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.
