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The hepatitis C virus (HCV) viroporin p7 oligomerizes to form ion channels, which are required for the assembly and secretion of infectious viruses. The 63-amino acid p7 monomer has two putative transmembrane domains connected by a cytosolic loop, and has both N- and C- termini exposed to the endoplasmic reticulum (ER) lumen. NMR studies have indicated differences between p7 structures of distantly related HCV genotypes. A critical question is whether these differences arise from the high sequence variation between the different isolates and if so, how the divergent structures can support similar biological functions. Here, we present a side-by-side characterization of p7 derived from genotype 1b (isolate J4) in the detergent 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and p7 derived from genotype 5a (isolate EUH1480) in n-dodecylphosphocholine (DPC). The 5a isolate p7 in conditions previously associated with a disputed oligomeric form exhibits secondary structure, dynamics, and solvent accessibility broadly like those of the monomeric 1b isolate p7. The largest differences occur at the start of the second transmembrane domain, which is destabilized in the 5a isolate. The results show a broad consensus among the p7 variants that have been studied under a range of different conditions and indicate that distantly related HCVs preserve key features of structure and dynamics.

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 cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined 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 neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes 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 while 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 showed that these residues are indeed 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 molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Infection with the hepatitis C virus (HCV) has a big impact on global health. It is estimated that approximately 3 % of the world’s population carry HCV, putting more than 200 million people at risk of developing severe liver disease, including chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The HCV encoded viroporin p7 forms ion channels that are crucial for the assembly and secretion of infectious viruses, making it a potential drug target. Its hydrophobic nature makes p7 notoriously difficult to investigate in an untagged native form. A previously determined 16 Å electron microscopy single-particle reconstruction in detergent showed a hexameric, flower-shaped p7 protein. In conjunction with one hexameric and several monomeric p7 solution state NMR structures published, this constitutes the currently available structural information framework. An E. coli expression system is introduced, which is especially adapted to express isotopically labeled p7. For the first time, suitable solution-state NMR conditions at physiological pH and temperature were identified that gave rise to high quality spectra suitable to interrogate iminosugar drug interactions with untagged isotopically labeled J4 p7 (C27S) solubilised in detergent. A novel secondary structure topology was observed and preliminary iminosugar binding sites were determined. Further, a DIB (droplet interface bilayer) system to analyse p7 ion channel function was established, which is suitable to elucidate how inhibitors act on p7 genotypes and how different lipids influence the ion channel function of p7. The p7 oligomeric state was further investigated using native gel analysis, showing that isolates representing HCV genotypes 1 - 6 form oligomeric complexes. An ion channel defective dibasic mutant implicated in severely compromising viral fitness is also shown for the first time to form an oligomer, implicating that it is not an assembly problem that leads to the abrogated function.