Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

Abstract

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.

New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

“…We previously showed that a mutant containing proline at residue 55 of the B loop was impaired for fusion, and mutant V55P/S71P, with two B-loop mutations, displayed no fusion (21). Although these findings supported the importance of the spring-loaded conformational change, our prior study did not directly address coiled-coil formation and did not analyze critical conformational changes in HA2.…”

Lysolipids Do Not Inhibit Influenza Virus Fusion by Interaction with Hemagglutinin

“…The HA-mediated fusion is triggered by the acidic pH milieu of the endosomal lumen, which causes the HA ectodomain to convert into a fusogenic conformation. An extended trimeric coiled coil in the HA2 subunit is formed (5)(6)(7)(8). The reconstruction of the three-dimensional structure of the complete HA ectodomain revealed that the preserved trimeric shape of the ectodomain may direct the orientation of the coiled coil with the fusion peptides at its tip toward the target membrane (9).…”

The C Terminus of the B5 Receptor for Herpes Simplex Virus Contains a Functional Region Important for Infection

“…The first characterized of these are hemagglutinin (HA) of influenza virus (34) and gp41 of HIV (22). When these viruses bind to the cell, HA at low pH of an endosome or gp41 at neutral pH undergo detectable conformational changes that eventually involve the coiled coils.…”

“…Mutagenesis of apolar residues that are positioned to form a hydrophobic core in the ␣-helix of the heptad repeat (25,26) have been shown to alter ␣-helix conformation. Point mutations for influenza, human immunodeficiency virus (HIV) gp41 or other viral proteins alter ␣-helix formation and disrupt viral-induced membrane fusion (1,4,5,10,15,34,43). They have been identified as functional features in some cellular and viral fusion proteins (6,40).…”