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The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5′- and 3′-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5′-UTR, and subsequent viral RNA replication requires sequences in the 3′-UTR and in the 5′-UTR. Addressing previous conflicting reports on a possible function of the 3′-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3′-UTR function. A translation enhancer function of the HCV 3′-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3′-UTR with a precise 3′ terminus. The 3′-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U · C) tract, and the most 3′ terminal stem-loop 1 of the highly conserved 3′ X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3′ X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3′-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3′-5′ end interaction and a switch between translation and RNA replication.

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.

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3′ region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB–IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.

MicroRNAs (miRNAs) are involved in the regulation of translation of many mRNAs and affect cell cycle progression, differentiation and metabolism. The liver-specific microRNA-122 (miR-122) contribut...