The virus-specific nature of the HN-F interaction suggests the existence of one or more domains on the NDV HN and F proteins that mediate a specific interaction between the two proteins. Analysis of the fusion-promoting activity of NDV-hPIV3 HN chimeras has demonstrated that the specificity of HN for its homologous F protein segregates with the stalk of HN (6
). However, the actual determinants of the HN-F interaction on either protein have not been firmly established.
We previously identified two highly conserved amino acids, P93 and L94, in the intervening region between the two heptad repeats in the stalk of NDV HN (36
). Here, we have investigated the effects of amino acid substitutions for these residues on fusion. The introduction of any of three different substitutions, alanine, leucine, or serine, for residue P93 results in a significant reduction in fusion promotion and the ability of HN to interact with the F protein at the cell surface. This phenotype is not due to changes in either expression or receptor recognition activity, both of which are comparable to those of wt HN. However, consistent with our earlier findings (36
), substitutions for P93 do impair NA activity. The replacement of P93 may induce a subtle structural change in the HN stalk that is transmitted to its globular domain, thus affecting the NA active site.
The effect of substitutions for P93 on NA activity calls into question the basis for their effect on fusion. These substitutions could indirectly affect HN′s fusion promotion function by altering the transmission of a signal from the receptor recognition site in the globular domain to the F-specific region in the stalk, thereby abrogating the HN-F interaction. P93-mutated proteins have the same phenotype as proteins carrying substitutions at the heptadic positions, although the effect of the latter on the HN-F interaction was not determined (32
Our results for P93-mutated NDV HN are in agreement with the phenotype of a mutated protein carrying a serine substitution at the corresponding residue (P111) in hPIV3 HN. This substitution, initially identified in a NA-deficient hPIV3 variant virus (25
), induces an F-triggering defect, despite wt receptor recognition activity and avidity for receptors (26
). However, F insertion into the target membrane progressed to fusion more slowly with P111S-HN than with the wt HN protein. Based on this finding, it was proposed that this triggering-defective protein may have a diminished capacity to interact with F. It may be that the P111S-HN protein requires a longer period of contact with its cellular receptor in order to induce the conformational change in HN required for the conversion of F to its fusion-ready form. Perhaps P93-mutated NDV HN proteins also have a F-triggering defect, resulting in the weak fusion detected in the content mixing assay. Consistent with this, P93-mutated NDV HN proteins are unable to coimmunoprecipitate with F, suggesting that these substitutions interfere with their ability to interact with F.
A surprising result is the 65% increase in HAd activity over the wt level exhibited by L94P-HN. This is the only substitution introduced at this position or anywhere else in this domain that gives this phenotype. Evidently, the presence of the two prolines in succession in the stalk somehow stabilizes the receptor binding activity of HN, possibly by providing added rigidity to the peptide backbone.
Although substitutions for P93 appear to alter the structure of HN, some substitutions for the neighboring residue L94, as well as A89 and L90, modulate fusion and the HN-F interaction with no detectable effect on the other HN functions. When residue L94 is replaced with alanine, glycine, or proline, the mutated HN proteins display a significant reduction in fusion promotion activity while maintaining wt levels of NA and receptor recognition activities. These L94 substitutions also cause loss of the ability of HN to be coimmunoprecipitated by the anti-F MAb. Interestingly, L94R HN promotes a significant level of fusion, approximately 50% of the wt level. Consistent with this, we were able to detect an interaction between this protein and F. The L94I substitution has a minimal effect on fusion and the HN-F interaction, consistent with the conservative nature of this substitution. Thus, substitutions for L94 appear to directly affect fusion, since they have no significant effect on any other HN function. They may modulate fusion via a direct effect on the ability of HN to interact with the F protein. Apparently, the size of the residue at this position carries some importance to the HN-F interaction and fusion. A larger, though charged amino acid, such as arginine, is more functionally conservative than the smaller glycine or alanine.
