title: Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.
abstract: Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two 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 the 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 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular 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 nonexchangeable 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 that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified.
  
  
  
  The recent discovery of new relatives of hepatitis C virus (HCV) enables for the first time the study of cross-species determinants shaping hepaciviral pathogenesis. Nonprimate hepacivirus (NPHV) was described to infect horses and represents so far the closest homolog of HCV. Both viruses encode the same viral proteins; however, NPHV protein functions remain poorly understood. In this study, we aimed to dissect NPHV p7 on a structural and functional level. By using various NMR structures of HCV p7 as templates, three-dimensional homology models for NPHV p7 were generated, highlighting conserved residues that are important for ion channel function. A p7-based trans-complementation approach and the construction of NPHV/HCV p7 chimeric viruses showed that the N terminus and transmembrane domains were nonexchangeable. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious viral particles. These results identify species-specific constraints as well as exchangeable determinants in hepaciviral assembly.
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Walter
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bollenbach
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Doerrbecker
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pfaender
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Richard J P
authors.initials: RJP
authors.lastname: Brown
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Vieyres
authors.affiliation: Leeds Institute of Cancer & Pathology, University of Leeds, St James' University Hospital, Leeds, Great Britain.
authors.forename: Claire
authors.initials: C
authors.lastname: Scott
authors.affiliation: Leeds Institute of Cancer & Pathology, University of Leeds, St James' University Hospital, Leeds, Great Britain.
authors.forename: Richard
authors.initials: R
authors.lastname: Foster
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, Great Britain.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, Great Britain.
authors.forename: Nicole
authors.initials: N
authors.lastname: Zitzmann
authors.affiliation: Leeds Institute of Cancer & Pathology, University of Leeds, St James' University Hospital, Leeds, Great Britain.
authors.forename: Stephen
authors.initials: S
authors.lastname: Griffin
authors.affiliation: Institut de Biologie et Chimie des Protéines, CNRS, UMR 5086, Laboratoire International Associé CNRS-UIUC, Labex Ecofect, University of Lyon, Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pietschmann
authors.affiliation: Institute for Experimental Virology, TWINCORE, Hannover, Germany Eike.Steinmann@twincore.de.
authors.forename: Eike
authors.initials: E
authors.lastname: Steinmann
mesh_terms: D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005816:Genetic Complementation Test; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D006736:Horses; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes; D008958:Models, Molecular; D010539:Permeability; D013045:Species Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication; D000086082:Viroporin Proteins
chemical_list: D007473:Ion Channels; D008081:Liposomes; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins