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.

The C-HA1 antibody is an antipeptide antibody against the COOH-terminal residues of HA1. The C-HA1 antibody reacts preferentially with low pH-treated HA (White and Wilson, 1987) and efficiently precipitates low pH-treated HA from crude cell lysates. Immunoprecipitations with the C-HA1 antibody were conducted as follows: Plates of metabolically labeled cells were treated with trypsin to cleave HA0, incubated at 37 Њ C for 15 min at the indicated pH in MES-saline buffer, and then reneutralized with MES-saline buffer, pH 7. Cells were lysed with cell lysis buffer containing protease inhibitors (Kemble et al., 1993) and immunoprecipitated with the C-HA1 antibody for 1 h at 4 Њ C. Immunecomplexes were bound to protein A agarose (Boehringer Mannheim GmbH, Mannheim, Germany) for 1 h at 4 Њ C and washed extensively as described (Kemble et al., 1993) . Samples were analyzed by SDS-PAGE and phosphorimager analysis.