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 tested the ability of the single point mutant HAs to mediate full fusion, the transfer of small aqueous contents from RBCs. For this purpose RBCs were preloaded with the small (molecular weight 995) soluble fluorescent content probe, calcein AM. The fusion protocol was identical to that described for the outer leaflet mixing assay. As seen in Fig. 10 and Table III , the extent of con- Figure 5 . Reactivity of wt-, Ala-and Pro-substituted HAs with the C-HA1 antibody. Cells transfected with plasmids encoding (A) Ala-and (B) Pro-substituted HAs were metabolically labeled, treated with 5 g/ml trypsin, incubated at different pH values for 15 min at 37ЊC, reneutralized, and lysed in an NP-40 cell lysis buffer. Cleaved cell lysates were immunoprecipitated with the C-HA1 Ab and analyzed by 12% SDS-PAGE followed by phosphorimager analysis.% HA ppt., the HA precipitated by C-HA1 when total HA precipitated at pH 5 is considered as 100%. tent mixing with the mutant HAs paralleled their phenotype for lipid mixing: all of the mutants except V55P facilitated full fusion at comparable levels to wt-HA. Although the content-mixing fusion activity of V55P was significantly impaired, it was not abolished.