key: cord-0769499-m40s6uw9
authors: Sakamoto, Naoya; Tanabe, Yoko; Yokota, Takanori; Satoh, Kenichi; Sekine‐Osajima, Yuko; Nakagawa, Mina; Itsui, Yasuhiro; Tasaka, Megumi; Sakurai, Yuki; Cheng‐Hsin, Chen; Yano, Masahiko; Ohkoshi, Shogo; Aoyagi, Yutaka; Maekawa, Shinya; Enomoto, Nobuyuki; Kohara, Michinori; Watanabe, Mamoru
title: Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA
date: 2007-08-07
journal: J Gastroenterol Hepatol
DOI: 10.1111/j.1440-1746.2007.05076.x
sha: 60dcb26d949f1db7d90267eafa9e200118dcc1a5
doc_id: 769499
cord_uid: m40s6uw9

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. Methods: We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon‐expressing cells in vitro and transgenic mice in vivo. Results: Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by ∼10(−3). Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/loxP switching system resulted in specific suppression of virus protein synthesis in the liver. Conclusion: Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.

Hepatitis C virus (HCV), which affects 170 million people worldwide, is one of the most important pathogens causing liver-related morbidity and mortality. 1 The difficulty in eradicating HCV is attributable to limited treatment options against the virus and their unsatisfactory efficacies. Even with the most effective regimen with pegylated interferon (IFN) and ribavirin in combination, the efficacies are limited to less than half of the patients treated. 2 Given this situation, the development of safe and effective anti-HCV therapies is one of our high-priority goals.

RNA interference (RNAi) is a process of sequence-specific, post-transcriptional gene silencing that is initiated by doublestranded RNA. 3, 4 Because of its potency and specificity, RNAi rapidly has become a powerful tool for basic research to analyze gene functions and for potential therapeutic applications. Recently, successful suppression of various human pathogens by RNAi have been reported, including human immunodeficiency viruses, 5, 6 poliovirus, 7 influenza virus, 8 severe acute respiratory syndrome (SARS) virus 9 and hepatitis B virus (HBV). [10] [11] [12] [13] We and other researchers have reported that appropriately designed small interfering RNA (siRNA) targeting HCV genomic RNA can efficiently and specifically suppress HCV replication in vitro. [14] [15] [16] [17] [18] [19] We have tested siRNA designed to target the wellconserved 5′-untranslated region (5′-UTR) of HCV-RNA, and identified the most effective target, just upstream of the translation initiation codon. Furthermore, transfection of DNA-based vectors expressing siRNA was as effective as that of synthetic siRNA in suppressing HCV replication. 14 In this study, we explored the further possibility that efficient delivery and expression of siRNA may be effective in suppression and elimination of HCV replication and that delivery of such HCV-directed siRNA in vivo may be effective in silencing viral protein expression in the liver. Here, we report that HCV replication was suppressed in vitro by recombinant retrovirus and adenovirus vectors expressing short hairpin RNA (shRNA) and that the delivery of the adenovirus vector to mice in vivo specifically inhibited viral protein synthesis in the liver.

Huh7 and Retro Pack PT67 cells (Clontech, Palo Alto, CA, USA) were maintained in Dulbecco's modified minimal essential medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum at 37°C under 5% CO2. To maintain cell lines carrying the HCV replicon, G418 (Wako, Osaka, Japan) was added to the culture medium to a final concentration of 500 mg/mL.

