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.

Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Lipid mix, pH 5 ϩ ϩ ϩ ϩ* ϩ* ϩ ϩ Ϯ ϩ ϩ Ϫ pH, Lipid mix 5.2 5.2 5.2 5.4 ND ND ND 5.2 5.2 5.2 NA Content mix, pH 5 ϩ ϩ ϩ ϩ* ϩ* ϩ ϩ Ϯ ϩ ϩ Ϫ ND indicates not done. NA indicates not applicable (no fusion detected at any pH). In the seventh row, data are for both lipid and content mixing. Asterisks in the eighth and tenth rows indicate a decrease in fusion that correlated with a reduced level of cleaved HA at the cell surface. In the eighth and tenth rows, Ϯ indicates decreased fusion activity for V55P after normalization for cell surface expression (see text for details). ‡ These values are upper limits. The pH values for 50% proteinase K sensitivity or reactivity with C-HA1 antibody will be between pH 5.6 and 6.2 (see Figs. 4 and 5). Figure 10 . Fusion activity of Pro-substituted HAs: content mixing. Cells transfected with plasmids encoding wt-and Pro-substituted HAs were prepared for fusion, incubated with calcein AM-labeled RBCs, and inspected by fluorescence microscopy as described in the legends to Figs. 6 and 9. membrane fusion proteins. Many viral fusion proteins, including those of retro-, paramyxo-, filo-, and coronaviruses contain or are predicted to contain coiled-coil regions (Chambers et al., 1990; Fass et al., 1996; Gallaher, 1996; Weissenhorn et al., 1997) . Moreover, a variety of peptide inhibition and mutagenesis studies have supported the hypothesis that the (predicted) coiled-coil regions of these proteins are important for fusion (Buckland et al., 1992; Chen et al., 1993; Ramsdale et al., 1996; Reitter et al., 1995; Wild et al., 1994a,b; Lambert et al., 1996; Yao and Compans, 1996) .