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 rationalize that L80P shows more fusion activity than expected as follows: (a) HA2 80 is in the first turn of the long ␣ helix in native wt-HA. The fact that L80P is well expressed at the cell surface as a trimer with biochemical properties similar to wt-HA implies that the Pro substitution at position 80 was accommodated in the pH 7 structure. Hence the segment containing HA2 80 in L80P likely did not have to convert from a random into a helical 293T cells were transfected with the indicated amounts of pCB6 HA or pCB6 V55P-HA and analyzed for cell surface HA expression by FACS ® using the site A mAb (60 g/ml) and an FITC-conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR). "Percent of cells" indicates the percent of cells expressing HA; "Intensity" describes the mean intensity of each sample. Initial fusion rates were calculated from the data displayed in Fig. 8 and are given in U/s. structure at low pH. (b) If the loop to helix transition is nucleated at the NH 2 -terminal end of the loop (HA2 55), then a residue at the COOH-terminal end, especially one that is already in part of an ␣-helical segment, may not significantly influence the transition to the low-pH form. (c) Independent evidence (next paragraph) supports the importance of the NH 2 -terminal end of the spring-loaded conformational change region. Furthermore (d), L80P appears to be somewhat impaired in syncytia formation. We recently isolated an influenza virus mutant with the substitution HA1 K27E. This mutant was selected (four times independently) by growth in the presence of diiodofluorescein, a small molecule that is predicted to bind in the spring-loaded coiled-coil region (Hoffman et al., 1997) . Diiodofluorescein affects the conformational change in X:31 HA and inhibits fusion and infection in tissue culture. The mutation would promote the formation of a salt link between HA1 E27 and HA2 R54 (the most NH 2 -terminal residue of the loop; see Fig. 8, C and D in Hoffman et al., 1997) and thereby stabilize the molecule (i.e., make it harder to undergo the spring-loaded conformational change). Biochemical data support this prediction (see Table IV in Hoffman et al., 1997) . The proximity of HA1 27 to HA2 54 suggests the importance of the NH 2 -terminal end of the spring-loaded coiled-coil region for fusion activation.