HCV replicon plasmids, pRep-Feo, pRep-Fluc and pRep-BSD were constructed from were constructed from a virus, HCV-N strain, genotype 1b. 21 The pRep-Feo expressed a chimeric reporter protein of firefly luciferase (Fluc) and neomycin phosphotransferase. 14, 20 The pRep-Fluc and the pRep-BSD expressed the Fluc and blasticidin S (BSD) resistance genes, respectively (Fig. 1) . The replicon RNA synthesis and the transfection protocol have been described previously. 22

The design and construction of HCV-directed siRNA vectors have been described. 14 Briefly, five siRNA targeting the 5′-UTR of HCV RNA were tested for their efficiency to inhibit HCV replication, and the most effective sequence, which targeted nucleotide position of 331 though 351, was used in the present study. To construct shRNA-expressing DNA cassettes, oligonucleotide inserts were synthesized that contained the loop sequence (5′-TTC AAG AGA-3′) flanked by sense and antisense siRNA sequences (Fig. 2a) . These were inserted immediately downstream of the human U6 promoter. To avoid a problem in transcribing shRNA because of instability of the DNA strands arising from the tight palindrome structure, several C-to-T point mutations, which retained completely the silencing activity of the shRNA, were introduced into the sense strand of the shRNA sequences (referred to as 'm'). 23 A control plasmid, pUC19-shRNA-Control, expressed shRNA directed towards the Machado-Joseph disease gene, which is a mutant of ataxin-3 gene and is not normally expressed. We have previously described the sequence specific activity of the shRNA-Control. 24 Prior to construction of the virus vectors, we tested silencing efficiency of five shRNA constructs of different lengths that covered the target sequence (Fig. 2a) . The shRNA-HCV-19, shRNA-HCV-21 and shRNA-HCV-27 had target sequences of 19, 21 and 27 nucleotides, respectively. Transfection of these shRNA constructs into Huh7/pRep-Feo showed that shRNA with longer target sequences had better suppressive effects (Fig. 2b ). Therefore, we used shRNA-HCV-27m (abbreviated as shRNA-HCV) in the following study.

The U6-shRNA expression cassettes were inserted into the StuI/ HindIII site of a retrovirus vector, pLNCX2 (Clontech) to construct pLNCshRNA-HCV and pLNCshRNA-Control (Fig. 2c) . The plasmids were transfected into the packaging cells, Retro Pack PT67. The culture supernatant was filtered and added onto Huh7 cells with 4 mg/mL of polybrene. Huh7 cell lines stably expressing shRNA were established by culture in the presence of 500 mg/mL of G418.

Recombinant adenoviruses expressing shRNA were constructed using an Adenovirus Expression Vector Kit (Takara, Otsu, Japan). The U6-shRNA expression DNA cassette was inserted into the SwaI site of pAxcw to construct pAxshRNA-HCV and pAxshRNA-Control. The adenoviruses were propagated according to the manufacturer's protocol (AxshRNA-HCV and AxshRNA-Control; Fig. 2c ). A 'multiplicity of infection' (MOI) was used to standardize infecting doses of adenovirus. The MOI stands for the ratio of infectious virus particles to the number of cells being infected. An MOI = 1 represents equivalent dose to introduce one infectious virus particle to every host cell that is present in the culture.

pISRE-TA-Luc (Invitrogen, Carlsbad, CA, USA) contained five copies of the consensus interferon stimulated response element (ISRE) motifs upstream of the Fluc gene. pTA-Luc (Invitrogen), which lacks the enhancer element, was used for background determination. The pcDNA3.1 (Invitrogen) was used as an empty vector for mock transfection. pRL-CMV (Promega, Madison, WI, USA), which expresses the Renilla luciferase protein, was used for normalization of transfection efficiency. 25 A plasmid, peGFPneo (Invitrogen), was used to monitor percentages of transduced cells. 

Total cellular RNA was extracted from cultured cells or liver tissue using ISOGEN (Nippon Gene, Tokyo, Japan). Total cellular RNA (2 mg) was used to generate cDNA from each sample using the SuperScript II reverse-transcriptase (Invitrogen). The mRNA expression levels were measured using the Light Cycler PCR and detection system (Roche, Mannheim, Germany) and Light Cycler Fast Start DNA Master SYBR Green 1 mix (Roche).

