Replicons of a rodent hepatitis C model virus permit selection of highly permissive cells

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Raphael Wolfisberg, Kenn Holmbeck, Louise Nielsen, Amit Kapoor, Charles M Rice, Jens Bukh, Troels K H Scheel

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 viral 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 bi-cistronic 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 importance of this HCV host-factor also 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 inducing 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, 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 viral 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.

Original languageEnglish
JournalJournal of Virology
ISSN0022-538X
DOIs
Publication statusE-pub ahead of print - 10 Jul 2019

ID: 225426627