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.

We next created and assessed the phenotypes of two double Pro mutants, V55P/L80P and V55P/S71P. The V55P/ L80P mutant was not expressed at a detectable level on the cell surface (demonstrated by both indirect immunofluorescence and by RBC binding; data not shown). The V55P/S71P mutant was expressed at the cell surface. Calculations for V55P/S71P indicated acceptable phi and psi angles, an unaltered force field score, and a COILS2 score of 1.31. In terms of its biochemical properties V55P/S71P was well expressed at the cell surface and was readily cleaved by trypsin from HA0 to HA. It bound RBCs, reacted with an antibody against the major antigenic site, cosedimented with wt-HA in a sucrose density gradient (suggesting that it formed a trimer), and changed conformation at low pH, at ‫6.0ف‬ pH units higher than wt-HA (Figs. 3 B, 4 C, and 11) . Nonetheless, V55P/S71P appeared to be completely impaired in its ability to mediate lipid mixing, the first stage of fusion (Fig 11) .