Luciferase activity was measured using a luminometer, Lumat LB9501 (Promega) and the Bright-Glo Luciferase Assay System (Promega) or the Dual-Luciferase Reporter Assay System (Promega).

Total cellular RNA was separated by denaturing agaroseformaldehyde gel electrophoresis, and transferred to a nylon membrane. The membrane was hybridized with a digoxigenin-labeled probe specific for the full-length replicon sequence, and subsequently with a probe specific for beta-actin. The signals were detected by chemiluminescence reaction using a Digoxigenin Luminescent Detection Kit (Roche), and visualized by Fluoro-Imager (Roche). For the western blotting, 10 mg of total cell lysate was separated on NuPAGE 4.12% Bis-TrisGel (Invitrogen), and blotted onto an Immobilon PVDF Membrane (Roche). The membrane was incubated with monoclonal antibodies specific for HCV-NS5A (BioDesign, Saco, ME, USA), NS4A (Virogen, Watertown, MA, USA), or beta-actin (Sigma), and detected by a chemiluminescence reaction (BM Chemiluminescence Blotting Substrate; POD, Roche).

A replicon, pRep-Fluc, was transfected into cells and the luciferase activities of the cell lysates were measured serially. To correct the transfection efficiency, each value was divided by the luciferase activity at 4 h after the transfection.

Cells were transfected with a replicon, pRep-BSD, and were cultured in the presence of 150 mg/mL of BSD (Invitrogen). BSDresistant cell colonies appeared after~3 weeks of culture, and were counted. 

Transgenic mice, CN2-29, inducibly express mRNA for the HCV structural proteins (genotype1b, nucleotides 294-3435) by the Cre/loxP switching system. 28 The transgene does not contain fulllength HCV 5′-UTR, but shares the target sequence of the shRNA-HCV. Although the transgenic mouse CN2 has been previously reported as expressing higher levels of the viral proteins, the expression levels of the viral core protein in the CN2-29 mice are modest and similar to that in the liver of HCV patients. Thus, we chose CN2-29 mice in the present study.

The mice were infected with AxshRNA-HCV or controls (AxshRNA-Control or AxCAw1) in combination with AxCAN-Cre, which expressed Cre recombinase. Three days after the infection, the mice were killed and HCV core protein in the liver was measured as described below. The BALB/c mice were maintained in the Animal Care Facility of Tokyo Medial and Dental University, and transgenic mice were in the Tokyo Metropolitan Institute of Medical Science. Animal care was in accordance with institutional guidelines. The review board of the university approved our experimental animal studies and all experiments were approved by the institutional animal study committees.

The amounts of HCV core protein in the liver tissue from the mice was measured by a fluorescence enzyme immunoassay (FEIA) 29 with a slight modification. Briefly, the 5F11 monoclonal anti-HCV-core antibody was used as the first antibody on the solid phase, and the 5E3 antibody conjugated with horseradish peroxidase was the second antibody. This FEIA can detect as little as 4 pg/mL of recombinant HCV-core protein. Contents of the HCV core protein in the liver samples were normalized by the total protein contents and expressed as pg/mg total protein.

Liver tissue was frozen with optimal cutting temperature (OTC) compound (Tissue Tek; Sakura Finetechnical, Tokyo, Japan). The sections (8 mm thick) were fixed with a 1:1 solution of acetone : methanol at -20°C for 10 min and then washed with phosphate-buffered saline (PBS). Subsequently, the sections were incubated with the IgG fraction of an anti-HCV core rabbit polyclonal antibody (RR8) 28 in blocking buffer or antialbumin rabbit polyclonal antibody (Dako Cytomation, Glostrup, Denmark) in PBS overnight at 4°C. The sections were incubated with secondary antibody, Alexa-antirabbit IgG (Invitrogen) or TRITIC-antirabbit IgG (Sigma), for 2 h at room temperature. Fluorescence was observed using a fluorescence microscope.

