Department of Biochemistry, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
J Virol. 2023 Jul 27;97(7):e0085821. doi: 10.1128/jvi.00858-21. Epub 2023 Jun 20.
The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome forms RNA structures that regulate virus replication and translation. The region contains an internal ribosomal entry site (IRES) and a 5'-terminal region. Binding of the liver-specific microRNA (miRNA) miR-122 to two binding sites in the 5'-terminal region regulates viral replication, translation, and genome stability and is essential for efficient virus replication, but its precise mechanism of action is still unresolved. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5' UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable replication of wild-type HCV genomes in cell culture, several viral variants with 5' UTR mutations exhibit low-level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Further, we provide evidence that translation regulation is the major role for miR-122 and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5' UTR mutations that stimulate translation and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Finally, we show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in promoting HCV. The unusual and essential role of miR-122 in promoting HCV propagation is incompletely understood. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside the liver.
丙型肝炎病毒（HCV）基因组的 5'非翻译区（UTR）形成调节病毒复制和翻译的 RNA 结构。该区域包含内部核糖体进入位点（IRES）和 5'末端区域。肝脏特异性 microRNA（miRNA）miR-122 与 5'末端区域中的两个结合位点结合，调节病毒复制、翻译和基因组稳定性，并且对病毒的有效复制至关重要，但它的确切作用机制仍未解决。目前的假设是，miR-122 结合通过促进病毒 5'UTR 形成翻译活性 HCV IRES RNA 结构来刺激病毒翻译。虽然 miR-122 是细胞培养中检测到野生型 HCV 基因组复制所必需的，但具有 5'UTR 突变的几种病毒变体在没有 miR-122 的情况下仍能进行低水平复制。我们表明，能够独立于 miR-122 复制的 HCV 突变体表现出增强的翻译表型，这与其独立于 miR-122 复制的能力相关。此外，我们提供了证据表明翻译调控是 miR-122 的主要作用，并表明通过联合影响刺激翻译的 5'UTR 突变和通过敲低降解基因组的宿主外切核酸酶和磷酸酶来稳定病毒基因组，可以将 miR-122 非依赖性 HCV 复制恢复到 miR-122 依赖性水平。最后，我们表明，能够独立于 miR-122 复制的 HCV 突变体也独立于通过经典 miRNA 合成途径产生的其他 microRNA 复制。因此，我们提供了一个模型，表明翻译刺激和基因组稳定是 miR-122 促进 HCV 的主要作用。miR-122 在促进 HCV 传播中的异常和重要作用尚不完全清楚。为了更好地了解其作用，我们分析了能够独立于 miR-122 复制的 HCV 突变体。我们的数据表明，病毒独立于 miR-122 复制的能力与病毒翻译增强相关，但需要基因组稳定才能恢复有效的 HCV 复制。这表明病毒必须同时获得这两种能力才能逃避对 miR-122 的需求，并且影响 HCV 进化到在肝脏外复制的可能性。
