title: Treatment of influenza virus with beta-propiolactone alters viral membrane fusion.
abstract: Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000μM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.
authors.affiliation: University of Bordeaux, CBMN UMR 5248, IPB, IECB, F-33600 Pessac, France; CNRS, CBMN UMR 5248, F-33600 Pessac, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bonnafous
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Nicolaï
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Taveau
authors.forename: Michel
authors.initials: M
authors.lastname: Chevalier
authors.forename: Fabienne
authors.initials: F
authors.lastname: Barrière
authors.forename: Julie
authors.initials: J
authors.lastname: Medina
authors.forename: Olivier
authors.initials: O
authors.lastname: Le Bihan
authors.forename: Olivier
authors.initials: O
authors.lastname: Adam
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ronzon
authors.forename: Olivier
authors.initials: O
authors.lastname: Lambert
mesh_terms: D000547:Amantadine / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D020285:Cryoelectron Microscopy; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000737:chemistry; D006389:Hemagglutinins, Viral / Q000737:chemistry*; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D008985:Monensin / Q000737:chemistry / Q000494:pharmacology; D010539:Permeability; D011420:Propiolactone / Q000737:chemistry* / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D005452:Fluoresceins; D006389:Hemagglutinins, Viral; D008081:Liposomes; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus; D011420:Propiolactone; D008985:Monensin; D000547:Amantadine; C007740:fluorexon
keywords: Beta-propiolactone; Cryo electron microscopy; Cryo electron tomography; Hemagglutinin; Influenza virus; Membrane fusion