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Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

ABSTRACT: 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.

SUBMITTER: Tanaka T

PROVIDER: S-EPMC5972865 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

Tanaka Tomohisa T Otoguro Teruhime T Yamashita Atsuya A Kasai Hirotake H Fukuhara Takasuke T Matsuura Yoshiharu Y Moriishi Kohji K

Journal of virology 20180314 7

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 i ...[more]

PMID: 29343570

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Project description: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.

Project description:The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5'-untranslated region (5'UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5'UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon.The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression.

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Dataset Information

Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

Publications

Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

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