MicroRNAs are small RNA molecules that regulate messenger RNA (mRNA) expression. MicroRNA 122 (miR-122) is specifically expressed and highly abundant in the human liver. We show that the sequestration of miR-122 in liver cells results in marked loss of autonomously replicating hepatitis C viral RNAs. A genetic interaction between miR-122 and the 5′ noncoding region of the viral genome was revealed by mutational analyses of the predicted microRNA binding site and ectopic expression of miR-122 molecules containing compensatory mutations. Studies with replication-defective RNAs suggested that miR-122 did not detectably affect mRNA translation or RNA stability. Therefore, miR-122 is likely to facilitate replication of the viral RNA, suggesting that miR-122 may present a target for antiviral intervention.

Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

Infections with hepatitis C virus (HCV) are marked by frequent viral persistence, chronic liver disease, and extraordinary viral genetic diversity. Although much has been learned about HCV since its discovery, progress has been slowed by a lack of permissive cell culture systems supporting its replication. Productive infections have been achieved recently with genotype 2a virus, but cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. We describe production of infectious genotype 1a HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most secreted RNA was noninfectious, banding in isopycnic gradients at a density of 1.04–1.07 gm∕cm³, but infectivity was associated with H77-S particles possessing a density of 1.13–1.14 gm∕cm³. The specific infectivity of H77-S particles (5.4 × 10⁴ RNA copies per focus-forming unit) was significantly lower than JFH-1 virus (1.4 × 10² RNA copies per focus-forming unit). Infection with either virus was blocked by CD81 antibody. Sera from genotype 1a-infected individuals neutralized H77-S virus, but had little activity against genotype 2a virus, suggesting that these genotypes represent different serotypes. The ability of this genotype 1a virus to infect cultured cells will substantially benefit antiviral and vaccine discovery programs.

Mitochondrial antiviral signaling protein (MAVS) is an essential component of virus-activated signaling pathways that induce protective IFN responses. Its localization to the outer mitochondrial membrane suggests an important yet unexplained role for mitochondria in innate immunity. Here, we show that hepatitis A virus (HAV), a hepatotropic picornavirus, ablates type 1 IFN responses by targeting the 3ABC precursor of its 3C ^pro cysteine protease to mitochondria where it colocalizes with and cleaves MAVS, thereby disrupting activation of IRF3 through the MDA5 pathway. The 3ABC cleavage of MAVS requires both the protease activity of 3C ^pro and a transmembrane domain in 3A that directs 3ABC to mitochondria. Lacking this domain, mature 3C ^pro protease is incapable of MAVS proteolysis. HAV thus disrupts host signaling by a mechanism that parallels that of the serine NS3/4A protease of hepatitis C virus, but differs in its use of a stable, catalytically active polyprotein processing intermediate. The unique requirement for mitochondrial localization of 3ABC underscores the importance of mitochondria to host control of virus infections within the liver.

MiR-122 is a liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious virus is dependent upon two miR-122-binding sites near the 5′ end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious virus in transfected cells, while virus production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus life cycle.

There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction (“H-NS2/NS3-J”) and at a site of natural, intergenotypic recombination within NS2 [“H-(NS2)-J”] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.