Pore Formation in Target Liposomes by Viral Fusion Proteins

Abstract

This chapter introduces methodologies to characterize plasma-membrane and viral-membrane interactions in a model system using liposomes as target membranes. It focuses on hemagglutinin (HA), a trimeric integral membrane protein that protrudes from the viral membrane as a spike, with a long stem and a globular tip. Low endosomal pH causes a conformational change, which moves the fusion peptide to the outside of the trimer. Experiments with a photoactivatable phospholipid derivative have shown that on mixing influenza virus with liposomes at low but not at neutral pH, the fusion peptide enters the liposomal membrane. The interaction of this peptide with the liposomal membrane is most likely the starting point for membrane permeabilization. Using liposomes as target membranes, it was found that whole virus, as well as bromelain-released HA (BHA) ectodomains or intact purified HA, induce the complete leakage of small water-soluble fluorescent probes across the liposomal membrane, and that a single BHA trimer suffices to empty the liposome completely. BHA induces pores of a defined size, rather than leading to membrane rupture. If the pores have a defined size and prolonged lifetime, they are not simply lipid defects, given the flexibility and fast diffusion of lipids, but are most likely proteinaceous. The chapter describes the measurement of pore size and lifetime, determination of the number of proteins required to form a pore, and related methodologies.