Unusual architecture of the p7 channel from hepatitis C virus

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 i12 and i13 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 wholecell 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 p7mediated cation conductance and its inhibition by adamantane derivatives. Many viruses have developed integral membrane proteins to transport ions and other molecules across the membrane barrier to aid various steps of viral entry and maturation. These membrane structures, known as viroporins, usually adopt minimalist architectures that are significantly different from those of bacterial or eukaryotic ion channels. Therefore, understanding the structural basis of how viroporins function broadens our knowledge of channels and transporters while generating new opportunities for therapeutic intervention. The viroporin formed by the HCV p7 protein has been sought after as a potential anti-HCV drug target. p7 is a 63-residue membrane protein that oligomerizes to form ion channels with cation selectivity, for Ca over K and Na (refs 2, 3, 12, 13), and a more recent study has also reported p7-mediated H intracellular conductance. The p7 channel is required for viral replication; it has been shown to facilitate efficient assembly and release of infectious virions, although the precise mechanism of these functions remains unclear. The channel activity can be inhibited by adamantane and long alkyl chain iminosugar derivatives and hexamethylene amiloride in vitro, with varying reported efficacies. In addition to ion conduction, p7 has been shown to specifically interact with the non-structural HCV protein NS2, indicating that its channel activity could be regulated. There is not yet a detailed structure of the p7 channel, although a number of NMR studies showed that the p7 monomer has three helical segments: two in the amino-terminal half of the sequence and one near the carboxy terminus. A single-particle electron microscopy (EM) study obtained a 16 Å resolution electron density map of the p7 oligomer using the random conical tilting approach. The map shows that the p7 channel is a 42-kDa hexamer and adopts a flower-like shape that does not resemble any of the known ion channel structures in the database. It is not known how the small p7 polypeptide assembles into what appears to be a complex channel structure, and whether the viroporin has adopted novel structural elements for cation selectivity and channel gating. Amantadine or rimantadine blocks the influenza M2 channel by binding to the small pore formed by four transmembrane helices, but the pore of the p7 hexamer is expected to be much bigger and it is thus unclear how these small molecules would fit. We sought to address these questions by determining detailed structures of the p7 hexamer and its drug-binding site. We systematically tested p7 amino acid sequences from various HCV genotypes and found that the sequence from genotype 5a (EUH1480 strain) generated samples that were sufficiently soluble for structure determination (Supplementary Fig. 1). This p7 construct, designated here as p7(5a), could be efficiently reconstituted in dodecylphosphocholine (DPC) micelles at near physiological pH and generated high-quality NMR spectra (Supplementary Fig. 2). Negativestain EM of the DPC-reconstituted p7(5a) in NMR buffer showed hexameric, flower-shaped particles that are similar to those in the electron micrographs of the p7 (JFH-1 strain, genotype 2a) hexamer in dihexanoyl-phosphatidyl-choline (DHPC) micelles used earlier for single-particle reconstruction (Supplementary Fig. 3). Moreover, isothermal titration calorimetry and NMR chemical shift perturbation analyses of p7(5a)–rimantadine interaction showed that the drug binds specifically to the reconstituted protein with a binding constant (Kd) from 50 to 100mM at 3 mM detergent concentration (Supplementary Figs 4 and 5). The above results together indicate that the p7(5a) polypeptides reconstituted in DPC micelles form structurally relevant hexamers. Structure determination of the p7(5a) hexamer by NMR used an approach taken earlier for oligomeric membrane proteins, which involves: (1) determination of local structures of the monomers; and (2) assembly of the oligomer with intermonomer distance restraints and orientation constraints. The NMR-derived restraints define an ensemble of structures with backbone root mean squared deviation (r.m.s.d.) of 0.74 Å (Fig. 1a). Each monomer consists of an N-terminal helix (H1) from residues 5–16, a middle helical segment (H2), with a