Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs - PubMed (original) (raw)

Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs

Takanori Yokota et al. EMBO Rep. 2003 Jun.

Abstract

Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5' untranslated region (5' UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5' UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5' UTR at which approximately 80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

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Figures

Figure 1

Design of small interfering RNAs and target constructs. (A) Predicted secondary structure of the 5′ internal ribosomal entry site (IRES) in the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome (nucleotides 1–341) and target sites of small interfering RNAs (siRNAs; Brown et al., 1992). (B) Structures of the HCV IRES reporter and replicon plasmids. BGH pA, bovine growth hormone polyadenylation site; CMV P/E, cytomegalovirus early promoter/enhancer; ΔC, truncated HCV core region (nucleotides 342–377); EMCV, encephalomyocarditis virus; _FLu_c, firefly luciferase gene; NPT, neomycin phosphotransferase gene; NS3, NS4, NS5A and NS5B, genes that encode HCV nonstructural proteins; pT7, T7 promoter; Rep-Feo, replicon that expresses a chimeric protein consisting of NPT and Fluc.

Figure 2

Effects of small interfering RNA oligonucleotides on the internal ribosomal entry site reporter and the hepatitis C virus replicon. (A) 293T cells were transfected with pIRES–FLuc, pRL-RSV and small interfering RNAs (siRNAs) at the concentrations indicated, or with a control siRNA (NC). The cells were harvested 48 h after transfection, and luciferase activities were measured. (B) Huh7 Rep-Feo cells were transfected with siRNA oligonucleotides or control siRNAs (NC). The internal luciferase activities were measured 48 h after transfection. Values are shown as percentages of the siRNA negative control, as the mean ± s.d. Asterisks indicate p < 0.05. FLuc, firefly luciferase; IRES, internal ribosomal entry site; pRL–RSV, Renilla luciferase expression plasmid; Rep-Feo, replicon that expresses a chimeric protein, consisting of NPT and Fluc.

Figure 3

Reversal of suppression by negative-control small interfering RNAs. Two small interfering RNA (siRNA) controls were used to analyse further the effects of siRNA 331. The controls used were an siRNA with a shuffled sequence of siRNA 331 (331-shuffle) and an siRNA with mutations at the tenth and eleventh nucleotides from the 5′ end of the siRNA 331 sequence (331-mutant). siRNAs 331, 331-shuffle and 331-mutant were cotransfected with pIRES-Fluc into 293 cells (A) or were transfected into Huh7 Rep-Feo cells (B). Luciferase assays were performed 48 h after transfection. pIRES–FLuc, a reporter-gene vector that expresses messenger RNA consisting of the hepatitis C virus 5′ untranslated region and the upstream part of the core region, connected in-frame with firefly luciferase.

Figure 4

Suppression of replicon RNA and replicon-derived hepatitis C virus non-structural-protein synthesis by small interfering RNA 331. (A,B) Human hepatoma Huh7 Rep-Feo cells (which stably express the hepatitis C virus (HCV) Feo replicon) were mock-transfected (lane 1) or were transfected with 2.5 nM (lane 2), 25 nM (lane 3) or 125 nM (lane 4) of small interfering RNA (siRNA) 331. Lane 5, untransfected Huh7 cells. The cells were harvested 48 h after transfection. (A) Northern blotting of the HCV replicon and β-actin RNA. Numbers below the replicon fluorogram show the results of densitometric analysis shown as a percentage of the mock-transfected control. (B) Western blotting using the monoclonal anti-NS5A, anti-NS3 and anti-NS4A antibodies. Densitometry readings (as percentages of the mock-transfected control) for lanes 1–4 were 100, 30, 10 and 10, respectively, for NS5A, 100, 25, 56 and 43 for NS3, and 100, 4, 10, 0 for NS4A.

Figure 5

Effects of expressing small interfering RNA 331 from a DNA-based vector on hepatitis C virus internal-ribosomal-entry-site-mediated translation and replication. The structures of small interfering RNA (siRNA) expression vectors are shown in (A). Tandem- or stem–loop-type siRNA 331 expression vectors were co-transfected with pIRES-Fluc and pRL–RSV (a _Renilla_-luciferase expression plasmid) into 293T cells (panel (B), or were transfected into Huh7 Rep-Feo cells (C). Luciferase activities were measured 48 h after transfection. Values are shown as percentages of the siRNA-negative control as the mean ± s.d. Asterisks indicate p<0.05. pIRES–FLuc, a reporter-gene vector that expresses messenger RNA consisting of the hepatitis C virus 5′ untranslated region and the upstream part of the core region, connected in-frame with firefly luciferase.

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Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs - PubMed (original) (raw)