Abstract
The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5′ terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.
Highlights
The liver-specific microRNA-122 recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA
We generated two variants of these constructs: One version followed the regular architecture of the HCV genome, in which translation of the polyprotein is initiated at the HCV internal ribosome entry site (IRES) (Luc-SG, luciferase reporter genes (Luc)-full length (FL)), causing RNA stability, replication, and translation to be dependent on miR-122
Reconstitution of miR-122 by co-transfection of a miR-122 duplex rendered Hep3B cells permissive for HCV replication to similar levels as those measured in Huh7.5 cells[17] (Fig. 1b, d), whereas no replication was observed upon transfection of a miR-122 mutant with a point mutation in the seed region (Fig. 1c, d, miRmut)
Summary
The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. Our results suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures. The 5′ UTR contains a set of secondary structures that compose the internal ribosomal entry site (IRES), allowing a cap-independent translation of the viral genome. DII can adopt an L-shaped tertiary structure, which is essential for IRES function as it mediates the displacement of eukaryotic initiation factor 2 (eIF2) complex to facilitate attachment of the 60S ribosomal subunit. Mir-122 binding further masks the HCV RNA 5′ end, conferring resistance to the degradation by host nucleases Xrn[1] and Xrn[2] and thereby contributing to increased protein production[14,15]
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