The promotion of membrane fusion by most paramyxoviruses, including NDV, is dependent on the recognition of receptors by HN (18
). Indeed, the strength of the HN-receptor interaction can determine the extent of fusion (12
). However, lectins cannot replace HN in fusion (22
). Also, MAbs to antigenic sites 3 and 4 on NDV HN inhibit fusion but not attachment (13
). Thus, HN′s role in fusion clearly involves something more than attachment. A second role for HN in fusion is mediated by a virus-specific interaction between it and the homologous F protein (8
However, the relationship between HN′s recognition of receptors and its interaction with F is not clear. At present, there are two hypotheses. One is that the two proteins interact intracellularly. In this model, HN is thought to maintain F in a prefusion state until receptor recognition at the cell surface induces a conformational change in F (29
). An intracellular interaction between the two proteins is supported by data indicating that coexpression of HN with F alters the conformation of the latter independent of receptor binding (19
). On the other hand, the demonstration that paramyxovirus HN or F proteins tagged for retention in the endoplasmic reticulum do not affect transport of the other protein argues against a strong interaction between the two proteins, at least in this cellular compartment (24
). The alternative hypothesis proposes that the HN-F interaction takes place only after the two proteins have arrived at the cell surface, triggered by HN′s recognition of receptors (17
). In this model, HN and F would not interact either intracellularly or in the absence of receptor binding. Of course, the two hypotheses are not mutually exclusive, and the possibility exists that HN and F interact intracellularly and the intracellular interaction is not fusion related.
HN proteins deficient in receptor recognition activity provide an opportunity to begin to discriminate between these two mechanisms. If the two proteins do arrive at the cell surface already associated with each other, the interaction would presumably take place independent of receptor recognition and might even be stabilized in its absence. In this case, one would expect attachment-deficient HN and F to be coimmunoprecipitated from the cell surface. On the other hand, if the HN-F interaction is triggered at the cell surface by HN′s recognition of receptors, this would result in a lack of HN-F complex formation with an HN protein that fails to bind receptors.
By the introduction of single-amino-acid substitutions in the NA active site, we have produced several NDV HN proteins with single-amino-acid substitutions which lack detectable receptor recognition and fusogenic activity. Four of these mutants, carrying substitutions of D198R, K236R, Y526L, and E547Q, fail to interact with the NDV F protein in a coimmunoprecipitation assay. This strongly suggests that HN and F do not arrive at the cell surface as a complex, at least one stable under the conditions used for the coimmunoprecipitation. The simplest explanation for these findings is that the HN-F interaction takes place at the cell surface, triggered by HN′s recognition of receptors.
Among HN active site mutations, I175E appears to be unique with respect to its relationship to F. Similar to the others, this NA active site mutation virtually eliminates attachment, NA, and fusion. However, unlike the others, the I175E substitution converts HN to a form of the protein that interacts with the F protein. This mutation separates the HN-F interaction from receptor recognition. It apparently converts HN to a fusion-ready conformation, though it is still unable to promote fusion due to the lack of receptor recognition activity. The reduced amount of I175E-HN coimmunoprecipitating with F relative to wt HN indicates that the mutant complex is either formed less efficiently or is less stable.
An interaction between I175E-AV-HN and F is consistent with previous findings obtained with I175E-Kansas HN (1
). The latter mutant promotes fusion 50% more effectively than the parent wt protein, despite exhibiting a more than 50% reduction in HAd activity. It was postulated that the I175E mutation in this protein acts by promoting a conformational change in HN that is propagated to the dimer interface through residue R174. This change in the dimer association would then trigger the HN-F interaction necessary for fusion.
The mechanism by which this change in the structure of the dimer converts HN to its F-interactive form is still not clear. An understanding of this process is dependent on the precise location of the site in HN that mediates the interaction with F. It has been postulated that residues in the dimer interface, exposed by the change in dimer association, may directly interact with the F protein (1
). Subsequently, mutations in the HN dimer interface were identified that severely reduced fusion (31
). However, a mutational analysis of a domain at the membrane-proximal end of the dimer interface has revealed that loss of fusion-promoting activity correlates with a destabilization of the interaction between HN and its receptor(s) (2
). This suggests that the fusion deficiency resulting from at least some mutations in the dimer interface may be due to a defect in receptor binding, rather than to a direct effect on fusion.
