Dataset Information

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

ABSTRACT: UNLABELLED: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. IMPORTANCE: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.

SUBMITTER: Walter S

PROVIDER: S-EPMC4859704 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

Walter Stephanie S Bollenbach Alexander A Doerrbecker Juliane J Pfaender Stephanie S Brown Richard J P RJP Vieyres Gabrielle G Scott Claire C Foster Richard R Kumar Abhinav A Zitzmann Nicole N Griffin Stephen S Penin François F Pietschmann Thomas T Steinmann Eike E

Journal of virology 20160429 10

<h4>Unlabelled</h4>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 N ...[more]

PMID: 26962224

Similar Datasets

Project description:The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Project description:The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.

Dataset Information

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

Publications

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets