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.

To explore further the ability of V55P to mediate outer leaflet lipid mixing, we conducted a quantitative analysis of its level of cell surface expression and its ability to mediate transfer of R18 (Fig. 7) . To do this, cells transfected with different amounts of wt-HA and V55P-HA cDNA with plasmids encoding wt-and Ala-substituted HAs were metabolically labeled with 35 S-TransLabel, treated with 5 g/ ml trypsin, and then incubated at the indicated pH for 15 min at 37ЊC, reneutralized, and lysed in an NP-40 cell lysis buffer. Cell lysates were then digested with proteinase K. The proteins were immunoprecipitated with the site A mAb, resolved by SDS-PAGE, and subjected to phosphorimager analysis. The amount of HA in each sample was calculated as described in Materials and Methods and expressed as percent sensitive to proteinase K. (B) Cells were transfected with plasmids encoding wt-, Pro-and Gly-substituted HAs and then treated with trypsin and low pH as described in A. Cell lysates were then digested with proteinase K, precipitated with Con A-agarose, reduced, subjected to 10% SDS-PAGE, and analyzed on Western blots as described in the legend to Fig. 2 . In C, cells were transfected with a plasmid encoding the double mutant V55P/S71P and processed as in B, except that they were treated with 10 g/ml trypsin before low-pH treatment.