Inhibition of Hepatitis C Virus Infection in Cell Culture by Small Interfering RNAs

Abstract

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. In this study, we investigated RNA interference (RNAi), a specific gene silencing process mediated by small interfering RNA (siRNA) duplexes, as an antiviral strategy against HCV. Synthetic siRNAs were designed to target conserved sequences of the HCV 5' nontranslated region (NTR) located in a functional, stem-loop structured domain of the HCV internal ribosome entry site (IRES), which is crucial for initiation of polyprotein translation. Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. One siRNA, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. These results indicate that the HCV life cycle can be efficiently blocked by using properly-designed siRNAs that target functionally important, highly conserved sequences of the HCV IRES. This finding offers a novel approach towards developing IRES-based antiviral treatment for chronic HCV infections.

Repression of the Internal Ribosome Entry Site-dependent Translation of Hepatitis C Virus by an Engineered PUF Protein

“…Although the detailed mechanism is yet to be defined, binding of mHsPUM1-HD to its target appears to interfere with eIF-3 binding and subsequent interactions with 40S ribosomal subunit. While some cur- rent strategies, such as the use of RNAi or aptamers, can reduce the translation level to some extent (13,(20)(21)(22), our approach differs from these methods, as it can be effective in organisms or cellular compartments where aptamers or RNA interference machinery is not available (17).…”

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

“…To verify the specificity of siRNA gene silencing on HCV RNA replication, we established a robust control for comparative purposes. From previous reports, HCV replication is repressed by siRNA-targeting of regions within NS3 (Kapadia et al, 2003;Prabhu et al, 2005), NS5B (Kapadia et al, 2003;Ng et al, 2007;Prabhu et al, 2005) or the 59 untranslated region (UTR) (Chevalier et al, 2007;Kanda et al, 2007;Kronke et al, 2004;Ng et al, 2007;Seo et al, 2003;Yokota et al, 2003). The 59 UTR is the most conserved segment of the HCV genome and contains a viral IRES for cap-independent translation of the viral polyprotein (Tsukiyama-Kohara et al, 1992;Wang et al, 1993).…”

“…The IRES harbours RNA structures that can be divided into four domains (termed I-IV). A siRNA that targets domain III/IV is particularly effective at reducing HCV RNA replication (referred to as si313 in Chevalier et al, 2007). Furthermore, this region is conserved across several HCV genotypes, including JFH1.…”

siRNA and RNAi optimization

“…A promising approach for cancer and viral therapy came from the development of immunostimulatory siRNAs (is-siRNAs) 86,94 . These bifunctional molecules, linking potent gene silencing properties to suitable production of IFNs, can effectively control chronic viral diseases such as HIV-AIDS, HBV and HCV infections [95][96][97] . Furthermore, the application of this new siRNA design can not only overcome the cancer drug resistance, but also strengthen the immune surveillance usually evaded by cancer cells [98][99][100][101][102] …”