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J. Cell Biol.,
Volume 141, Number 6, June 15, 1998 1335-1347
* Department of Cell Biology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908; We tested the role of the "spring-loaded"
conformational change in the fusion mechanism of the
influenza hemagglutinin (HA) by assessing the effects
of 10 point mutants in the region of high coiled-coil
propensity, HA2 54-81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double
proline substitution at residues 55 and 71. Mutants were
expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for
their ability to change conformation and induce fusion
at low pH. We found the following: Specific mutations
affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed
trimers that could be expressed at the cell surface in a
form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the
major antigenic site and bound red blood cells. Seven
out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational
change. V55P displayed a substantial reduction (~60-
80%) in the initial rate of lipid mixing. The other single
mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved
trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both
V55P and V55P/S71P was at the level of outer leaflet
lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational
change is required for fusion. An alternate model is discussed.
Departments of
Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94143
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