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

TY - JOUR

T1 - Replicons of a Rodent Hepatitis C Model Virus Permit Selection of Highly Permissive Cells

AU - Wolfisberg, Raphael

AU - Holmbeck, Kenn

AU - Nielsen, Louise

AU - Kapoor, Amit

AU - Rice, Charles M.

AU - Bukh, Jens

AU - Scheel, Troels K.H.

PY - 2019/10/1

Y1 - 2019/10/1

N2 - 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 liverderived cells did not readily support the complete RHV life cycle, but repliconcontaining 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 RHVrn1 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.

AB - 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 liverderived cells did not readily support the complete RHV life cycle, but repliconcontaining 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 RHVrn1 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.

KW - animal models

KW - antiviral agents

KW - hepacivirus

KW - hepatitis C virus

KW - micro-RNA

KW - replication

KW - replicon

UR - http://www.scopus.com/inward/record.url?scp=85072152750&partnerID=8YFLogxK

U2 - 10.1128/jvi.00733-19

DO - 10.1128/jvi.00733-19

M3 - Journal article

C2 - 31292246

AN - SCOPUS:85072152750

SN - 0022-538X

VL - 93

JO - Journal of Virology

JF - Journal of Virology

IS - 19

ER -