Similarly, A89Q-HN and L90N-HN have the same phenotype as the weakly fusogenic L94-mutated proteins. They modulate fusion promotion and the HN-F interaction with no significant effect on NA or receptor recognition activity. Thus, these substitutions also appear to directly affect fusion promotion and the HN-F interaction.
Our data indicate that the co-IP assay can detect quantitative differences in the HN-F interaction at different levels of fusion. Mutated proteins that exhibit a significant level of fusion can be shown to interact with the F protein in the assay. For example, L94I-HN and L94R-HN, which fuse at 77.9 and 43.3% of the wt level, respectively, coimmunoprecipitate with an anti-F MAb at 51.9 and 31.8% of the wt level, respectively. Similarly, L90A-HN, which fuses at almost 50% of the wt level, is coimmunoprecipitated with an anti-F MAb at 27.6% of the wt amount. A89I-HN and L90I-HN coimmunoprecipitate 66.6 and 87.3% of the wt amount, consistent with their levels of fusion of approximately 90% of the wt level.
Thus, the co-IP assay can detect differences in the HN-F interaction that are consistent with differences in fusion promotion. However, the amount of co-IP of HN is not directly proportional to the extent of fusion. This is very likely a reflection of the limits of the co-IP assay. Of the P93-mutated HN proteins, an interaction can be detected, albeit at less than 2%, only with P93A-HN. This protein fuses at almost 20% of the wt level. An interaction between the other two P93-mutated HN proteins and F was not detected, presumably because these proteins promote fusion below 14% of the wt level. Similarly, L94A-HN, L94G-HN, and L94P-HN do not coimmunoprecipitate with F, consistent with their low fusion-promoting activities. Also, neither A89Q-HN nor L90N-HN, both of which fuse at less than 20% of the wt level, interacts with F in the co-IP assay. Thus, taken together, these data suggest that the limit of detection of the HN-F interaction coincides with a level of fusion of approximately 20% of the wt level. The one notable exception to this is I175E-HN, which is capable of interacting with F, despite a lack of receptor recognition activity (20
). Thus, the co-IP assay has proven extremely useful in evaluating the relative effects of amino acid substitutions in both HN and F on the ability of the two proteins to interact with each other in the promotion of fusion.
Our results identify the intervening region in the NDV HN stalk as being critical to its ability to complement F in the promotion of fusion. These findings are consistent with NDV-hPIV3 HN chimera studies, which assign the F-specific region of NDV HN to its stalk (8
). However, these findings are inconsistent with a recent peptide-based study, which asserts that residues 124 to 152 of HN directly mediate its interaction with the homologous F protein (11
). It is suggested that this span of residues in the globular head of HN directly interacts with a heptad repeat domain in NDV F situated just outside the transmembrane. Point mutations in this domain were evaluated for their effect on HN function. While substitutions for residues I133 and L140 do result in a 70 to 80% reduction in fusion, they also impair NA activity by as much as 60% and attachment by as much as 50% (11
). Thus, these mutated HN proteins are analogous to our P93-mutated HNs. The effect of the I133 and L140 substitutions on fusion may be a reflection of structural changes in HN rather than a direct effect on its interaction with F. Also, the effect of these two substitutions on the HN-F interaction was not tested. In addition, the peptide data contrast with the fact that several chimeras with NDV residues 124 to 152 intact not only fail to fuse with NDV F but fuse very efficiently with hPIV3 F (37
In summary, the substitutions we have introduced at positions 89, 90, and 94 are the only ones for which a correlation has been demonstrated between fusion deficiency and decreased ability of HN to interact with F, with no other detectable negative effect on HN′s structure or function. But it still remains to be determined how these substitutions modulate the HN-F interaction. The intervening region may directly mediate an interaction with a complementary domain in the stalk region of the F protein. P93 may introduce a kink in the helix, so that the side chains of the surrounding amino acids can protrude away from the backbone in a bulge-like structure. Alternatively, we cannot strictly rule out the possibility that the domain does not make direct contact with F but may be critical to the conversion of HN to its F-interactive form.