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.

As the first phase of our analysis, we assessed the synthesis, glycosylation, and cell surface expression of the single point mutant HAs. wt-and mutant HA cDNAs were expressed in COS 7 cells. Parallel cultures were treated with dMM, an inhibitor of terminal glycosylation, since recent experiments indicate that certain HA mutants acquire excess terminal carbohydrates in HA1 (Kemble et al., 1993) . As seen in Fig. 2 A , when produced in the absence of dMM, the HA0s from the mutants V55A, S71A, S71G, and S71P comigrated with wt-HA0. The HA0s from the other mutants, L80A, V55G, L80G, V55P, and L80P, ran as multiple higher molecular weight species. When grown in the presence of dMM, however, all mutant HA0s comigrated with wt-HA0. These findings suggested that specific substitutions within the region of high coiled-coil propensity (Gly and Pro substitutions at position 55 as well as Ala, Gly, and Pro substitutions at position 80) affected the initial folding of HA0 such that it was excessively glycosylated. In view of our previous analysis of glycosylphosphatidylinositol-anchored HA (Kemble et al., 1993) , the most likely explanation is that the head domains of these mutants are not packed exactly as in wt-HA, such that the N-linked carbohydrate addition sites at HA1 positions 165 and 285 are excessively glycosylated. Since dMM does not affect the biological functions of wt-HA (Kemble et al., 1994) , all subsequent analyses were performed with HAs produced in the presence of dMM.