The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein

The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity.     

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.     

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.     

Overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV) fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. We have previously shown that free thiols are present in cell surface-expressed NDV F protein and that blocking the production of free thiols by thiol-disulfide exchange inhibitors inhibited the membrane fusion mediated by F protein (J Virol. 81:2328-2339, 2007). Extending these observations, we evaluated the role of the overexpression of two disulfide bond isomerases, protein disulfide isomerase (PDI) and ERdj5, in cell-cell fusion mediated by NDV glycoproteins. The overexpression of these isomerases resulted in significantly increased membrane fusion, as measured by syncytium formation and content mixing. The overexpression of these isomerases enhanced the production of free thiols in F protein when expressed without hemagglutination-neuraminidase (HN) protein but decreased free thiols in F protein expressed with HN protein. By evaluating the binding of conformation-sensitive antibodies, we found that the overexpression of these isomerases favored a postfusion conformation of surface-expressed F protein in the presence of HN protein. These results suggest that isomerases belonging to the PDI family catalyze the production of free thiols in F protein, and free thiols in F protein facilitate membrane fusion mediated by F protein.     

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.     

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.     

Mutations in the stalk of the measles virus hemagglutinin protein decrease fusion but do not interfere with virus-specific interaction with the homologous fusion protein

The hemagglutinin (H) protein of measles virus (MV) mediates attachment to cellular receptors. The ectodomain of the H spike is thought to consist of a membrane-proximal stalk and terminal globular head, in which resides the receptor-binding activity. Like other paramyxovirus attachment proteins, MV H also plays a role in fusion promotion, which is mediated through an interaction with the viral fusion (F) protein. The stalk of the hemagglutinin-neuraminidase (HN) protein of several paramyxoviruses determines specificity for the homologous F protein. In addition, mutations in a conserved domain in the Newcastle disease virus (NDV) HN stalk result in a sharp decrease in fusion and an impaired ability to interact with NDV F in a cell surface coimmunoprecipitation (co-IP) assay. The region of MV H that determines specificity for the F protein has not been identified. Here, we have adapted the co-IP assay to detect the MV H-F complex at the surface of transfected HeLa cells. We have also identified mutations in a domain in the MV H stalk, similar to the one in the NDV HN stalk, that also drastically reduce fusion yet do not block complex formation with MV F. These results indicate that this domain in the MV H stalk is required for fusion but suggest either that mutation of it indirectly affects the H-dependent activation of F or that the MV H-F interaction is mediated by more than one domain in H. This points to an apparent difference in the way the MV and NDV glycoproteins interact to regulate fusion.     

Conserved glycine residues in the fusion peptide of the paramyxovirus fusion protein regulate activation of the native state

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.     

Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding.     

新城疫病毒F蛋白细胞融合活性位点中保守氨基酸基因突变分析

为了确定新城疫病毒融合蛋白(F)分子上活性位点中保守氨基酸在F蛋白的细胞融合作用,弄清F细胞融合的分子机理,采用基因定点突变法,创造一个酶切位点,用酶切反应初步筛选突变株,然后用DNA序列分析进一步确定,并于真核细胞内进行表达,Giemsa染色定性和指示基因法定量检测细胞融合功能,荧光强度分析(FACS)检测表达效率情况。结果表明,NDV F第117位苯丙氨酸(F)突变成亮氨酸(L)时对细胞融合作用没有显著影响。R112和K115同为保守序列,分别突变为G时,细胞融合活性只有原来的44%,下降了56%。细胞表面表达效率没有明显的改变。N147突变为K时,细胞融合活性明显下降,只有原来的15%,而细胞表面表达效率没有明显的改变。L154为保守序列,突变为K时,细胞融合活性消失,说明L154是一个非常关键的氨基酸,对维持F蛋白的细胞融合活性非常重要。细胞表面表达效率也有所下降(为原来的94%)。D462属于高度保守氨基酸,当突变为N时,细胞融合活性消失,但经细胞表面表达效率分析证明,此突变蛋白未表达于细胞表面,证明在细胞浆转运至细胞表面的过程中发生了问题。当突变为R和E时,细胞融合活性未发生改变,但细胞表面表达效率有所下降,分别为野毒株的63%和44%。说明NDV F分子上与HN相互作用的特异性区域中的某些保守氨基酸在细胞融合中发挥着重要作用,对F蛋白的折叠、加工、转运等,发挥着不同作用,从而影响F蛋白的细胞融合作用和/或在细胞表面的表达量。     

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.     

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.     

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.     

Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses.

The fusion glycoprotein (F) and hemagglutinin-neuraminidase (HN) genes of human parainfluenza virus type 2 (PI2) were molecularly cloned and expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Expression of the F and HN proteins was detected by using immunoprecipitation and surface immunofluorescence staining. Although the F protein was found to be cleaved into F1 and F2 and expressed on cell surfaces, no cell fusion was observed. However, cotransfection of the F-protein gene together with the P12 HN gene resulted in significant levels of cell fusion. Cell fusion was also observed when separate cell cultures were transfected with the HN and F genes and the F-expressing cells were mixed with the HN-expressing cells. Surprisingly, when the PI2 F protein was expressed together with the parainfluenza virus type 3 (PI3) HN protein, no fusion was detectable in the transfected cells. Similarly, no fusion was found upon coexpression of the PI2 HN and PI3 F proteins. However, coexpression of the PI3 F and HN proteins resulted in extensive cell fusion, which resembled the PI2 coexpression result. These results indicate that under the conditions used, the F protein is unable to cause fusion by itself and the HN protein provides a specific function in cell fusion which cannot be provided by another paramyxovirus attachment protein. Further, the results suggest that a type-specific functional interaction between the F and HN proteins is involved in mediating cell fusion.     

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.     

Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion

The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37 degrees C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.     

Mutated form of the Newcastle disease virus hemagglutinin-neuraminidase interacts with the homologous fusion protein despite deficiencies in both receptor recognition and fusion promotion

The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt alpha-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.