Abstract
Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.
Highlights
Afflicting around 170 million people worldwide, Hepatitis C virus (HCV) infection and associated liver diseases are significant health concerns in developing world and even in developed countries [1]
We demonstrated a significant decrease in mRNA and protein expression of HCV 1a genotype nonstructural proteins (NS2, NS4A & NS5A) at mRNA and protein levels in stable cell lines
Stable Cell Line Generation Expressing NS2, NS4A and NS5A Proteins in Huh-7 Cells Stable Huh-7 cell clones persistently expressing (NS2, NS4A & NS5A proteins up to 30th days post-transfection was produced under the selection pressure of antibiotic Gentamycin (G418)
Summary
Afflicting around 170 million people worldwide, Hepatitis C virus (HCV) infection and associated liver diseases (hepatic cirrhosis, fibrosis and hepatocellular carcinoma) are significant health concerns in developing world and even in developed countries [1]. HCV is a small, enveloped, positive sense single-stranded RNA (ssRNA) virus of approximately 9.6 kb long [3]. The viral single polyprotein is proteolytically cleaved by host and viral encoded proteases into four mature structural and six nonstructural proteins [5]. The structural proteins are actively involved in virion formation while the nonstructural proteins are essential for viral replication, translation and polyprotein processing [6]. The proteins that significantly contribute to viral replication and translation (i.e., NS5B and NS3) are the potential active target sites for the development of novel direct-acting antivirals and genome-based therapeutics [7]. Subgenomic replicons, HCV pseudo-particles, infection based cell culture systems and small animal models are currently available to study HCV replication and for the development of anti-HCV treatment strategies [10] [11]
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