Correction: microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation.

[This corrects the article DOI: 10.1371/journal.pone.0056272.].

The role of microRNAs in hepatitis C virus RNA replication.

Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.

MicroRNA-122 dependent binding of Ago2 protein to hepatitis C virus RNA is associated with enhanced RNA stability and translation stimulation.

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.

Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases.

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the Internal Ribosome Entry Site (IRES) in the 5'-untranslated region (5'-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5'-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G0 and G1 phases of the cell cycle but significantly drops during the S phase and even more in the G2/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during the G0, G1 and G2/M phases but is lower during the S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in the S phase, indicating that the predominant G0/G1 state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet unknown factors involved in the differential extent of stimulation by miR-122.