Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392 Giessen, Germany.
Virus Res. 2012 Aug;167(2):343-52. doi: 10.1016/j.virusres.2012.05.022. Epub 2012 Jun 5.
Translation of the Hepatitis C Virus (HCV) positive strand RNA genome is directed by an internal ribosome entry site (IRES) in the viral RNA's 5'-untranslated region (5'-UTR). HCV propagates preferentially in the liver, and HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) acting on two target sites in the 5'-UTR. This stimulation is effective in living cells containing miR-122 and also in the rabbit reticulocyte lysate in vitro-translation system after addition of miR-122. Another RNA sequence located in the Core protein coding sequence can base-pair in a long-range RNA-RNA interaction to the HCV 5'-UTR, overlapping with the miR-122 target sites and the short spacer between them, and thereby inhibits HCV translation. Here we show genetic evidence that in reticulocyte lysate single-stranded miR-122 interferes with this inhibitory long-range RNA-RNA interaction and thereby contributes to enhanced HCV translation, involving not only the 5'-seed sequence of miR-122 but also sequences at its 3'-end. Also RNA oligonucleotides shorter than a typical microRNA stimulate HCV translation, confirming that in the reticulocyte lysate the stimulation of HCV translation functions by displacement of the inhibitory long-range interaction by miR-122. In contrast, in transfected HuH-7 hepatoma cells and in HeLa cells this interference of miR-122 with the inhibitory long-range RNA-RNA interaction plays not a major role, but only duplex miR-122 RNAs of the correct length stimulate HCV translation. These results suggest that: (1) the processing of the microRNA precursors and (2) the events occurring at the HCV RNA differ between cells and reticulocyte lysate.
丙型肝炎病毒（HCV）正链 RNA 基因组的翻译由病毒 RNA 的 5'-非翻译区（5'-UTR）中的内部核糖体进入位点（IRES）指导。HCV 主要在肝脏中繁殖，肝脏特异性 microRNA-122（miR-122）作用于 5'-UTR 中的两个靶位点，刺激 HCV 翻译。这种刺激在含有 miR-122 的活细胞中有效，并且在体外翻译系统兔网织红细胞裂解物中加入 miR-122 后也有效。另一个位于核心蛋白编码序列中的 RNA 序列可以通过长距离 RNA-RNA 相互作用与 HCV 5'-UTR 碱基配对，与 miR-122 靶位点及其之间的短间隔重叠，从而抑制 HCV 翻译。在这里，我们提供遗传证据表明，在网织红细胞裂解物中单链 miR-122 干扰这种抑制性长距离 RNA-RNA 相互作用，从而有助于增强 HCV 翻译，不仅涉及 miR-122 的 5'-种子序列，还涉及其 3'-末端的序列。此外，短于典型 microRNA 的 RNA 寡核苷酸也刺激 HCV 翻译，这证实了在网织红细胞裂解物中，miR-122 通过置换抑制性长距离相互作用来刺激 HCV 翻译的功能。相比之下，在转染的 HuH-7 肝癌细胞和 HeLa 细胞中，miR-122 与抑制性长距离 RNA-RNA 相互作用的这种干扰不起主要作用，只有正确长度的双链 miR-122 RNA 才能刺激 HCV 翻译。这些结果表明：（1）miRNA 前体的加工和（2）细胞和网织红细胞裂解物中 HCV RNA 发生的事件不同。
