Detection of Anti-Hepatitis C Virus Effects of Interferon and Ribavirin by a Sensitive Replicon System

Abstract

Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dosedependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 g/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 g/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46-to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. HCV causes chronic liver diseases, including cirrhosis and hepatocellular carcinoma, because most patients fail to clear the virus and the persistent infection that follows (1,11,20). Current therapy for HCV-related chronic hepatitis is based on the use of interferon (IFN). However, virus clearance rates are limited to approximately 10 to 20% of cases treated with IFN only (9,23,26). Combination therapy with IFN and ribavirin improves the HCV clearance rate, although the molecular mechanism responsible for this improvement is not yet fully understood (23,25,26). However, some direct antiviral mechanisms of ribavirin have been proposed (19). One possible mechanism is the direct inhibition of HCV RNA-dependent RNA polymerase, and another possibility is the RNA mutagen effect that drives a rapidly mutating RNA virus over the threshold to "error catastrophe." The detection of these direct anti-HCV effects has been hampered by the lack of an appropriate sensitive system for evaluating HCV replication.Although HCV belongs to the Flaviviridae family and has a genome structure similar to those of the other flaviviruses (3, 27), efficient cell culture systems a...

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

“…pcDNA3-myc-HuR was obtained by cloning the HuR coding sequence in pcDNA3-myc vector. The HCV monocistronic replicon construct pFKi383hygubiNS3-3=5.1 (31), the bicistronic replicon constructs SGR-JFH1/Luc (where Luc is luciferase) and SGR-JFH1/Luc-GND (32), and the full-length HCV-JFH1 construct (33) (generous gifts from Ralf Bartenschlager and Takaji Wakita) were used in the present study.…”

“…Huh7.5 cells were transfected with HCV-JFH1 RNA, followed by (Adapted from reference 39 with permission.) (B) 32 P-labeled HCV 3= UTR riboprobe was used in UV cross-linking reactions with either recombinant HuR, PTB, or La proteins. Unlabeled RNA corresponding to the variable region, poly(U/UC) region, 3= X region, and the full-length 3= UTR were used in 50-and 100-fold excess concentrations (corresponding to the first and second lanes, respectively, of each pair of lanes) to identify the region which effectively competes with the radiolabeled RNA to bind to each of these proteins.…”

Isolation and Characterization of an Huh.7.5.1-Derived Cell Clone Highly Permissive to Hepatitis C Virus

“…To identify steps in the HCV life cycle that were enhanced in Huh7.5.1-8 cells, transient-replication assays were performed using a subgenomic replicon carrying the luciferase reporter gene SGR-JFH1/Luc-wt (21). As shown in Fig.…”

Mechanisms of action of interferon and ribavirin in chronic hepatitis C: Summary of a workshop

“…61 When infectivity was used as a functional readout for mutagenesis, diminished infectivity was found with ribavirin treatment of HCV cultures.58 , 60 The concentrations of ribavirin needed to achieve mutagenic effects were high (up to 400 uM) and may exceed those achieved in vivo. These concentration differences may help to explain the discordance observed between cell culture and human studies 54,55,[62][63][64][65][66][67] (Table 3).…”