Membrane Perturbation and Fusion Pore Formation in Influenza Hemagglutinin-mediated Membrane Fusion: A NEW MODEL FOR FUSION

Pierre Bonnafous,Toon Stegmann

doi:10.1074/jbc.275.9.6160

Abstract

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.

Highlights

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a “fusion peptide” domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion
By taking into account the available evidence for lipidic intermediates containing a hemifusion diaphragm in the literature, we propose that lipidic hemifusion intermediates are involved in influenza virus-induced fusion, the perturbation of the target membrane at the site of fusion initially leads to formation of a large pore in the target membrane, and we discuss the role of this pore in initiating fusion
We show that a variety of HA preparations (BHA, HA rosettes, and virus) all induce pores of less than 26 Å in diameter in liposomal membranes at low pH

Summary

A NEW MODEL FOR FUSION*

(Received for publication, August 13, 1999, and in revised form, November 24, 1999). From the Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 Route de Narbonne, 31077 Toulouse, France. Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a “fusion peptide” domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. Lipidic intermediates could be “stalks” of fused outer membrane leaflets, formed after a focal perturbation of the target membrane bilayer by the inserted viral fusion peptides [11, 12]. This “hemifusion” intermediate would expand laterally, be followed by breakthrough and merger of the inner leaflets at this point, leading to complete fusion. By taking into account the available evidence for lipidic intermediates containing a hemifusion diaphragm in the literature, we propose that lipidic hemifusion intermediates are involved in influenza virus-induced fusion, the perturbation of the target membrane at the site of fusion initially leads to formation of a large pore in the target membrane, and we discuss the role of this pore in initiating fusion

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION