A Specific Point Mutant at Position 1 of the Influenza Hemagglutinin Fusion Peptide Displays a Hemifusion Phenotype

Abstract

We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

“…The G1E mutation in intact HA also inhibits HA-mediated fusion (22,23). Our results indicate that studies of membrane fusion activity using liposomes that are at the brink of spontaneous lipid mixing, such as those of DOPC:DOPE: cholesterol (1:1:1), are not as discriminatory of the fusogenic activity of different protein constructs with different intrinsic fusogenic activity.…”

“…Our results indicate that studies of membrane fusion activity using liposomes that are at the brink of spontaneous lipid mixing, such as those of DOPC:DOPE: cholesterol (1:1:1), are not as discriminatory of the fusogenic activity of different protein constructs with different intrinsic fusogenic activity. This is also indicated by the fact that there was some fusion activity of the G1E-HA2* with these liposomes, but no fusion was observed with the intact G1E mutant in the experiments on cell fusion (22).…”

“…2A). This mutation in the first position of the HA2 is known to abolish HA-mediated fusion (22,23). The difference between HA2* and G1E-HA2* became greater in the less fusogenic liposomes composed of DOPC:cholesterol (1:1) (Fig.…”

Analysis of a Membrane Interacting Region of Herpes Simplex Virus Type 1 Glycoprotein H

“…Several mutagenesis studies indicate that the glycine at the N terminus of influenza virus HA2 fusion peptide is particularly critical, and substitution of this residue with more polar or more hydrophobic residues results in a complete loss of activity; the only tolerated change in this position appears to be an alanine. A particularly interesting mutant is G1S of HA2, because replacing the glycine with a serine facilitates lipid mixing but not content mixing (65,66). Also the ability of human immunodeficiency virus type 1 gp41 to promote membrane fusion can be completely abolished by a single mutation G10V within the fusion peptide (67).…”

A bundling of viral fusion mechanisms

“…Mutational studies of class I viral fusion proteins have shown that simply pulling the two membranes together is not sufficient (1)(2)(3) to catalyze fusion in a physiological context: in these systems, a specific activity of the membrane-inserted peptides is required for full fusion. The precise mechanism of membrane activity has remained elusive.…”