Membrane fusion by an RGD-containing sequence from the core protein VP3 ofhepatitis A virus and the RGA-analogue: Implications for viral infection

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by bi ophysical methods. Two types of model membrane were used: vesicles and mono layers spread as the air/water interface, with a composition that closely r esembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15 ; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine SM: sphingomyelin fro m chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alp ha -phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/ SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-diale oyl-3-trimethylammonium-propane) membranes were also prepared in order to d issect the electrostatic and hydrophobic components in the interaction. Cha nges in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three t ypes of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are trigge red by acidification at pH 4-6, characteristic of the endosome. Fluorescenc e experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and lea kage or mixing of aqueous contents in anionic and cationic vesicles at pH 4 -6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/ PE) results in leakage without lipid mixing, indicating pore formation. Rep lacement of aspartic acid in the RGD motif by alanine maintains the membran e-destabilizing properties of the the peptide at low pH, but not its antige nicity. Since the RGD tripeptide is related to receptor-mediated cell adhes ion and antigenicity results suggest that receptor binding is not a molecul ar requirement for fusion. The possible involvement of peptide-induced memb rane destabilization in the mechanism of hepatitis A virus infection of hep atocytes by the endosomal route is discussed. (C) 2000 John Wiley & Sons, I nc.