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 use three lines of reasoning to reconcile our support for the spring-loaded conformational change model with the countering evidence from the electron microscopic and low temperature studies cited above: (a) Most of the experiments in the latter studies were performed in the absence of target membranes. However, recent studies with both HA (Tatulian et al., 1995; Gray and Tamm, 1997 ) and with a model retrovirus (Hernandez et al., 1997) have provided clear evidence for differences in the conformational changes of viral fusion proteins at membrane surfaces. (b) Some separation of the globular head domains is required for fusion, at least with X:31 HA at 37ЊC (Godley et al., 1992; Kemble et al., 1992) . However, it is not clear to us that the globular head domains must separate to an extent that would be readily visible in the electron microscope to elicit the loop to helix transition of HA2 55-76 (see Shangguan et al., 1998 for an alternate viewpoint). (c) It may be that only a few trimers, those at the fusion site (Danieli et al., 1996) , adopt the spring-loaded conformational change during fusion, and that these few molecules have escaped detection in the electron microscopic (and antibody) studies. The bulk population of virion HAs may undergo the change at a later time (Weber et al., 1994; Wharton et al., 1995) . In addition, we have noted a change in one of the neutral specific epitopes (N1) analyzed in the low temperature study (performed on HA; Stegmann et al., 1990) when we analyzed bromelain-released hemagglutinin (BHA) treated on ice at pH 5.0. With BHA, loss of the N1 epitope appeared to occur to the same extent and with the same time course on ice as at 37ЊC (Bodian, 1992) .