Hoffman Brett, Li Zhubing, Liu Qiang
VIDO-InterVac, Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
VIDO-InterVac, Vaccinology and Immunotherapeutics, Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
J Gen Virol. 2015 Aug;96(8):2114-2121. doi: 10.1099/vir.0.000141. Epub 2015 Apr 10.
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.
丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）对病毒复制至关重要；然而，其对HCV RNA翻译的影响仍存在争议，部分原因是使用了缺乏3'UTR的报告基因，而NS5A会与该区域的聚（U/UC）序列结合。我们使用一个含有海肾荧光素酶基因且两侧为HCV UTR的单顺反子RNA，研究了NS5A在HCV翻译中的作用。我们发现NS5A以剂量依赖的方式下调病毒RNA翻译。这种下调需要HCV的5'和3'UTR，因为用肠道病毒71的5'和3'UTR或5'端的帽结构替换任何一个序列都会消除NS5A对翻译的影响。NS5A也下调了HCV基因组RNA的翻译。NS5A对HCV翻译的抑制需要3'UTR中的聚（U/UC）序列，因为删除该序列后NS5A不影响翻译。此外，我们表明，虽然NS5A的两亲性α螺旋对病毒翻译没有影响，但NS5A的三个结构域可以独立抑制翻译，这也依赖于3'UTR中聚（U/UC）序列的存在。这些结果表明，NS5A通过一种涉及3'UTR中聚（U/UC）序列的机制下调HCV RNA翻译。
