Abstract
BackgroundHepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed.ResultsAlthough previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN) against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4.ConclusionWe have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326–348) and S-ODN-2 (nt 264–282)], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.
Highlights
Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy
Several antisense oligonucleotide (ASO) that have been designed to bind to the stem-loop structures in the hepatitis C virus (HCV) Internal Ribosome Entry Sites (IRES) have been effective in inhibiting HCV replication in cell-culture assays and the expression of HCV luciferase reporter gene in the livers of mice infected with recombinant vaccinia virus expressing the reporter construct [7]
Studies on HCV using ASOs have utilized antisense phosphorothioate oligonucleotides (S-ODN) that were designed as complementary to sequences present in the 5' non coding region (5'-NCR) of IRES of the viral genome. These studies were carried out using inhibition of gene expression in HCV-luciferase reporter constructs as a readout, or using inhibition of viral replication using subgenomic HCV containing 5'-NCR, core, and part of the envelope proteins components driven by HCMV immediate early promoter [8]
Summary
Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. Several ASOs that have been designed to bind to the stem-loop structures in the HCV Internal Ribosome Entry Sites (IRES) have been effective in inhibiting HCV replication in cell-culture assays and the expression of HCV luciferase reporter gene in the livers of mice infected with recombinant vaccinia virus expressing the reporter construct [7]. Studies on HCV using ASOs have utilized antisense phosphorothioate oligonucleotides (S-ODN) that were designed as complementary to sequences present in the 5' non coding region (5'-NCR) of IRES of the viral genome These studies were carried out using inhibition of gene expression in HCV-luciferase reporter constructs as a readout, or using inhibition of viral replication using subgenomic HCV containing 5'-NCR, core, and part of the envelope proteins components driven by HCMV immediate early promoter [8]. The viral replication but not the biologically relevant infectious viral particles can be demonstrated by such an approach
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