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 invoke the phenotype of the double Pro-substituted mutant, V55P/S71P, as our strongest evidence for the importance of the spring-loaded conformational change: no fusion is observed with V55P/S71P for at least 1 h at pH 5 and 37ЊC. Nonetheless, V55P/S71P behaves like wt-HA in all of its biochemical properties analyzed: trimer formation, cell surface expression, cleavage of HA0, RBC binding, reactivity with an antibody against the major antigenic site, and resistance to proteinase K at neutral pH. The only noted biochemical difference in V55P/S71P is an upward shift ‫6.0ف(‬ U) in its pH dependence of the conformational change, a phenotype seen previously for other fusion-competent HA mutants (Steinhauer et al., 1996) . The lack of fusion activity of V55P/S71P correlates well with its predicted (COILS 2 score ϭ 1.31) and observed (see below) impairment in helix formation.