Formation of the 6HB, as detected by ELISA, was prevented by substitution of residues in HRB by alanines. Probably, mutation of HRB abrogates its interaction with HRA. Nevertheless, antigenic site I, which is not available for antibody binding in the prefusion structure, was readily accessible for MAb 131-2a after the introduction of the alanine residues in HRB. This result thus indicates that the formation of the 6HB is not required nor is the driving force for the conformational changes leading to the exposure of antigenic site I. Thus, antigenic site I becomes available for antibody binding prior to the formation of the 6HB. Previously, Russell and coworkers showed that mutations in HRB of paramyxovirus SV5 F protein that destabilized the 6HB structure may result in hyperactive fusion phenotype. Hence, also for the SV5 F protein the 6HB stability alone does not appear to dictate conformational changes in F. Preventing the formation of the 6HB did not necessarily IMD 0354 inhibited the exposure of antigenic site I. The display of antigenic site I was prevented, however, by complete deletion of HRB, in the context of the fulllength protein or when the non-cleaved soluble protein was extended with a trimerization domain. It is likely that linkage of the GCN4 domain directly to the body of the F molecule is more effective at stabilizing the protein in the prefusion form than linking it to HRB, which is itself not trimerized and therefore may allow triggering. The GCN4-extended F protein lacking HRB showed a very similar antibody recognition profile as that of the recombinant soluble F protein that was stabilized in the prefusion conformation via the introduction of inter subunit disulfide bridges, indicative of these proteins adopting a similar prefusion-like conformation. Higher expression levels were observed, however, for the protein lacking HRB than for Flys. Phthalylsulfacetamide HRBcys-GCN. We hypothesize that the stalk formed by HRB of RSV F is very unstable and has a strong tendency to dissociate resulting in the exposure of antigenic site I. For other paramyxovirus F proteins, residues immediately upstream of HRB have been shown to play a crucial role in the stability of the prefusion conformation.