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 tested the ability of wt-and mutant HAs to induce lipid mixing using RBCs labeled with the fluorescent lipid probe octadecylrhodamine (R18). HA-expressing cells were prepared for RBC binding and fusion as described in Materials and Methods. RBC-cell complexes were incubated at pH 5 for 2 min at 37ЊC, reneutralized, and observed with a fluorescence microscope. Like those expressing wt-HA, cells expressing all of the mutant HAs were able to bind RBCs. As shown previously (Kemble et al., 1994; Danieli et al., 1996) , cells expressing wt-HA induced transfer of R18 at pH 5 (Fig. 6, A and B) . S71P and L80P induced efficient transfer of R18 at pH 5 (Fig. 6 A) . The extent of R18 transfer with V55P was quite low at 2 min ( Fig. 6 A) but appeared to increase at 10 min (Fig. 6 B) of acidification. As expected, neither wt-HA nor any of the mutant HAs induced transfer of R18 at neutral pH nor at low pH if they had not been pretreated with trypsin (data not shown). The extent of syncytia formation with V55P and L80P appeared less than for wt-HA and the other mutants.