Structural Insights into the Mechanism of microRNA Modulated Viral Translation.

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that control a vast array of cellular processes by repressing mRNA translation. Liver expressed miR-122 is a miRNA that has been co-opted by hepatitis C virus (HCV) to enhance viral translation. Recently, miR-122 antagomir therapy in non-human primates has been shown to suppress HCV viremia; this proof-of-concept study demonstrates the considerable potential of this novel antiviral strategy. The mechanism by which miR-122 modulates HCV translation, however, is unclear. To examine the structural changes that miR-122 exerts on the HCV internal ribosomal entry site (IRES), we developed an advanced Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) method of analyzing RNA architecture. SHAPE determines RNA secondary structure at single-nucleotide resolution, with an accuracy far superior to other mapping methods. Using the above strategy, we show that binding of miR-122 to one of its target sites within the 5' UTR of HCV induces a conformational shift in the HCV IRES at the distant AUG translation start site. Surprisingly, binding of miR-122 to its second target site in HCV is mediated by a number of non-canonical base-pairings. Mutation of the 3' half of miR-122 (tail) disrupted these non-canonical interactions and its ability to induce a conformational shift at the AUG start site. We also observed that, in vitro, the first miR-122 target site in HCV is part of a putative triple-strand RNA motif. These results provide the first demonstration that the tail of this liver-encoded miRNA can directly alter the RNA conformation of the HCV IRES, and thereby provide new insights into the mechanism by which miR-122 influences viral translation. We also show that the 5' UTR of HCV contains a triplex structure important for viral translation.