Moreover, the possibility that dimer interface residues in the globular domain could directly mediate the interaction with F is inconsistent with data obtained by the analysis of chimeras composed of segments from heterologous HN proteins. These studies have shown that specificity for the homologous F protein segregates with the stalk region of HN (5
). HN chimeras with heterologously derived globular domains fuse only with the F protein homologous to the HN stalk segment. It is difficult to reconcile how dimer interface residues could mediate specificity in these chimeras. They are neither conserved nor located in the domain that determines F specificity. To this end, it will be informative to determine the F-interactive capacity of fusion-deficient HN carrying mutations in the dimer interface. It is quite possible that changes at the dimer interface could be part of the conformational change that takes place in HN in its conversion from a form that does not interact with F to one that does.
A more recent peptide-based study suggests that the NDV HN-F interaction is mediated by amino acids 124 to 152 in the globular domain of HN and the membrane-proximal heptad repeat in F (7
). This finding may be consistent with the demonstration that an NDV-hPIV3 HN chimera with 141 NDV-derived N-terminal residues complements NDV F in fusion and that one with only 125 NDV-derived N-terminal residues does not (5
). However, it is inconsistent with the failure of several other chimeras with residues 124 to 152 intact to fuse with NDV F. In fact, chimera CH1(−6), which has NDV-derived residues 120 to 571 intact, not only does not fuse with NDV F but fuses quite efficiently with hPIV3 F (5
). While it is possible that the F-interactive domains in the NDV and hPIV3 HN proteins could be totally different, it seems unlikely that the F-interactive residues reside in the stalk in one HN protein and in the globular domain in the other. Clearly, to understand the changes in HN that take place upon its conversion to the F-interactive form, we must first more definitively identify the region of the protein that mediates that interaction.
One question that can be answered is why I175E-mutated HN fails to promote fusion, despite its efficient interaction with the F protein. This appears to be the result of its lack of receptor recognition activity, which is, in turn, a result of its lack of NA activity. When the protein is supplied with NA activity, it gains both significant attachment (11
) and fusion-promoting (Fig. ) activities.
Thus, perhaps a potentially more informative question to pose is why I175E-NDV HN lacks attachment activity in the first place. NDV-Kansas, carrying the I175E mutation, retains almost 50% of wt HAd activity and promotes fusion 50% more efficiently than wt HN (1
). We have shown that the amino acid differences between the HN proteins from the two strains of the virus that are responsible for their phenotypic differences with respect to HAd and fusion are at positions 193, 214, 219, and 228. When I175E-HN is further mutated at these positions to those residues present in NDV-Kansas HN (F193L, S214T, V219I, and N228S), the phenotype of the protein becomes virtually indistinguishable from that of I175E-Kansas HN, including enhanced fusogenic activity. The importance of these residues to HN function was also supported by the analysis of other strains of the virus.
Figure shows the position in one monomer of each of these residues relative to both residue I175 and the dimer interface. All four residues reside at the interface, with residues 193 and 228 at one end and residues 214 and 219 at the other. With their close proximity to the dimer interface, our data argue for the importance of the integrity of the dimer interface to the receptor recognition activity of NDV HN. This is consistent with earlier conclusions based on a mutational analysis of residues 218 to 226 (2
). Substitutions at positions 220, 222, and 224 destabilize the interaction between HN and its receptor(s). In addition, a V219A substitution results in a 60% enhancement of HAd activity at 37°C relative to wt HN (2
FIG. 8. Locations of HN residues 193, 214, 219, and 228 relative to the dimer interface and residue 175. The figure shows a top view of the Kansas HN dimer with the pH 6.5 structure of Crennell at al. (3). The two monomers in the dimer are shown in ribbon mode (more ...)
A role for residue 193 in the receptor recognition activity of HN is also consistent with our previous findings. AV-HN is unique in having a phenylalanine at residue 193, with leucine being much more common at this position in NDV HN from different strains (26
). Substitutions at this position can have profound effects on the strength of the HN-receptor interaction. MAb-selected variants and temperature-sensitive mutants of NDV-AV carrying an F193L or I substitution in HN (often in combination with a mutation at I175) can be shown to exhibit increased avidity for receptors and acquisition of the ability to promote fusion from without, an activity normally lacking in the parent wt NDV-AV virus (12
). This supports the idea that residues 175 and 193 are functionally related.
In conclusion, we have shown that four different mutations that abolish the receptor recognition activity of NDV HN also abolish its ability to interact with the homologous F protein at the cell surface. This is consistent with the idea that HN interacts with F only after binding to its receptor. Also, we have identified a point mutation (I175E) in the NA active site that converts HN to a form that is capable of interacting with the homologous F protein at the cell surface. The ability of the latter to complement F in fusion is still dependent on its ability to bind receptors. Thus, the I175E-HN-F complex could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion.