Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

Abstract

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 biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-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-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes 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 types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage 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. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.