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 next assessed whether the single point mutant HA0s were delivered to the cell surface and whether they could be proteolytically processed by the addition of trypsin. Cells expressing wt-and mutant HA0s were treated with either trypsin or chymotrypsin. As shown in Fig. 2 B , all of the mutant HA0s were accessible at the cell surface for proteolytic cleavage by trypsin, as evidenced by the appearance of a comigrating HA1 band. Most of the mutant HA1s were generated at approximately equal levels to wt-HA (V55A, S71A, S71G, S71P, and L80P), but for V55G, V55P, L80A, and L80G, the intensity of the HA1 band was less. These results suggested that all of the mutant HA0s were transported to the cell surface in a form that could be cleaved to HA1 and HA2. The latter mutants, however, were either less efficiently delivered to the cell surface or less efficiently cleaved from HA0 to HA1 and HA2. We consider the latter possibility likely since all Structure of HA at neutral pH displaying the residues present in TBHA2, a fragment of low pH-treated HA (Bullough et al., 1994) . (B) Structure of TBHA2. (C) Residues HA2 54-81 in TBHA2. The region of high coiled-coil propensity (Carr and Kim, 1993) , HA2 54-81, is shown in black. Wild-type sidechains for the residues mutated are displayed and labeled.