Ion-channel function and cross-species determinants in viral assembly of nonprimate hepacivirus p7
Walter S., Bollenbach A., Doerrbecker J., Pfaender S., Brown R., Vieyres G., Scott C., Foster R., Kumar A., Zitzmann N., Griffin S., Penin F., Pietschmann T., Steinmann E.
Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though both viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 by using various NMR structures were generated highlighting conserved residues important for ion-channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited similar liposome permeability features than HCV p7 indicative of similar ion-channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar sub-cellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N-terminus and transmembrane domains were non-exchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C-terminus of NPHV p7 were readily exchangeable allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins including liposome permeability and broadly acting determinants were identified which modulate hepaciviral virion assembly and contribute to the host-species barrier.