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.

RBCs (0.06%) were labeled with R18 as described above; for this assay, R18-labeled RBCs were only used if their fluorescence was 80-90% quenched. Labeled RBCs were bound to 293T cells that had been treated with neuraminidase and trypsin (or chymotrypsin) as described above. The RBC-cell complexes were harvested in 2 ml PBS (Ca 2 ϩ , Mg 2 ϩ free) with 0.5 mM EDTA, 0.5 mM EGTA, and 5 mM glucose and added to 8 ml of cold pH 7 fusion buffer. Cells were centrifuged at 800 rpm for 5 min, resuspended in a small volume (200 l) of fusion buffer, pH 7, kept on ice, and used within 2 h. Fusion experiments were conducted at RT using an LS-5B fluorimeter (Perkin-Elmer, San Jose, CA) as described previously (Danieli et al., 1996) . A quantity of RBC-cell complexes that gave ‫ف‬ 1.0 OD ( ‫ف‬ 15 l) was added to 3 ml fusion buffer (pH 7) in a cuvette, and the solution was agitated with a magnetic stirrer. After a baseline was obtained, the samples were brought to pH 5.2 with a predetermined amount of 1 M citric acid. The increase in fluorescence (fluorescence dequenching) was monitored, and the percent fluorescence dequenching was calculated relative to the total fluorescence obtained after solubilizing each sample in 0.5% NP-40.