Replicons of a Rodent Hepatitis C Model Virus Permit Selection of Highly Permissive Cells

Abstract

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems.IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.

Keywords: animal models; antiviral agents; hepacivirus; hepatitis C virus; micro-RNA; replication; replicon.

FIG 1

Rodent liver-derived cell lines do not readily support productive RHV infection. (A to D) Quantification of liver-specific markers in hepatic cell lines. Secreted rat (A), mouse (B), and human (C) albumin was measured in cell culture supernatant. (D) Quantification of miR-122 in relevant cells. HLCs, hepatocyte-like cells derived from human induced pluripotent stem cells; MEF, mouse embryonic fibroblasts (negative control). Data points represent means ± standard deviations (SD) from duplicates. (E) Intracellular viral RNA after transfection of McA-RH7777 cells with full-length genomic RHV-rn1 RNA or the replication-deficient GNN control. Data points represent means ± SD from duplicates. Schematic of the full-length RHV genome depicting predicted protein coding regions and UTR structures is shown above (ORF drawn to scale; genes encoding structural [red] and nonstructural [blue] proteins are indicated). (F) Fluorescence microscopy after transfection of McA-RH7777 cells with full-length genomic RHV-rn1-eGFP RNA. A nonrepresentative picture taken 5 days posttransfection at ×40 magnification shows expression of eGFP in a few clusters of cells. Viral spread could not be detected. A genome schematic of the RHV-rn1-eGFP reporter virus is shown above.

FIG 2

Selection for RHV replication in rodent hepatocytes. (A) Schematic representation of the bicistronic selectable subgenomic RHV replicon construct without (I) or with FLuc (FEO) (II) or eGFP (III) fused to the N terminus of the NPTII selection cassette. The first 12 amino acids of the core region (red) were fused in-frame with NPTII or reporters to ensure optimal translation of the first cistron (26). (IV) Monocistronic FEO replicon having both the reporter/selection cassette and the RHV replicase genes under the control of the RHV 5′ UTR. A short sequence encoding the ribosome-skipping 2A peptide from porcine teschovirus-1 ensures separation of NPTII and NS3. (B) Cell colonies stained using crystal violet following electroporation with RHV-SGR or RHV-SGR-GNN RNA. Representative images of G418-resistant McA-RH7777 rat hepatoma colonies after 2 weeks of selection are shown. (C) Number of selected colonies after electroporation of the RHV-SGR replicon into rat McA-RH7777 or mouse Hepa1-6 or AML12 cells. The number of rat and mouse cell colonies harboring RHV-SGR substitutions identified by sequence analysis is shown. (D) Validation of RHV NS3-4A protease activity in selected replicon-harboring Hepa1-6 cells using the fluorescent cell-based reporter system, leading to nuclear translocation of RFP upon NS3-4A cleavage of a MAVS target sequence (32). Diagram of native MAVS and the reporter is shown above with amino acid numbering. TMD, transmembrane domain; NLS, nuclear localization signal. (E) Comparison of the number of colonies after selection between the original replicon (wt) and those with single and combined mutations. Data from McA-RH7777 and Hepa1-6 cells are shown as fold change compared to levels for the wt in the respective cell line. Data points represent means ± SD from duplicates.

FIG 3

Luciferase reporter replicon systems enable direct quantification of RHV replication. (A) Absolute FLuc activity per well after transfection of parental McA-RH7777 cells with bicistronic FEO replicons with and without dinucleotide optimization. (B) FLuc activity after transfection of McA-RH7777 cells as described for panel A but with comparison of single and combined substitutions. (C) Comparison of FLuc activity per well after transfection of McA-RH7777 cells with mono- or bicistronic versions of the FEO replicon. (D) Replication efficiency of expanded rat McA-RH7777 (red) and mouse Hepa1-6 (blue) FEO cell clones. FLuc activity per cell was measured 4 weeks after electroporation with RHV-adFEO-10 RNA and subsequent G418 selection. (E) Absolute FLuc activity per well in parental rat McA-RH7777 (red) and mouse Hepa1-6 (blue) cells, measured 4 h after electroporation with a replication-deficient FEO-GNN replicon. Different passage numbers are shown, and the background luminescence level is indicated by the dashed line. All data points in panels A to E represent means ± SD from triplicates.

FIG 4

Selection of rat McA-RH7777 hepatoma cells with increased permissiveness to RHV replication. (A) Cells containing the eGFP replicon were expanded in selective media for 2 weeks and analyzed by flow cytometry. Mock, parental McA-RH7777 hepatoma cells. (B) Replication permissiveness of cured and parental McA-RH7777 cells as measured by FLuc activity per well 4 h and 5 days after electroporation with adFEO-10 or GNN as a control. Data points represent means ± SD from triplicates. For the parental and GFP-sorted cell populations, different passage numbers are shown. The background luminescence level is indicated by the dashed line. (C) Cell colonies stained using crystal violet following electroporation with RHV-SGR. Representative images of G418-resistant parental or clone 2E1 McA-RH7777 rat hepatoma colonies after 2 weeks of selection in gelatin-coated dishes are shown. (D) Quantification of miR-122 in highly permissive rat cells relative to the parental McA-RH7777 cell line. Quantification of the small noncoding RNA RNU6B was used as an endogenous reference for normalization. Data points represent means ± SD from duplicates.

FIG 5

HCV antivirals inhibit RHV replication. Treatment of RHV replicon-containing McA-RH7777 (red) or Hepa1-6 (blue) cells with escalating doses of HCV antivirals targeting the NS3-4A protease (A), NS5A (B), or the NS5B RdRp (C). Replication levels after treatment are shown as percentages of FLuc activity compared to levels for the untreated controls. Data points derived from drug treatment above cytotoxic concentrations were excluded, and the highest nontoxic concentration was tested for all drugs. (D) Escalating concentrations of the miR-122 antagonist miravirsen or a scrambled LNA (scLNA) were transfected into RHV or HCV replicon-containing McA-RH7777 and Huh-7.5 cells, respectively. All data are from representative experiments, and data points represent means ± SD from triplicates.