Dataset Information

Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

ABSTRACT: Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from other sources. Photoactivable amino acid analogs used for p7 protein synthesis enabled oligomerization state analysis in liposomes by cross-linking. Therefore, these findings emphasize the quality of the cell-free produced p7 proteoliposomes which can benefit the field of the hepatitis C virus (HCV) protein production and characterization and also provide tools for the development of new inhibitors to reinforce our therapeutic arsenal against HCV.

SUBMITTER: Soranzo T

PROVIDER: S-EPMC5113752 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

Soranzo Thomas T Cortès Sandra S Gilde Flora F Kreir Mohamed M Picart Catherine C Lenormand Jean-Luc JL

Protein expression and purification 20151022

Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from ...[more]

PMID: 26477501

Similar Datasets

Project description:The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Dataset Information

Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

Publications

Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets