Structure-function analysis of the equine hepacivirus 5' untranslated region highlights the conservation of translational mechanisms across the hepaciviruses.
Structure-function analysis of the equine hepacivirus 5' untranslated region highlights the conservation of translational mechanisms across the hepaciviruses.
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马肝炎病毒 5 非翻译区的结构功能分析强调了肝炎病毒翻译机制的保守性。
DOI：
10.1099/jgv.0.001316
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Lattimer J
中科院分区：
文献类型：
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作者：
Lattimer J
马肝炎病毒（EHcV）（现在也被归类为甲型肝炎病毒）是与丙型肝炎病毒（HCV）最接近的遗传亲缘关系，并被认为是在过去1000年内从丙型肝炎病毒中分化出来的。HCV和EHcV的5 ‘非翻译区（utr）都表现出内部核糖体进入位点（IRES）活性，允许不依赖于帽的翻译起始，但目前只对HCV的5 ’非翻译区进行了系统分析。在这里，我们报告了EHcV 5'UTR的详细结构和功能分析。利用引物延伸（SHAPE）分析的选择性2′羟基酰化分析确定了其二级结构，揭示了与HCV相似的4个茎环，分别为SLI、SLIA、SLII和SLIII。这指导了对EHcV 5'UTR的突变分析，使我们能够研究茎环在IRES功能中的作用。该方法显示，EHcV ires介导的翻译不需要SLI。相反，SLIII是必需的，特别是SLIIIb， SLIIId和在肝病毒科中保守的GGG基序。进一步的SHAPE分析证明，GGG基序介导了与40S核糖体亚基的相互作用，而SLIIIb顶端环中的CUU序列介导了与eIF3的相互作用。此外，我们发现位于SLIA和SLII之间的microRNA122靶序列在亚基因组复制子的背景下介导了翻译的增强。综上所述，这些结果强调了肝病毒翻译机制的保守性，尽管不同的初级序列。
Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across theHepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.