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.

Our mutational analysis focused on HA2 54-81, the segment of HA predicted to have a high propensity to form a coiled-coil (Carr and Kim, 1993) . The choice of which residues to mutate was based on several criteria (Table I) . First, we chose residues that are not absolutely conserved among naturally occurring influenza viruses. We did this to lower the probability that the structure of the mutant HAs would be severely altered. Second, we excluded residues that form salt bridges in the native structure, since loss of such contacts can elevate the pH of fusion by facilitating the conformational change (Steinhauer et al., 1996) . Third, one residue was chosen to represent each of three general locations within HA2 54-81: NH 2 -terminal (HA2 55), central (HA2 71), and COOH-terminal (HA2 80). The final criterion involved the location of the residue in the final coiled-coil (Bullough et al., 1994) . HA2 55 and 80 lie in "d" positions, which are critical for coiled-coil formation (Lupas et al., 1991; Lupas, 1996) . Conversely, HA2 71 lies in a "b" position, which is not critical for coiled-coil formation. The locations of HA2 residues 55, 71, and 80 in the native HA structure are shown in Fig. 1 A . Their positions in the final low-pH structure are shown in Fig. 1 , B and C . Note that HA2 80 is in a coiled-coil in both the pH 7 (in the first turn) as well as the pH 5 structure.