Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

Qiao, Hui; Pelletier, Sandra L.; Hoffman, Lucas; Hacker, Jill; Armstrong, R. Todd; White, Judith M.

Currently, there are two differing views of the fusion-active conformation of the influenza HA. The first view proposes that the fusion-active form is not significantly altered in three-dimensional structure from native HA. This model is based on two sets of findings. The first is that fusion can proceed at low pH at 0ЊC (albeit slowly) under conditions where changes in the head domain of HA were not detected either by electron microscopy (Stegmann et al., 1987) or by Figure 9 . pH profile of fusion for Pro-substituted mutants. Cells transfected with plasmids encoding wt-and Pro-substituted HAs were treated with neuraminidase and 10 g/ml trypsin for 10 min at RT. R18-labeled RBCs (0.05%) were bound for 25 min at RT. After unbound RBCs were removed, the cells were incubated at the indicated pH for 2 min at 37ЊC, reneutralized, and observed with a fluorescence microscope.