Yoko Tanabe

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

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 ∼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.

Synergistic Inhibition of Intracellular Hepatitis C Virus Replication by Combination of Ribavirin and Interferon-α

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication

Summary.  Type‐I interferons (IFNs) and the interferon‐stimulated genes (ISGs) play a major role in antivirus responses against hepatitis C virus (HCV) infection. In this study, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real‐time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon‐stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF‐9, GBP‐1, IFI‐6‐16, IFI‐27, 25OAS and IRF‐1. Knock down of GBP‐1, IFI‐6‐16 and IFI‐27 by short hairpin RNA resulted in increase of HCV replication. Thus, we conclude that downregulation of ISG expression is required in the host cells supporting HCV replication and that several ISGs directly suppress HCV replication. The search for ISGs that regulate HCV replication may help to elucidate the cellular antiviral defence mechanisms against HCV infection.

Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1

ABSTRACT Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.

Summary.  The number of amino acid substitutions in the interferon sensitivity‐determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon‐based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy

Background and Aim:  Combination treatments of interferon‐alpha (IFN) and ribavirin (RBV) are more effective than those of IFN alone in hepatitis C virus (HCV) infection. However, mechanisms of the action of the combination regimen are not well understood. To elucidate the viral genetic basis of IFN plus RBV combination therapy, genetic variabilities of HCV‐1b were analyzed.

Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA

Background and Aim:  We have reported previously that synthetic small interfering RNA (siRNA) and DNA‐based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. In this study, we investigated the effects of the siRNA targeting HCV‐RNA in vivo.

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.

Summary.  Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh‐7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC‐J4, one of chimpanzee‐infectious HCV clones. An HCV replicon derived from HC‐J4 (RpJ4) consists of HCV‐5′ untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV‐3′ untranslated region. The adaptive mutations known to be required for HCV‐Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC‐J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4‐S2197P, RpJ4‐S22001del, and RpJ4‐S2204I). RpJ4/ISDR mutant and RpJ4‐S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh‐7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418‐resistant cells were transduced efficiently by RpJ4‐S2197P, RpJ4‐S2204I, RpJ4‐S2201del and RpJ4‐S2201del/ISDR mutant, with the RpJ4‐S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC‐J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.

Amino acid substitutions in PKR-eIF2 phosphorylation homology domain (PePHD) of hepatitis C virus E2 protein in genotype 2a/2b and 1b in Japan and interferon efficacy

Recently, the envelope protein 2 (E2) of hepatitis C virus (HCV) was reported to interact with double stranded RNA-dependent protein kinase (PKR) through an element homologous to the phosphorylation site of PKR and its target, eukaryotic translation initiation factor (eIF) 2alpha (PKR-eIF2alpha phosphorylation homology domain: PePHD). Inhibition of the kinase activity of PKR by this interaction was postulated as a mechanism for the resistance to interferon (IFN) therapy. The aim of this study was to clarify whether the variation of PePHD amino acid sequences affects IFN efficacy in Japanese population. Amino acid sequences of PePHD (aa. 659-670 in genotype1b, aa. 663-674 in genotype 2a and 2b) were determined in randomly selected 112 patients with chronic hepatitis C (genotype 1b; 83 patients, 2a; 14 patients, 2b; 15 patients) prior to IFN monotheray. In 21 out of the 23 genotype 1b sustained responders (SR) (91%) and 55 out of the 60 non-SR (92%), PePHD sequences were identical to that of the HCV-1b consensus sequence. Only two SR showed one amino acid substitution in PePHD, and five non-SR showed amino acid substitutions in PePHD. Among 14 genotype 2a patients, only two SR had one amino acid substitution comparing to the consensus HCV-2a sequence. Likely, only one SR had an amino acid substitution in PePHD among 15 genotype 2b patients. In conclusion, our study revealed no clinical relationship between the amino acid sequence of PePHD and the outcome of IFN therapy. PePHD polymorphism was not suggested to play a role in predicting IFN efficacy.

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3

BackgroundInterferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system.MethodsAn HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE).ResultsThe baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% ± 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels.ConclusionsIRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.