrates syncytia forming activity of F and fusion promotion activity of HN molecules

For functional studies, the hemagglutinin-neuraminidase (HN) and the fusion protein (F) of Newcastle disease virus (NDV) were expressed in BHK cells using two vectors which are based on the Semliki Forest virus (SFV) replicon. The first system of high protein expression works by transfection of RNA which before has been in vitro transcribed from a vector containing the gene for the SFV self-amplifying replicase (REP) and a foreign gene using the SP6 promoter. A high level of protein (HN or F) expression was detected 18-20 h after transfection. To study the host range of this expression system, a panel of different cell lines were compared for transfections with SFV RNA. A wide range of expression efficiency was observed, the highest being BHK cells. The second system is based on a DNA plasmid in which the SFV-REP and a foreign gene are expressed in cells under the transcriptional and translational control of the cytomegalovirus immediate-early enhancer T7 promoter. DNA-electroporated BHK cells expressed also high levels of the recombinant proteins but at a delayed time point (40-48 h) as compared with the corresponding RNA. Co-expression of the two NDV proteins, HN and F, via this DNA vector in the same cells led to syncytium formation in the cell monolayer, showing that both proteins expressed in this way, were functionally active. F alone, expressed via this vector, displayed residual fusion activity suggesting its proteolytic cleavage and its functional independence from HN.

Sialidase, receptor-binding and fusion-promotion activities of Newcastle disease virus haemagglutinin-neuraminidase glycoprotein: a mutational and kinetic study.

Mutations were generated in residues at the putative catalytic site of the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus Clone 30 strain (Arg498, Glu258, Tyr262, Tyr317 and Ser418) and their effects on its three associated activities were studied. Expression of the mutant proteins at the surface of HeLa cells was similar to that of the wild-type. Sialidase, receptor-binding and fusion-promotion activities were affected to different degrees for all mutants studied. Mutant Arg498Lys lost most of its sialidase activity, although it retained most of the receptor-binding activity, suggesting that, for the former activity, besides the presence of a basic residue, the proximity to the substrate molecule is also important, as Lys is shorter than Arg. Proximity also seems to be important in substrate recognition, since Tyr262Phe retained most of its sialidase activity while Tyr262Ser lost most of it. Also, Ser418Ala displayed most of the wild-type sialidase activity. However, a kinetic and thermodynamic study of the sialidase activity of the Tyr262Ser and Ser418Ala mutants was performed and revealed that the hydroxyl group of these residues also plays an important role in catalysis, since such activity was much less effective than that of the wild-type and these mutations modified their activation energy for sialidase catalysis. The discrepancy of the modifications in sialidase and receptor-binding activities in the mutants analysed does not account for the topological coincidence of the two sites. These results also suggest that the globular head of HN protein may play a role in fusion-promotion activity.

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

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

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

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

Importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses.

The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcDelta30), as well as an MV with an H tail of 14 residues (rMV-HcDelta20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.

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

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

Mutational analysis of heptad repeats in the membrane-proximal region of Newcastle disease virus HN protein.

For most paramyxoviruses, syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (X. Hu et al., J. Virol. 66:1528-1534, 1992). The stalk region of the Newcastle disease virus (NDV) HN protein has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (R. Buckland et al., J. Gen. Virol. 73:1703-1707, 1992; T. Sergel-Germano et al., J. Virol. 68:7654-7658, 1994; J. Reitter et al., J. Virol. 69:5995-6004, 1995). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a-position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.

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.

A Single Amino Acid Change Enhances the Fusion Promotion Activity of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Glycoprotein

Clinical isolates of human parainfluenza virus type 1 in our laboratory were found to induce significantly different degrees of syncytium formation in CV-1 cells. Sequence analysis of high- and low-fusion strains suggested that the hemagglutinin-neuraminidase (HN) protein was responsible for the differences in fusion activity. We exploited the strain differences to define the specific amino acid residues of the HN protein which were responsible for the low and high fusion activities The HN proteins of the two low-fusogenic strains 8389 and 45785, and the highly fusogenic strain C35, were expressed in HeLa T4+ cells and their fusion promotion activities were compared. When coexpressed with C35 F, HNs from the low-fusogenic viruses were associated with much lower fusion activity then was C95 HN, suggesting that the HN proteins modified the fusogenicity of the viruses. To identify the region of the HN protein responsible for this difference, we constructed a series of chimeric HN cDNAs combining 8389 and C35 sequences. All chimetic HNs that contained C35 sequence in the central 36% of the protein exhibited high fusion promotion activity. Further analysis by site-directed mutagenesis showed that a single Asn-to-Lys substitution at position 242 converted 8389 HN to a highly fusion-promoting molecule. Thus, the globular head of the FIN molecule is involved in fusion promotion activity.

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.

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

The hemagglutinin-neuraminidase (HN) proteins of viruses in the Paramyxouirus 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 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.