Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region

Abstract

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.RNA interference (RNAi) is a biological process in which double-stranded RNA within the cell induces specific degradation of mRNA with homologous sequences. Protein components of the RNA interference machinery first cleave the long double-stranded RNA into 19-to 21-base pair short interfering RNA (siRNA) and then use the short RNA molecules as guides to target homologous RNA species (reviewed in references 7 and 30). The introduction or expression of siRNA in mammalian cells to target disease-causing genes or virus-specific sequences for degradation represents a potential new therapeutic strategy. Previous reports have shown that RNAi induction has promising antiviral activity against positive-and negative-stranded RNA viruses and DNA viruses in model systems (12, 23). A major concern regarding the use of RNA interference activity against virus infections, particularly RNA viruses, is the development of resistance. Many RNA viruses encode polymerase enzymes that lack proofreading abilities and as a result have high rates of mutation. Thus, there is a high probability that viruses with resistance to RNA interference induced by a particular siRNA will evolve during virus replication through the incorporation of nucleotide mutations within the target sequence of the siRNA. Previous reports have described the selection of human immunodeficiency virus and poliovirus escape mutants in response to prolonged RNA interference activity (2, 4).We have analyzed the potential of hepatitis C virus (HCV) to escape siRNA treatments. HCV infections can lead to the development of liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment ava...

Effective Suppression of Human Immunodeficiency Virus Type 1 through a Combination of Short- or Long-Hairpin RNAs Targeting Essential Sequences for Retroviral Integration

“…Recently, it was shown that accumulation of several point mutations is required for siRNA resistance in an HCV replicon system (40). Several studies have suggested that shRNA-resistant virus can emerge not only by escaping the siRNA-mediated degradation of mRNA but also by micro RNA-mediated translational inhibitory pathways (8,19,24,37,41).…”

Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates

“…Huh7-Lunet or Huh7 cells were coelectroporated with 10 g yeast RNA, 1 g of the corresponding replicon, and siRNA. For HCVrepLuc, 1 M of the corresponding siRNAs was used whereas for HCVrep-Neo, either 1 or 4 M of siRNAs was used (27). Replication was measured either in colony-formation assays (HCVrep-Neo) or by quantification of intracellular replicon-encoded Luciferase (HCVrep-Luc and HCVrep-Luc-GND) as described (26,28,29).…”

RNA interference and its therapeutic potential

“…The infection is often asymptomatic, but once establish, it can lead to the fibrosis and advanced cirrhosis. RNA interference activity has been used as a treatment by using of multiple siRNAs to reduce the devastating effects of HCV replication on the liver [70]. It was also reported that RNAi has been specifically inhibit HCV RNA replication and protein expression in Huh-7 cells by using a selectable sub-genomic HCV replicon cell culture system [39].…”