Hepatitis C virus (HCV) is a single-stranded plus-sense RNA virus that is transmitted by blood-to-blood contact, and infects the human liver. HCV has a unique dependence on the liver-specific microRNA miR-122, where miR-122 binds the 5´ un-translated region of the viral RNA at two tandem sites and increases viral RNA abundance. The mechanisms of augmentation are not yet fully understood, but the interaction is known to stabilize the viral RNA, increase translation from the viral internal ribosomal entry site (IRES), and result in increased viral yield. In an attempt to create a small animal model for HCV, we added miR-122 to mouse cell lines previously thought non-permissive to HCV, which rendered these cells permissive to the virus, additionally showing that miR-122 is one of the major determinants of HCV hepatotropism. We found that some wild-type and knockout mouse cell lines – NCoA6 and PKR knockout embryonic fibroblasts – could be rendered permissive to transient HCV sub-genomic, but not full-length, RNA replication upon addition of miR-122, and that other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122. These knockout cell lines demonstrated varying permissiveness phenotypes between passages and isolates and eventually completely lost permissiveness, and we were unable to achieve sub-genomic RNA replication in PKR knockout primary hepatocytes. Knockdown of NCoA6 and PKR in Huh7.5 cells did not substantially impact sub-genomic replication, leading us to conclude that there are additional factors within the cell lines that affect their permissiveness for HCV replication such as epigenetic regulation during passage or transformation and immortalization. We also added miR-122 to Hep3B cells, a human hepatoma cell line lacking expression of miR-122 and previously thought to be non-permissive to HCV replication. Added miR-122 rendered the cells as highly permissive to HCV replication as the Huh7-derived cell lines commonly used to study the virus. In these cells, we were also able to observe miR-122-independent replication of sub-genomic HCV RNA. This was verified by use of a miR-122 antagonist that had no impact on the putative miR-122-independent replication, and by mutating the miR-122 binding sites to make them dependent on a single nucleotide-substituted microRNA. This replication in the absence of miR-122 was not detected in full-length HCV RNA, but was detectable using a bi-cistronic full-length genomic replicon, suggesting that the addition of a second IRES in sub-genomic and full-genomic replicons altered replication dynamics enough to allow detectable RNA replication without miR-122 binding. Because miR-122 has been implicated in protecting the viral RNA from destabilization and degradation by Xrn1, the main cytoplasmic 5´ to 3´ RNA exonuclease, we employed our miR-122-independent system to test this miR-122-mediated protection. We verified that miR-122 functions to protect the viral RNA from Xrn1, but this was insufficient to account for the overall impact of miR-122 on replication, meaning that miR-122 has further functions in the virus’ life cycle. We showed that the effect of miR-122 on translation is due to stabilization of the RNA by protecting it from Xrn1, through binding at both sites. We further evaluated the role of each miR-122 binding site (S1 and S2) in the virus life cycle, and found that binding at each site contributes equally to increasing viral RNA replication, while binding at both sites exerts a co-operative effect. Finally, we determined that binding of miR-122 at site S2 is more important for protection from Xrn1, suggesting that miR-122 binding at S1 is more important for the additional functions of miR-122 in enhancing HCV RNA accumulation. Altogether, we have shown that miR-122 is partially responsible for the hepatotropic nature of Hepatitis C virus, and that supplementation with this microRNA can render non-permissive cells permissive to viral replication. We have also identified and confirmed replication of both sub-genomic and full-length HCV RNA in the absence of miR-122. Finally, we have characterized the impact of the host RNA exonuclease Xrn1 on the HCV life cycle, and determined the roles of each miR-122 binding site in shielding the viral RNA from this host restriction factor.