miR-122 is more than a shield for the hepatitis C virus genome.

S ince the discovery in 2005 of the essential role of the liver-specific microRNA, miR-122, in HCV replication (1), the mechanism by which it stimulates this process has proved elusive. In PNAS, Li et al. demonstrate thatmiR-122 acts to shield the HCV genome against degradation by the cytosolic RNA exonuclease, Xrn1 (2). Although this may be one way in which this microRNA promotes HCV replication, the authors also show that loss of Xrn1 is not enough to promote miR-122 independent HCV replication, indicating that miR-122 exerts yet another function in the HCV lifecycle. Most microRNAs function to suppress gene expression, either through transcript cleavage and degradation, when complete complementarity is present between the microRNA and target, or suppression of translation, whenmismatches exist between these species (3). This scenario is completely reversed for HCV and miR-122. Here, the binding of miR-122 to two sites at the far 5′ end of the HCV positive-strand RNA genome does not result in RNA degradation or in translational arrest, but instead miR-122 is essential for HCV replication (Fig. 1). MicroRNAs usually bind to specific RNAs by complementarity between the so-called seed sequence of the microRNA and the target RNA sequence. Enhancement of HCV RNA replication requires both miR-122 seed sequence interactions, as well as interactions outside of their seed sequences (4). TheHCV genome does not contain a cap structure at the 5′ end, which functions on cellular RNAs to promote translation, by recruiting ribosomal components, and stability, by shielding the normally single-stranded 5′ end from exonuclease digestion (5). Indeed, it was recently shown that miR-122 increases HCV RNA stability in much the same manner as a cap (6). However, the manner of degradation that miR-122 prevents and the extent to which this increase in stability plays into the overall impact of miR-122 on HCV replication was not determined. These are the questions that Li et al. set out to answer in their PNAS publication (2). Li et al. first tackle the question how HCV RNA is degraded by host cells (2). In eukaryotes, the exosome and Xrn1 represent two potential mRNA decay pathways (5). The exosome degrades transcripts in a 3′ to 5′ direction following deadenylation. Xrn1 is a cytoplasmic 5′ to 3′ exonuclease that degrades transcripts after the cap is removed by a decapping complex. In initial experiments, the authors find that both pathways impact the stability of HCV RNA when transfected into host cells, and HCV RNA was particularly sensitive to exosomal degradation in cell lysates. However, when examined in cells bearing actively replicating HCV RNA, only Xrn1 impacted viral RNA stability. The authors then show that miR-122 acts to protect the HCV RNA from Xrn1 degradation, as addition of extra miR-122 to cells increased HCV RNA stability to the same extent as Xrn1 silencing, and the combination of both did not exhibit an additive effect. Based on this finding, Li et al. then proceed to test the impact of Xrn1 on HCV replication and how this may be modulated by the presence of miR-122 (2). Indeed, silencing of Xrn1, but not components of the exosome, enhanced HCV RNA replication about twofold, indicating that the Xrn1 degradation pathways negatively regulate HCV RNA replication. However, supplementing cells with additional miR-122 enhanced HCV replication even when Xrn1 was silenced. Furthermore, a mutant HCV genome bearing changes to both miR-122 binding sites, which can be efficiently rescued by expression of a mutant miR-122 with complementary changes, was not rendered replication competent by Xrn1 silencing. The authors conclude from these results that miR-122 acts to shield theHCV RNA from Xrn1 degradation and to perform another Xrn1-independent function in enhancing HCV replication (2). Regardless of the mode of action of miR-122 in the HCV lifecycle, dissection of the way HCV RNA decays is an important advance toward understanding how HCV interacts with its host. Li et al. (2) speculate that the distinct role that Xrn1 plays in the turnover of HCV RNA in replicating cells may reflect the specific location where viral RNA replication takes place, i.e., membrane compartments that encapsulate the HCV RNA replication complex. Nucleases may not have access to this compartment, and therefore viral RNA may only be degraded when these compartments break down or when RNA is released for other events such as packaging into virions. Xrn1 is specifically concentrated in P bodies, but the authors show that HCV RNA is not present in these structures, and instead only colocalizes with Xrn1 in the cytoplasm of infected cells (2). Another interesting Fig. 1. miR-122 impacts HCV RNA stability and HCV RNA replication. Illustration of HCV RNA replication within membrane bound vesicles in the host cell cytoplasm. Also shown is a zoomed-in view of the 5′ end of the HCV genomic RNA with two bound miR-122 molecules. Cytoplasmic, but not P body-associated, Xrn1 degrades the HCV genomic RNA, perhaps after the 5′ triphosphate is modified by a pyrophosphatase. Although binding of miR-122 to the HCV RNA inhibits Xrn1 degradation, miR-122 also exerts another effect to enhance HCV RNA replication.