Dimer Organization of Membrane-Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane‐associated protein involved in multiple steps of the viral life cycle. Direct‐acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode‐of‐action of these drugs is poorly understood. This is due to the lack of information on the membrane‐bound NS5A structure. Herein, we present the structural model of an NS5A AH‐linker‐D1 protein reconstituted as proteoliposomes. We use highly sensitive proton‐detected solid‐state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self‐interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH‐linker‐D1 in a lipidic environment shedding light onto the mode‐of‐action of clinically used NS5A inhibitors.
The membrane orientation of the hepatitis C virus NS5A protein was assessed by combining a cell‐free protein synthesis approach with highly sensitive 1H‐detected solid‐state NMR. Insertion of lipids chelated with a paramagnetic Gd3+ ion allowed to orient the protein with respect to its membrane anchor using PRE. This information allowed to propose a model for the interaction of NS5A with a direct acting antiviral.