Poster abstracts
Poster number 99 submitted by Stephanie Hicks
The Apical Loop of the F2 Peptide in the Respiratory Syncytial Virus Fusion Protein Plays an Essential Role in Membrane Fusion
Stephanie Hicks (OSBP), Supranee Chaiwatpongsakorn (The Research Institute at Nationwide Childrens Vaccines and Immunity), Mark E. Peeples (The Research Institute at Nationwide Childrens Vaccines and Immunity)
Abstract:
Despite high morbidity and mortality, no vaccine is available for protection against RSV disease. The fusion (F) protein is produced in a metastable, prefusion conformation that refolds to the stable, postfusion conformation when triggered by an unidentified stimulus. This conformational change serves to mediate fusion between an F protein expressing membrane and an adjacent membrane. The prefusion conformation was recently co-crystalized with monoclonal antibody (mAb) D25 that specifically recognizes the prefusion form and is more highly neutralizing than mAbs that do not bind to the apex of the F protein. The ability of D25 to stabilize the prefusion form and its high neutralizing activity suggests its apical binding site is important for protein function. We hypothesize that residues in this binding site, including those in the apical F2 loop, are required for F protein triggering and, therefore, its function. Twelve residues in the apical loop of the F2 domain were mutated to assess their importance in fusion. Using a mAb that recognizes both protein conformations, it was determined these mutants were expressed on the cell surface. The ability of these mutants to cause fusion was determined with a cell-cell fusion assay. Six of the twelve alanine-scanning mutants were deficient in their ability to cause fusion, five of which were loss of charge mutants. Conservative mutations that retain the charges were able to cause cell-cell fusion, while non-conservative, opposite charge mutations were not. These results suggest the natural charge of each residue, and of the F2 apical loop, in general, is required for F protein function. Each mutant’s cell surface conformation was determined using mAbs specific to the prefusion or postfusion form. Despite an inability to function, most mutants reached the cell surface in the prefusion conformation, indicating premature triggering is not the reason for their loss of function.
Keywords: RSV , Fusion Protein