Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre 2650, Denmark.
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark.
Nucleic Acids Res. 2020 Jun 4;48(10):5555-5571. doi: 10.1093/nar/gkaa300.
Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was also described for the related pestiviruses, which are important livestock pathogens. The liver-specific miR-122 binds within the HCV 5' untranslated region (UTR), whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2, respectively) in the pestiviral 3' UTR. Here, we dissected the mechanism of miRNA dependency of the pestivirus bovine viral diarrhea virus (BVDV). Argonaute 2 (AGO2) and miR-17 binding were essential for viral replication, whereas let-7 binding was mainly required for full translational efficiency. Furthermore, using seed site randomized genomes and evolutionary selection experiments, we found that tropism could be redirected to different miRNAs. AGO cross-linking and immunoprecipitation (CLIP) experiments and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Interestingly, we also identified miRNA-independent variants that were obtained through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Altogether, our findings suggest that pestiviruses, although capable of miRNA-independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation.
宿主 microRNA（miRNA）依赖性是人类病原体丙型肝炎病毒（HCV）的一个标志，也被描述为相关的瘟病毒，它们是重要的家畜病原体。肝脏特异性 miR-122 结合在 HCV 5'非翻译区（UTR）内，而广泛表达的 let-7 和 miR-17 家族分别结合在瘟病毒 3'UTR 的两个位点（S1 和 S2）。在这里，我们剖析了瘟病毒牛病毒性腹泻病毒（BVDV）的 miRNA 依赖性的机制。Argonaute 2（AGO2）和 miR-17 结合对于病毒复制是必需的，而 let-7 结合主要是为了充分的翻译效率。此外，我们使用种子位点随机化基因组和进化选择实验，发现对不同 miRNA 的嗜性可以被重新定向。AGO 交联和免疫沉淀（CLIP）实验以及 miRNA 拮抗作用表明，这些替代变体结合并依赖于相应的 miRNA。有趣的是，我们还鉴定了 miRNA 非依赖性变体，这些变体是通过在基因组 3'末端附近获得补偿性突变而获得的。挽救实验表明，miRNA 结合和 3'突变有助于通过相互排斥的机制进行复制。总之，我们的研究结果表明，瘟病毒虽然能够进行 miRNA 非依赖性复制，但利用 miRNA 作为必要的宿主因子，在进化适应过程中具有有利的途径。
