Determining the Role of NS4B in Membrane Remodelling during Hcv Replication

Abstract

Hepatitis C is an RNA virus that replicates in association with intracellular membranous structures call membranous webs (MWs). Viral protein NS4B is a key organizer of replication, one crucial function being the induction of MWs. The mechanisms of MW formation are unknown, but it clearly involves induction of membrane curvature, which may require NS4B oligomerisation and possibly hydrophobic wedging. NS4B is known to oligomerise, and the N-terminal amphipathic helix AH2 has been implied as a major determinant of self-association. In order to understand the process of MW induction, we aimed to determine AH2's capacity to remodel membranes by studying the interaction of AH2 with membranes mimicking those found within the cell using 2H and 31P solid-state NMR. Our results show changes in membrane morphology, induced by AH2 in negatively charged vesicles, an effect not observed in neutral bilayers indicating a requirement for negatively charged lipids. Chemical cross-linking studies of AH2 in lipids vesicles confirms AH2 homo-oligomerisation and suggests a charge dependency; with larger oligomers observed in neutral lipid bilayers compared to negatively charged lipid bilayers and lipid mixtures mimicking cellular membranes. These results suggest that AH2 plays a crucial role in NS4B's capacity to alter membrane morphology.