Establishment of stable cell lines expressing Proteins of HCV Genotype 3a Pakistani isolate and their Molecular Characterization

Abstract

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which progresses to hepatocellular carcinoma (HCC) afflicting > 170 million people worldwide. HCV 3a is the most common genotype (about 70% of all genotypes) circulating in Pakistan. Expression of HCV individual gene of 3a would facilitate therapeutic and vaccines strategies against chronic HCV and liver Cirrhosis. The aim of the present study was the establishment of stable Huh-7 cell lines expressing structural and non structural proteins of HCV Genotype 3a Pakistani isolate obtained from chronic HCV patients. Blood samples were obtained from chronic HCV-3a positive patients. HCV individual genes were amplified using PCR with gene specific primers having restriction sites and kozak sequence. These gene amplicons were cloned in TA and mammalian expression vector PcDNA3.1+. Restriction digestion analysis and sequencing results confirmed the successful cloning of HCV genes in PcDNA3.1 vector. Huh-7 cell lines were transfected with these constructed plasmids having structural or non-structural HCV genes in confluent cells with lipofectamine. Positive clones were selected with G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated genes were demonstrated by RT-PCR, sequencing and Western blot analysis. We successfully cloned and expressed seven HCV-3a genes in PcDNA3.1 mammalian expression vector. Results of western blot and sequencing PCR confirmed the stable expression of these seven genes. Various cytokines like Tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF- β), leptin and Angiotensin were used as indicator of disease progression in huh-7 cells stably expressing HCV structural and nonstructural proteins in vitro. Collectively, these observations suggest a significant involvement of HCV huh-7/Core, huh-7/E2 and huh-7/NS5A in the up-regulation of gene expression proinflammatory cytokine TNF-α and profibrotic TGF-β. No significant change in the expression of Angiotensin-II in vitro was observed. Up-regulation of leptin was observed in huh-7/E2 cell line only. The stable cell-lines expressing HCV-3a individual genes would be a useful tool to investigate the role of various HCV proteins on HCV disease outcome and testing of new therapeutic strategies against HCV.