Phospholipid-driven conformational switching of HCV NS5A links protein folding to replication membrane remodeling.

Phospholipids are essential for RNA virus replication, yet their role in modulating conformational dynamics of membrane-associated viral proteins remains poorly understood. For NS5A, a key replication factor of hepatitis C virus, previous crystallographic models fail to capture the lipid-driven conformational mechanics we uncover here. Using structural informatics and biochemical probing of pharmacophore-guided mutants in defined lipid environments, we evaluated competing NS5A domain 1 dimerization models. Our data reveal an alternative membrane-specific fold stabilized by polyproline hinges and phospholipids (PIPs) such as phosphatidylinositol-4-phosphate, a host lipid enriched at replication membranes. PIP binding promotes a conformational switch that drives dimerization, linking lipid sensing to membrane remodeling and host factor recruitment. This reciprocal mechanism-where a lipid allosterically modulates a viral protein that reshapes membranes-is blocked by the antiviral pibrentasvir. These findings define a lipid-driven structural switch that governs NS5A pleiotropy and highlight dynamic lipid-protein interfaces as targets for antiviral intervention.