Fig 2: Proposed mechanism of HCV genome replication. Competition between miR-122 and PCBP2 for binding to the 5′UTR determines whether the genome is engaged in translation or RNA synthesis. At the initial stage of HCV infection, PCBP2 binding to the 5′UTR promotes a closed-loop form of the viral genome by bridging the 5′ and 3′ UTRs that favors translation (left). By contrast, miR-122 binding to the 5′UTR both disrupts the closed-loop, circular form of the genome, and enhances affinity of the 5′ end of the RNA for the RNA-dependent NS5B RNA polymerase, thereby promoting viral RNA synthesis (right). The interaction of miR-122 with the HCV 5′UTR additionally protects the genome from 5′ dependent cellular exonucleases and stabilizes RNA structure optimal for IRES activity. Continued competition between miR-122 and PCBP2 with similar binding affinities for S2 allows for the dynamic regulation of viral protein versus viral RNA synthesis required for persistent infection with a nonlytic virus.

Fig 3: Location of miR-122 and PCBP2 binding sites at the 5′ end of the HCV genome. (A) Schematic of the HCV genome. The HCV genome consists of a single open reading frame (ORF) flanked by highly structured, untranslated regions at the 5′ and 3′ ends (5′ and 3′ UTRs). Stem-loop structures at the 5′ and 3′ UTRs are indicated. Structural proteins and non-structural proteins are colored by blue and orange, respectively. (B) The miR-122 and PCBP2 binding sites in the 5′ UTR of the HCV genome. Nucleotides 1–45 of the HCV genome contain two tandem miR-122 binding sites (blue) and a PCBP2 binding site (cyan) that overlaps with the second miR-122 binding site. (C) Two miR-122 molecules bind to the 45 nucleotides of HCV RNA. HCV45 (1 μM) was incubated with the increasing amounts of miR-122, 0–3 μM in steps of 0.5 μM in presence of 5 mM Mg2+, and analysed by non-denaturing gel electrophoresis mobility shift assay. The number of miR-122 in the HCV45 complex is indicated.

Fig 4: PCBP2 binds directly to 5′ terminal HCV RNA sequence in competition with miR-122. (A) Gel shift assay with HCV47 RNA and PCBP2. IRdye-800 labelled HCV47 RNA (10 nM) was incubated with recombinant PCBP2 protein or control BSA. RNA-protein complexes were resolved on a 5% native polyacrylamide gel and visualized with an Odyssey Imaging System. RNA monomers (*) and dimers (**) are evident as two discreet bands, along with more slowly migrating PCBP2-RNA complexes (arrow). (B) Gel shift assay with HCV47 and PCBP2 in the presence of miR-122. The IRdye-800 labelled HCV47 RNA was incubated with 50 nM of single-stranded miR-122 or miR-124 prior to addition of PCBP2. (C) MiR-122 prevents HCV and PCBP2 interaction. The gel shift assay shown in (B) was repeated using 250 nM PCBP2 and increasing quantities of single-stranded miR-122 or miR-124, and percent of RNA bound to PCBP2 was plotted. The EMSA image is shown in Supplementary Figure S2. (D) Coomassie stained SDS-PAGE gel with the purified recombinant PCBP2. (E) PCBP2 binding to HCV45 and the HCV45:miR-122 complex. PCBP2 was titrated into either fluorescein-labeled HCV45-F (30 nM), labeled miR-122-F (30 nM), or the HCV45-F (30 nM) and unlabeled miR-122 (90 nM) complex. Dissociation constants determined from the titration curves are listed on the right. (F) PCBP2 and Ago2 pull down assays with biotinylated RNA probes. Design of 3′-biotinylated HCV47 (HCV47-Bio) and related S2p6m-Bio, S2p3,4m-Bio, and Ins43AA-Bio RNA probes are shown (left). Potential PCBP2 binding site that overlaps with S2 is shown in green, and altered bases in the S2 seed sequence are shown in red. PCBP2 pull down assay was carried out with the biotinylated RNA probes and Huh-7.5 cell lysate. Co-precipitated PCBP2 or Ago2 protein was detected by immunoblot. Asterisk (*) indicates a non-specific Ago band. (G) Replication reporter assay for S2 mutant viruses. Luciferase activities were measured in supernatant fluids of cells transfected with the HJ3-5/GLuc2A or mutant reporter viral RNAs, with or without supplementation with complementary mutant miR-122s (p6 and p34). (H) Replication reporter virus assay for Ins43AA. Luciferase activities in supernatant fluids of cells transfected with the HJ3-5/GLuc2A and Ins43AA mutant reporter, with or without RNAi-mediated depletion of PCBP2. Immunoblots of PCBP2 and GAPDH (loading control) are shown on top. (I) Translation reporter assay for Ins43AA. The schematic of the HCV-ΔC/GLuc mini-genome translation reporter is shown on top. Luciferase activities were measured in PCBP2-depleted or control Huh-7.5 cells transfected with the HCV-ΔC/GLuc translation reporter or Ins43AA mutant RNA. Results shown represent the means from 3 independent experiments, each with 2–3 technical replicates, ±SD. P-value by two-sided t-test.

Fig 5: miR-122 regulates PCBP2-mediated circularization of positive-strand HCV RNA. (A) Schematic showing experimental design of the 5′-3′ interaction assay with 3′-biotinylated HCV 5′ end sequence RNA (nts 1–47) and 5′ [32P]-labelled HCV 3′UTR RNA (nts 9378–9646) probes. (B) The 5′-3′ interaction of the HCV genome. Biotin-tagged 5′ end RNA (HCV47-Bio) was incubated with [32P]-labelled 3′ end RNA in the presence and absence of PCBP2, and pulled down with streptavidin beads. Pulled down [32P]-labelled 3′UTR was visualized by phosphoimager. BSA was used as unrelated control. The percent input 3′UTR RNA bound to the 5′RNA is shown below each lane. (C) Inhibition of 5′-3′ interaction by miR-122. Addition of ss-miR-122 mimic inhibits the PCBP2-mediated 5′-3′ interaction, whereas miR-124 does not. (D) Images of double-stranded mini-genome RNA in the presence of PCBP2. Design of HCV mini-genome duplex is shown on top. Electron micrographs showing linear and circular forms of the mini-genome duplex observed with TEM when incubated with or without PCBP2. (E) Inhibition of PCBP2-mediated circularization by miR-122. Mini-genome duplex RNAs were annealed with 100 ng of miR-122 or miR-124 before the addition of 100 or 50 ng of PCBP2 protein (approximate miRNA:protein molar ratios of 5:1 or 10:1), then visualized by TEM. 100 molecules in each preparation were manually classified as linear, circular or circular + linear. miR-122 versus miR-124 P-values by X2 test.