title: Anionic lipids are required for vesicular stomatitis virus G protein-mediated single particle fusion with supported lipid bilayers.
abstract: Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero)phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.
authors.affiliation: Emory Children's Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Matos
authors.forename: Mariana
authors.initials: M
authors.lastname: Marin
authors.forename: Byungwook
authors.initials: B
authors.lastname: Ahn
authors.forename: Wilbur
authors.initials: W
authors.lastname: Lam
authors.forename: Nuno C
authors.initials: NC
authors.lastname: Santos
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Melikyan
mesh_terms: D004705:Endocytosis; D011992:Endosomes / Q000235:genetics / Q000378:metabolism / Q000821:virology; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D018116:Vesiculovirus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: C028391:G protein, vesicular stomatitis virus; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D010743:Phospholipids; D014759:Viral Envelope Proteins
keywords: Fluorescence; Fusion Pore; Hemifusion; Membrane Bilayer; Membrane Fusion; Microscopic Imaging; Single Virus Imaging; Virus Entry