Data from: Phospholipid-driven conformational switching of HCV NS5A links protein folding to replication membrane remodeling

Bulankina, Anna 1 ; Richter, Rebecca 1 ; Nettles, James 2 ; Yamane, Daisuke 3 ; Grimm, Christian1 ; Karami, Yasaman 4 ; Stanton, Richard 2 ; Introini, Bianca5 ; Hermann, Jonas1 ; Charif, Hanaa1 ; König, Mia 1 ; Stroß, Claudia1 ; Ortiz, Cristina1 ; Kraus, Nico 1 ; Wood, Daniel5 ; Galceran, Facundo 6 ; Abele, Rupert 1 ; Maigret, Bernard 4 ; Schinazi, Raymond 2 ; Zeuzem, Stefan1 ; Biondi, Ricardo 7 ; Yi, MinKyung8 ; Tampé, Robert 1 ; Kudryashev, Mikhail5 ; Welsch, Christoph 1

Published Mar 04, 2026 on Dryad. https://doi.org/10.5061/dryad.cvdncjtht

Data files

Mar 04, 2026 version files 10.31 GB

Data_NS5A_PIP_manuscript.zip

10.31 GB
README.md

3.01 KB

Abstract

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.

Dataset DOI: 10.5061/dryad.cvdncjtht

Description of the data and file structure

For the HCV protein NS5A, we predicted pharmacophore sites and tested them in several assays. Our data reveal how NS5A interacts and modulates its membrane lipid environment.

The dataset “Data_NS5A_PIP_manuscript.zip” contains folders that are named according to the method, some contain subfolders named after the construct and additional information like used lipid compositions.

Abbreviations used for labeling of files:

Ctrl – control (liposomes), SUVs – small unilamellar vesicles/liposomes, PC – Phosphatidylcholine, PE – Phosphatidylethanolamine, PI4P – Phosphatidylinositol 4-phosphate, Chol - Cholesterol
FL – full length protein, AH - amphipathic helix peptide of NS5A, DeltaAH contains domain 1-3 of NS5A and DeltaDelta corresponds to NS5A domain 1
PIB - NS5A inhibitor pibrentasvir

For membrane remodelling by NS5A, we used negative stain Transmission electron microscopy (folder TEM negative stain) and Cryo-electron microscopy (folder Cryo-EM). Each subfolder contains the mrc files which indicate the used construct and/or used lipid composition. Filenames indicate the magnification used for imaging. In addition, obtained tomographs are added (folder Tomograms). All data can be visualized and analyzed with e.g. ImageJ.

Liposome recruitment (LRA) and binding assays were assayed by SDS-PAGE and immunochemistry. Each experiment is in separated and labeled folders and can be analyzed by e.g. ImageJ (folders LRA AH peptide, LRA DeltaAH lipids, LRA DeltaAH PIB, LRA DeltaDelta, PI4P-beads recruitment). Different constructs were used as indicated in the folder name. The NS5A inhibitor pibrentasvir (PIB) was tested in LRA as well as Phosphatidylinositol 4-phosphate (PI4P) beads for protein-lipid-interaction. An overview of the data and sample affiliation is listed in a separate pdf-file in each folder. Controls were set to 100% as indicated in the manuscript.

The folder PLOA (protein-lipid-overlay assay) contains the replicates with the labels of the different NS5A constructs used to investigate lipid-protein interactions.

The stability and effect of relipidated proteins were analyzed by thermal shift assays (folder TSA). The samples/curves from each run used in this study are labeled in a separate excel-file which is named the same as the raw data file obtained from StepOne software. For each buffer/lipid composition the background of the buffer was run on the same plate and subtracted from the obtained emission curves. For better comparison, the curves were normalized to the highest signal obtained between the temperature range 30-75°C. The emission curves can be analyzed with the StepOne Software.

Code/software

ImageJ and StepOne Software is needed.

Access information

n/a