Abstract
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.
Highlights
Hepatitis C Virus (HCV) is the sole member of the genus Hepacivirus in the positive strand RNA virus family Flaviviridae
In contrast to most cellular mRNAs, the initiation of translation of the HCV RNA is directed by an internal ribosome entry site (IRES) element that is located in the viral RNAs 59-untranslated region (59-UTR)
Consistent with the hypothesis that miRNP complexes containing an Ago protein are involved in unwinding the duplex miRNA precursor and using the mature microRNA guide strand as a probe, we show that Ago2 protein is directly involved in the complex with the miR-122 that binds the HCV 59-UTR, whereas such a complex does not form with the consensus sequence in the 39-UTR
Summary
Hepatitis C Virus (HCV) is the sole member of the genus Hepacivirus in the positive strand RNA virus family Flaviviridae. In contrast to most cellular mRNAs, the initiation of translation of the HCV RNA is directed by an internal ribosome entry site (IRES) element that is located in the viral RNAs 59-untranslated region (59-UTR). This IRES recruits the ribosomes to the internal translation start site on the viral RNA [2,3]. Subsequent initiation steps require the binding of eIF3 to the apical regions of stem-loop III [8], while HCV translation initiation is independent of eIF4 group factors [9]. While the expression of cellular surface receptors involved in HCV binding and entry is not strictly limited to hepatocytes [10], a contribution to tissue selectivity can be attributed to the stimulation of HCV translation and genome accumulation by microRNA-122 (miR-122) [11,12,13,14] since this microRNA is expressed preferentially in liver cells or in the HuH-7 hepatoma cell line [15,16,17,18]
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