SPECIFIC SINGLE OR DOUBLE PROLINE SUBSTITUTIONS IN THE SPRING-LOADED COILED-COIL REGION OF THE INFLUENZA HEMAGGLUTININ IMPAIR OR ABOLISH MEMBRANE-FUSION ACTIVITY

We tested the role of the ''spring-loaded'' conformational change in t he fusion mechanism of the influenza hemagglutinin (HA) by assessing t he effects of 10 point mutants in the region of high coiled-coil prope nsity, 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 ce ll surface expression and structural features as well as for their abi lity to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate struct ure 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 cou ld be proteolytically cleaved from the pre cursor, HA0, to the fusion- permissive form, HA1-S-S-HA2. All mutants reacted with an antibody aga inst the major antigenic site and bound red blood cells. Seven out oft en mutants displayed a wild-type (wt) or moderately elevated pH depend ence for the conformational change. V55P displayed a substantial reduc tion (similar to 60-80%) in the initial rate of lipid mixing. The othe r single mutants displayed efficient fusion with the same pH dependenc e as wt-HA. The double proline mutant V55P/S71P displayed no fusion ac tivity despite being well expressed at the cell surface as a proteolyt ically cleaved trimer that could bind red blood cells and change confo rmation at low pH. The impairment in fusion for both V55P and V55P/S71 P was at the level of outer leaflet lipid mixing. We interpret our res ults in support of the hypothesis that the spring-loaded conformationa l change is required for fusion. An alternate model is discussed.