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.

Fluorimetric fusion assays were performed on transiently transfected 293T cells. 293T cells were maintained in DME and supplemented as described above for the COS 7 cells except that G418 was added to maintain T-antigen expression. The 293T cells were transfected with pCB6-HA using the calcium phosphate precipitation method (Wigler et al., 1977) when the plates were ‫ف‬ 50% confluent. A total of 5 g DNA (pCB6-HA plus carrier pCB6) was transfected per 6-cm plate. The transfected cells were treated with 10 mM NaButyrate ‫ف‬ 16 h before analysis to enhance expression. Unless stated, all cells were treated 16 h before analysis with 0.25 mM deoxymannojirimycin (dMM) (Calbiochem, San Diego, CA) as described in the Results section.