Jangra Rohit K, Yi Minkyung, Lemon Stanley M
Center for Hepatitis Research, Institute for Human Infections and Immunity, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-0610, USA.
J Virol. 2010 Jul;84(13):6615-25. doi: 10.1128/JVI.00417-10. Epub 2010 Apr 28.
miR-122 is a liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious virus is dependent upon two miR-122-binding sites near the 5' end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious virus in transfected cells, while virus production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus life cycle.
miR-122是一种肝脏特异性微小RNA，它正向调节丙型肝炎病毒（HCV）RNA丰度，并且对于传染性HCV的产生至关重要。通过遗传学方法，我们发现其增强传染性病毒产量的能力取决于HCV基因组5'端附近的两个miR-122结合位点，即S1和S2。在S1和S2中都具有碱基替换的病毒RNA在转染细胞中无法产生传染性病毒，而在补充了互补miR-122突变体的细胞中，病毒产量恢复到接近野生型水平。对仅在一个位点有替换的突变体进行比较发现，S1起主导作用，因为在补充野生型miR-122的细胞中，S2而非S1突变体产生高病毒产量。S1或S2中的突变使HCV RNA的翻译减少超过50%，并且通过转染互补miR-122突变体可部分恢复。然而，与病毒复制的情况不同，两个位点在调节翻译方面发挥同等作用。我们得出结论，miR-122通过直接结合基因组RNA中的两个位点来促进复制，并且至少部分是通过刺激内部核糖体进入位点（IRES）介导的翻译来实现的。但是，对双结合位点突变体和翻译存在定量等效缺陷的IRES突变体的复制能力进行比较表明，与miR-122结合丧失相关的翻译减少不足以解释双突变体在病毒产生方面的严重缺陷。因此，miR-122可能在病毒生命周期的另一个步骤中发挥作用。
