Construction of HCV minigenome containning IFN-β and investigation of its inhibitory effect on viral replication

Objective To construct minigenome containning IFN-β responsive to HCV and to study its inhibitory effect on viral replication. Methods Total RNA was extracted from lymphocytes stimulated by poly I:C and was reversely transcripted into cDNA. IFN-β gene was amplif ied by PCR and was cloned into pT7-5U△131-315rI3Urz in antisense direction to obtain pT7-5U△131-315rIFNI3Urz. The minigenome 5U△131-315rIFNI3URz was amplified and inserted into the plasmid pcDNA3.1 by positioning the minigenome immediately adjacent to the transcription start site of the cytomegalovirus (CMV) promoter, resulting in pCMV-5U△131-315rIFNI3URz. The pT7-5U△131-315rIFNI3Urz was transcripted into RNA in vitro, then transfected into Huh7.5BB7. After 48h, cell was harvested and total proteins were extracted for Western Blot, IFN-β antibody as the fi rst antibody. When the pCMV-5UrIFNI3URz was transfected into Huh7.5JFH-1 cell the real-time PCR assay was used to detect HCV RNA replication level. Results The Huh7.5-JFH-1 cell line which was developed by transfecting infectious RNA into Huh7.5 cell which can replicate autonomously and virus particle can be packed successfully. IFN-β could be specifi cally expressed after 5UrIFNI3Urz RNA and pCMV-5UrIFNI3URz was trasnfected into Huh7.5BB7 cell lines. The level of HCV RNA replication was distinctly decreased when Huh7.5-JFH-1 cell line was transfected with pCMV-5UrIFNI3URz. Conclusion The minigenome containning inverted IFN-β could specifi cally be expressed in HCV infected cell lines, the viral replication level was decreased to a certain extent. This study provides the new idea of anti-HCV infection.