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.

丙型肝炎病毒（HCV）的5'非翻译区（UTR）由四个域（I，II，III和IV）和假结组成，对于翻译和病毒复制至关重要。马非灵长类肝炎病毒（EHcV）包含5'UTR，该5'UTR由大5'末端结构域（I）组成；三个另外的结构域（I'，II和III），它们分别与HCV的结构域I，II和III同源；和假结，按列出的顺序。在这项研究中，我们调查了EHcV 5'UTR在翻译和病毒复制中的作用。由于域III的结构差异，EHcV 5'UTR的内部核糖体进入位点（IRES）活性低于HCV 5'UTR的内部核糖体进入位点。就IRES活性和亚基因组复制子（SGR）的复制而言，EHcV的结构域I和III在HCV 5'UTR中具有功能，尽管域II在SH复制的EHcV和HCV之间不可交换。此外，跨越EHcV域I和I'的区域（5'-近端EHcV特异性区域）提高了RNA稳定性，并为HCV SGR提供了不依赖microRNA 122（miR-122）的复制能力，而单独改善了EHcV域I SGR复制和RNA稳定性，与miR-122无关。这些数据表明，无论miR-122表达如何，跨越EHcV结构域I和I'的区域均可改善RNA稳定性和病毒复制。5'-近端EHcV特异性区域可能代表一种促进非肝组织中病毒复制的内在机制。跨越EHcV域I和I'的区域（5'-近端EHcV特异性区域）提高了RNA稳定性，并为HCV SGR提供了独立于microRNA 122（miR-122）的复制能力，而单独的EHcV域I则改善了SGR复制与RNA稳定性无关，与miR-122无关。这些数据表明，无论miR-122表达如何，跨越EHcV结构域I和I'的区域均可改善RNA稳定性和病毒复制。5'-近端EHcV特异性区域可能代表一种促进非肝组织中病毒复制的内在机制。跨越EHcV域I和I'的区域（5'-近端EHcV特异性区域）提高了RNA稳定性，并为HCV SGR提供了独立于microRNA 122（miR-122）的复制能力，而单独的EHcV域I则改善了SGR复制与RNA稳定性无关，与miR-122无关。这些数据表明，无论miR-122表达如何，跨越EHcV结构域I和I'的区域均可改善RNA稳定性和病毒复制。5'-近端EHcV特异性区域可能代表一种促进非肝组织中病毒复制的内在机制。这些数据表明，无论miR-122表达如何，跨越EHcV结构域I和I'的区域均可改善RNA稳定性和病毒复制。5'-近端EHcV特异性区域可能代表一种促进非肝组织中病毒复制的内在机制。这些数据表明，无论miR-122表达如何，跨越EHcV结构域I和I'的区域均可改善RNA稳定性和病毒复制。5'-近端EHcV特异性区域可能代表一种促进非肝组织中病毒复制的内在机制。

重要性在其他肝炎病毒中，EHcV是最接近HCV的病毒同源物。HCV表现出狭窄的宿主范围和特定于肝脏的嗜性，而流行病学报告表明EHcV感染马，驴和狗的肝脏和呼吸器官。但是，解释HCV和EHcV之间宿主或器官向性差异的机理尚不清楚。在这项研究中，我们的数据表明EHcV的5'非翻译区（UTR）由内部核糖体进入位点（IRES）元件组成，该元件可与HCV IRES元件功能上互换。此外，5'-近端EHcV特异性区域以miR-122独立的方式增强病毒复制和RNA稳定性。我们的数据表明，EHcV 5'UTR中结构域II的上游区域有助于这些肝炎病毒之间观察到的组织嗜性差异。