Anna V. Bulankina, R. Richter, James H. Nettles, Daisuke Yamane, Christian Grimm, Yasaman Karami, Richard A. Stanton, Bianca Introini, Jonas Hermann, Hanaa Charif, Mia König, Claudia Stroß, Cristina Ortíz, Nico Kraus, D. J. Wood, Facundo Galceran, Rupert Abele, Bernard Maigret, Raymond F. Schinazi, Stefan Zeuzem, Ricardo M. Biondi, MinKyung Yi, Robert Tampé, Mikhail Kudryashev, Christoph Welsch
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.