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 a second test of the ability of the mutant HAs to change conformation at low pH, we analyzed the ability of Figure 2 . Migration of wt-and mutant HA0s on SDS gels. (A) Effect of dMM: COS 7 cells were transfected with plasmids encoding wtand mutant HAs and grown in the absence (Ϫ) or presence (ϩ) of 0.25 mM dMM. Cell lysates were prepared, and glycoproteins were precipitated with Con A-agarose. Gel samples were prepared in sample buffer containing 100 mM DTT and separated by 10% SDS-PAGE. The gel was analyzed by Western blotting with a rabbit anti-HA antiserum as described in Materials and Methods. The antiserum reacts with HA0 and HA1. (B) Proteolytic processing: Cells transfected with plasmids encoding wt-and mutant HAs were grown in the presence of dMM and treated with either 5 g/ml chymotrypsin (Ϫ) or trypsin (ϩ) for 6 min at RT. Cell lysates were prepared and analyzed for HA protein as described above. Figure 3 . Sucrose gradient sedimentation analysis of wt-and Prosubstituted HAs. Cells transfected with plasmids encoding wtand (A) single or (B) double Pro-substituted HAs were treated with (A) 5 or (B) 10 g/ml trypsin, lysed in an NP-40 lysis buffer, and analyzed on 3-30% continuous sucrose gradients. The gradients were fractionated, and samples were precipitated with Con A-agarose, resolved by 10% SDS-PAGE, and analyzed for HA protein as described in the legend to Fig. 2 . Fraction 1 is the top of the gradient. The band shown is HA1. the single point mutant HAs, pretreated at the indicated pH, to react with an antibody against the COOH terminus of HA1 (C-HA1). This antibody efficiently detects low pH-treated wt-HA in cell lysates, and HA acquires reactivity to this antibody with a very similar time course and pH dependence to that with which it acquires reactivity to an antibody against the fusion peptide (which does not precipitate HA from crude cell lysates; Qiao, H., and J.M. White, unpublished results). Acquisition of reactivity with C-HA1 and sensitivity to proteinase K appear with similar time and pH dependencies (White and Wilson, 1987) .