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.

For the purpose of this study, we analyzed 10 mutant HAs with Ala, Gly, or Pro substitutions in the region of high coiled-coil propensity; one mutant contained prolines at two residues: HA2 55 and 71. All 10 mutant HAs were expressed at the cell surface, could be cleaved from HA0 to HA, comigrated with wt-HA on SDS gels (when produced in the presence of dMM), reacted with an antibody against the major antigenic site, bound RBCs, and, where tested (7 of 10), formed trimers. Hence, none of the 10 HA coiled-coil mutants appeared to be severely altered in pH 7 structure. Several of the mutations did, however, affect the precise structure of the HA trimer. Several mutants, notably those with Gly and Pro substitutions at positions 55 and 80, were excessively terminally glycosylated (in the absence of dMM), presumably because their globular head domains are not packed exactly as in wt-HA (Kemble et al., Figure 7 . Cell surface expression and normalized fusion activity of wt-and V55P-HA. Cells transfected with the indicated amounts of plasmids encoding wt-and V55P-HA (1ϫ ϭ 1.25 g) were metabolically labeled, treated with either 5 g/ml chymotrypsin (C) or trypsin (T), lysed with an NP-40 lysis buffer, and immunoprecipitated with the site A mAb. Samples were then resolved by 12.5% SDS-PAGE and analyzed with a phosphorimager. (A) Gel scan. (B) Quantitation of the intensity of the bands. (C and D) Parallel cultures of cells expressing different amounts of wtand V55P-HA at their surface were treated at 37ЊC with pH 5 buffer for (C) 2 or (D) 10 min and then analyzed for fusion as described in the legend to Fig. 6 . Fused cells in four or five fields ‫002ف(‬ cells/field) were counted and averaged. 1993). And, in contrast to wt-HA, four of the mutants, including the fusion-competent mutants V55G, L80G, and L80P, were sensitive to proteinase K (and for L80P reactive with an antibody against the COOH terminus of HA1) at neutral pH.