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 assessed the ability of the single point mutant HAs to undergo low pH-dependent conformational changes. Conformational changes in wt-HA have been assayed by reactivity to various antibodies as well as by sensitivity to proteases. We first tested the sensitivity of each single point mutant to proteinase K; wt-HA becomes sensitive to proteinase K during the first stage of the conformational change (White and Wilson, 1987) . As seen in Fig. 4 A , all of the Ala-substituted mutants showed a pH dependence of proteinase K sensitivity essentially identical to that of wt-HA, with the exception of L80A, whose pH dependence was higher. The Gly and Pro mutants were assessed for proteinase K sensitivity by a Western blot analysis (Fig. 4 B ) . Mutants with Gly or Pro at position 71 behaved identically to wt-HA. In contrast, the mutants with Gly or Pro at positions 55 or 80 (V55G, L80G, V55P, and L80P) were completely sensitive to proteinase K over the entire pH range examined. To our knowledge V55G, V55P, L80G, and L80P are the first HA mutants identified that are sensitive to proteinase K at pH 7.