Role of the lipid environment in NS5A structural rearrangements and drug interactions in hepatitis C

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Functional studies of viral proteins in their lipid environment are particularly difficult. The non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) is a most prominent example, that despite being a key target site of direct-acting antiviral agents (DAA), still is among the most enigmatic HCV proteins. Virus replication is closely tied to the lipid metabolism of liver cells with cholesterol and its precursor molecules known to modulate the biological activity of membrane-bound proteins. NS5A is critically involved in viral replication that takes place at newly formed membranes within the endoplasmic reticulum (ER; membranous web) and assists viral assembly in close vicinity of lipid droplets (LDs). Resistance mutations against NS5A inhibitors and related fitness-compensatory mutations exclusively occur at the NS5A-lipid interface, and potentially persist for years after treatment failure with adverse impact on retreatment responses. Despite the potential key role of lipids in the NS5A structure-function relationship, dynamic models to address the role of the lipid environment for structural rearrangements in NS5A are still in their infancy. We apply an integrated structural biology approach combining computational techniques with experimental approaches such as optical biosensor technology to assess the impact of specific subcellular lipid environments on NS5A conformation and lipid-triggered structural rearrangements. By using artificial membrane technology and synthetic biology, we mimic the ER and LD membrane lipid composition with purified NS5A protein embedded. We will identify specific NS5A-lipid binding events and study related structural rearrangements and their impact on NS5A self-interaction, oligomerization and membrane remodeling. Our aim is to understand mechanistic details in the interplay between membrane lipids and NS5A to explore fundamental biochemical principles in the biology of RNA viruses. In this way, we will also characterize the mechanisms of action of NS5A inhibitors and related viral escape mechanisms.