It has been hypothesized that, like other fusion proteins, the paramyxovirus F protein is folded into a metastable conformation, and, upon activation of fusion, the F protein undergoes a cascade of conformational changes resulting in the formation of a very stable, six-stranded coiled coil composed of HR1 and HR2 domains (2
). To explore potential conformational changes in the F protein during onset of fusion and the role of the HN protein in that process, we characterized the binding and fusion inhibition of antibody specific for the HR1 domain of the NDV F protein. This antibody immunoprecipitated the F0
forms of the protein from cell extracts solubilized with detergent, and precipitation of F protein was unaffected by expression of the HN protein. Antibody against a comparable domain of the simian virus 5 (SV5) F protein was raised and would precipitate only the uncleaved form of the protein (3
). This different result may be attributed to differences in the conformation of the NDV F protein and the SV5 F protein in cell lysates. The anti-HR1 antibody raised against NDV sequences did preferentially precipitate the uncleaved F protein. Furthermore, we observed that precipitation of F1
was preferentially lost as cell extracts “aged,” suggesting conformational changes in the cleaved F protein after cell lysis masked the HR1 domain in the cleaved form of the protein. For these reasons, immunoprecipitation was performed immediately upon cell lysis.
In contrast to results obtained with cell extracts, anti-HR1 antibody minimally detected F protein expressed on cell surfaces, either cleaved or uncleaved, in the absence of HN protein expression. This result suggested that without HN protein expression, the membrane-associated F protein was folded so that the HR1 domain was inaccessible to antibody binding. Perhaps in the absence of HN protein, the F protein folds into a more stable conformation in which the HR1 domain is masked rather than the metastable form that can initiate fusion. Such an interpretation is consistent with the observation that wild-type NDV F protein cannot initiate fusion, even at low levels, in the absence of HN protein expression (17
In striking contrast, the uncleaved F protein coexpressed with HN protein bound the anti-HR1 antibody, and this binding depended specifically upon the coexpression of NDV HN protein. Binding was not observed in the presence of the measles virus HA protein or the Sendai virus HN protein. This result suggests that F protein expressed with HN protein is folded so that the HR1 domain is accessible to antibody and is, therefore, in a conformation different from that of F protein expressed alone. Since fusion cannot occur prior to F protein cleavage, this result also suggests that the conformational differences in the F protein due to coexpression of HN protein are manifested prior to cleavage activation of fusion. Indeed, it has been reported that HN protein and uncleaved F protein coimmunoprecipitate, a result that suggests an interaction between HN protein and uncleaved F protein (31
The wild-type, cleaved F protein, coexpressed with HN protein, bound anti-HR1 antibody poorly, although significantly more than cleaved F protein expressed alone. The low level of binding may be due to residual uncleaved F protein on cell surfaces. Another possibility (not mutually exclusive with the first) was that only one of several forms of the cleaved F protein was accessible to anti-HR1 binding. Cleaved F protein in the presence of HN protein may exist in at least two forms on cell surfaces, one in a prefusion form and the other in a postfusion form. It is proposed that in the postfusion conformation, the HR1 domain forms the interior trimer of the six-stranded HR1-HR2 complex (1
); thus, it is reasonable to suppose that the HR1 domain is inaccessible to antibody in this conformation. Indeed, Dutch et al. have presented evidence suggesting that the SV5 HR1 domain is inaccessible to a peptide antibody in the six-stranded coiled-coil complex (3
). If much of the cleaved F protein exists in this postfusion conformation, there would be only low-level anti-HR1 binding, as was observed. To explore the possibility that the prefusion form of the protein could bind antibody, we determined if anti-HR1 antibody could block fusion. Indeed, RBC fused with syncytia expressing the HN and wild-type F proteins; thus, a population of F protein on syncytium surfaces is capable of directing fusion. Importantly, anti-HR1 antibody inhibited this fusion. This result is consistent with the idea that the fusion-competent, cleaved F protein on the syncytium surfaces was accessible to anti-HR1 antibody in the presence of HN protein.
Two models have been proposed for the role of HN protein in fusion (Fig. ), models that differ in the role of attachment in inducing conformational changes in F protein. Model 1 proposes that HN and F proteins interact only after HN protein receptor binding and this interaction initiates F protein conformational changes required for fusion (11
). In this model, conformational differences in F protein in the presence and absence of HN protein should be manifested only after HN protein attachment. An alternative model is that HN and F proteins form a metastable complex prior to HN protein attachment (31
), a complex that prevents the HR1-HR2 interactions that would occur in F proteins expressed without HN protein. HN protein attachment with a concomitant conformational change releases the F protein and thus stimulates the cascade of F protein conformational changes required for fusion, changes that result in the formation of the six-stranded coiled coil composed of HR1 and HR2 domains. In this model, conformational differences in F protein in the presence and absence of HN protein should be manifested prior to HN protein attachment.
FIG. 9. Proposed relationship of F protein conformational changes to HN protein attachment. (Model 1) HN and F proteins interact only after attachment of HN protein to a receptor. Interaction stimulates a conformational change in F protein that unmasks the HR1 (more ...)
Since anti-HR1 antibody detected a conformational difference between F proteins expressed with and without HN protein, we asked when this conformational difference was manifested, before or after HN protein attachment. We asked when the antibody could inhibit fusion relative to the binding of RBC. Clearly, inhibition occurred if antibody was bound prior to RBC addition. This result is consistent with the idea that the HR1 domain is accessible prior to attachment. Furthermore the inhibition was greater than that observed when antibody was added after the RBC, particularly during the earlier times of incubation at 37°C. That antibody added only prior to RBC binding could inhibit fusion is more consistent with model 2. However, we cannot exclude the possibility that antibody bound to postactivation forms of the F protein indirectly interferes with conformational shifts in the prefusion form of the protein.
These combined results are most consistent with model 2 (Fig. ). F protein, either cleaved or uncleaved and coexpressed with HN protein, is folded into a conformation in which the HR1 domain is accessible to antibody binding. Upon attachment of HN protein to its receptor, the F protein initiates conformational changes necessary for the close approach of the two membranes, conformational changes that mask the HR1 domain. In contrast, F protein expressed alone folds into a conformation in which the HR1 domain is inaccessible to anti-HR1 antibody.