New insights into the spring-loaded conformational change of influenza virus hemagglutinin

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-lielix spring-loaded conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63PJF70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited protcolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only similar to50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.