Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features

Abstract

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.

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

“…In both cases, solid-state NMR was compared to solution-state NMR data obtained in micelles, and Opella and coworkers first identified the localization of the transmembrane domains of p7 and Vpu in lipid bilayers. Unfortunately, the full p7 structure in lipids could not yet be solved, as this would help to resolve contrasting data between different detergent-based solution NMR studies [ 145 , 146 , 147 , 148 , 149 ]; the membrane might actually play a central role in determining p7 structure and oligomerization.…”

Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR

“…Solution NMR was also used to determine the structure of another helical membrane protein, the p7 cation channel from hepatitis C. Chou and colleagues determined a hexameric assembly of the p7 protein in DPC micelles, in which the protein formed a funnel-like structure with a flower shape [ 78 ]. However, this structural model has been disputed by Zitzman, Schell and colleagues, who have presented evidence that in DPC micelles, the protein is monomeric [ 79 , 80 ]. The latter investigation used a higher excess of DPC than Chou et al [ 81 ], who subsequently showed that p7 does form a hexameric structure in isotropic bicelles.…”

Solution NMR: A powerful tool for structural and functional studies of membrane proteins in reconstituted environments

“…Phospholamban (PNL), a homopentamer expressed in sarcoplasmic reticulum that controls intracellular Ca 2ϩ levels through its phosphorylation state, is another example where solution NMR structure determined in DPC micelles (PDB code 1ZLL) (143) differs significantly from the one obtained in lipid bilayer by a combination of solution and solid-state NMR methods (PDB code 2KYV) (136). The "bellflower" model originally proposed (Fig.…”