Combined HCV Cofactor Directed Sirnas Significantly Enhance the Inhibitory Effect of Single Sirnas on Hepatitis C Virus Subgenomic Replication and Translation

Background/Aims: Persistent infection with hepatitis C virus (HCV) leads to the development of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. SiRNAs are currently being evaluated as promising therapeutic agents for HCV infection. Unfortunately, the antiviral efficacy of HCV directed siRNAs is limited by emergence of escape variants that are resistant to siRNA because of the high mutation rate of HCV. Alternatively, cellular RNA binding proteins that play an important role in the replication cycle of hepatitis C virus could be more attractive targets for siRNAs to sustain antiviral effects. We have previously shown that PTB-directed siRNAs inhibited subgenomic HCV IRES-mediated translation by depletion of PTB (Polypyrimidine tract binding protein), while PSMA7 (proteasome alpha-subunit 7) and HuR (Human antigen R) directed siRNAs decreased both, HCV replication and translation. In this study we investigated whether the inhibitory effect on subgenomic HCV replication and translation by HCV cofactor directed siRNAs could be significantly increased by synergistic or additive effects of siRNA combinations.

Methods: Combinations of siRNA plasmids targeting PSMA7, PTB and HuR were transfected into Huh7 cells expressing a monocistronic subgenomic HCV replicon. HCV RNA and protein level of siRNA combinations were compared with levels of single transfected siRNAs targeting PTB, PSMA7 and HuR.

Results: Northern blot analysis revealed a reduction of up to 30% in HCV replicon RNA by siRNAs targeting PSMA7 and HuR. Combination of PSMA7 and HuR directed siRNAs enhanced the inhibitory effect and reduced HCV RNA level up to 60%. Level of HCV NS5B protein expression showed a reduction of up to 40% by each siRNA targeting PTB, PSMA7 and HuR. An additive inhibitory effect was observed by combination of HCV cofactor directed siRNAs. In Western blot analysis, a reduction of HCV NS5B protein expression up to 75% was detected by combined siRNAs.

Conclusions: Our results demonstrate that combinations of siRNAs targeting essential cellular cofactors of subgenomic HCV replication and translation provide additive inhibitory effects on HCV replication cycle, suggesting the potential for a successful dual antiviral strategy against HCV.

Keywords: HCV cofactors, RNA interference, hepatitis C virus