Elimination of Hepatitis C Virus from Hepatocytes by a Selective Activation of Therapeutic Molecules

Xiaoyu Wen,Nobuyuki Kato,Yoshio Mori,Yoshiharu Matsuura,Hiroshi Kukihara,Hideki Tani,Takayuki Abe,Shuhei Taguwa,Kohji Moriishi,Masashi Tatsumi,Tetsuro Suzuki,Paul Digard

doi:10.1371/journal.pone.0015967

Abstract

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.

Highlights

Hepatitis C virus (HCV) is a major cause of chronic liver diseases
HCV replicon cells and Huh7OK1 cells infected with HCV propagating in cell culture (HCVcc) were transfected with the plasmids encoding either wildtype or dominant active mutant of IRF3 or IRF7 together with the reporter plasmids encoding a luciferase gene under the control of the promoters of IFNa6, IFNb and ISRE, respectively
We observed significant activation of the promoters of IFNa6 and ISRE in the replicon and HCVccinfected cells compared with naıve and mock-infected cells upon expression of IRF7m, while we observed no activation of the IFNa6 promoter in cells expressing IRF3m

Summary

Introduction

Hepatitis C virus (HCV) is a major cause of chronic liver diseases. A high risk of chronicity is the major concern of HCV infection, since chronic HCV infection often leads to liver cirrhosis and hepatocellular carcinoma [1,2]. HCV belongs to the family Flaviviridae and possesses a single positive-stranded RNA genome that encodes a single polyprotein composed of about 3,000 amino acids. The HCV polyprotein is processed into 10 viral proteins by host and viral proteases. Laboratory strains of HCV propagating in cell culture (HCVcc) have been established based on the full-length genome of the genotype 2a JFH1 strain [4], establishment of a robust cell culture system capable of propagating serum-derived HCV from hepatitis C patients has not yet been achieved

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Results

Conclusion