Abstract
The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by (1)H and (13)C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.
Highlights
The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity
An alternative topology wherein the C terminus is exposed to the cytoplasm may be adopted [9]. p7 is not required for RNA replication in vitro but is essential for the late phase of assembly and the release of infectious Hepatitis C virus (HCV) in vitro [7, 10] as well as productive infection in vivo [11]. p7 probably acts in concert with other viral structural proteins and/or NS2, and its function may depend on interactions with other viral factors, such as core and/or NS2 [12,13,14]
Phospholipid Bilayer—The model of the complete p7 monomeric protein was built on the basis of the structural information acquired from Circular dichroism (CD) and NMR experiments in membrane mimetics and the p7 membrane topology with both N and C termini pointing toward the endoplasmic reticulum lumen, as determined previously [8]
Summary
Sequence Analyses—Sequence analyses were performed using tools available at the Institut de Biologie et Chimie des Proteines (IBCP) (i.e. the Network Protein Sequence Analysis (NPSA) Web site) [29]. Molecular Modeling of the Monomeric HCV p7 Protein—On the basis of the structural information acquired from both CD experiments in membrane mimetics and NMR structures in TFE/water solutions, a model of the complete monomeric protein was built. Because the latter experiments only supply fragments of the complete structure of p7 (see “Results”), a number of assumptions should be made when the segments of known structure are combined with those of undefined structure. A 40-ns trajectory was generated, over which the last 20 ns were considered for the analysis of the molecular assembly
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