The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10° and 25° relative to the bilayer normal. A truncated construct corresponding to the second transmembrane domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix segment tilted at 10° is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7.

Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug-protein and protein-protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.