Statistical analyses were performed using Student's t-test; P-values of less than 0.05 were considered to be statistically significant.

Retrovirus vectors propagated from pLNCshRNA-HCV and pLNCshRNA-Control were used to infect Huh7 cells, and cell lines were established that constitutively express shRNA-HCV and shRNA-Control (Huh7/shRNA-HCV and Huh7/shRNA-Control, respectively). There were no differences in the cell morphology or growth rate between shRNA-transduced and nontransduced Huh7 cells (data not shown). The HCV replicon, pRep-Fluc, was transfected into Huh7/shRNA-HCV, Huh7/ shRNA-Control and naive Huh7 cells by electroporation. In Huh7/ shRNA-Control and naive Huh7 cells, the initial luciferase activity at 4 h decreased temporarily, which represents decay of the transfected replicon RNA, but increased again at 48 h and 72 h, which demonstrate de novo synthesis of the HCV replicon RNA. In contrast, transfection into Huh7/shRNA-HCV cells resulted in a decrease in the initial luciferase activity, reaching background by 72 h (Fig. 3a) . Similarly, transfection of the replicon, pRep-BSD, into Huh7 cells and BSD selection yielded numerous BSDresistant colonies in the naive Huh7 (832 colonies) and Huh7/ shRNA-Control cell lines (740 colonies), while transfection of Huh7/shRNA-HCV, which expressed shRNA-HCV, yielded obviously fewer colonies (five colonies), indicating reduction of colony forming units by~10 2 (Fig. 3b) . There was no difference in shape, growth or viability between cells expressing the shRNA or not. These results indicated that cells expressing HCV-directed shRNA following retrovirus transduction acquired resistance to HCV replication.

We investigated subsequently the effects of recombinant adenovirus vectors expressing shRNA. AxshRNA-HCV and AxshRNA-Control were used separately to infect Huh7/pRep-Feo cells, and the internal luciferase activities were measured sequentially (Fig. 4a) . AxshRNA-HCV caused continuous suppression of HCV RNA replication. Six days postinfection, the luciferase activities fell to background levels. In contrast, the luciferase activities of the Huh7/pRep-Feo cells infected with AxshRNA-Control did not show any significant changes compared with untreated Huh7/ pRep-Feo cells (Fig. 4a) . The dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay showed no significant difference between cells that were infected by recombinant adenovirus and uninfected cells (Fig. 4b) . In the northern blotting analysis, the cells were harvested 6 days after infection with the adenovirus at an MOI of 1. Feo-replicon RNA of 9.6 kb, which was detectable in the untreated Huh7/pRep-Feo cells and in the cells infected with AxshRNA-Control, diminished substantially following infection with the AxshRNA-HCV (Fig. 4c) . Densitometries showed that the intracellular levels of the replicon RNA in the Huh7/pRep-Feo cells correlated well with the internal luciferase activities. Similarly in the western blotting, cells were harvested 6 days after infection with adenovirus. Levels of the HCV NS4A and NS5A proteins that were translated from the HCV replicon decreased following infection with the AxshRNA-HCV (Fig. 4d) . These results indicated that the decrease in luciferase activities was due to specific suppressive effects of shRNA on expression of HCV genomic RNA and the viral proteins, and not due to non-specific effects caused by the delivery of shRNA or to toxicity of the adenovirus vectors.

It has been reported that double-stranded RNA may induce interferon-stimulated gene (ISG) responses which cause instability of mRNA, translational suppression of proteins and apoptotic cell negative-or positive-control shRNA plasmids was transfected. (Fig. 5a) . Similarly, the expression levels of an interferoninducible MxA protein did not significantly change by transfection of shRNA-expression vectors (Fig. 5b) . These results demonstrate that the shRNA used in the present study lack induction of the ISG responses both in the form of the expression plasmids and the adenovirus vectors.

The effects of HCV-targeted siRNA-and shRNA-expressing adenoviruses were confirmed by using HCV-JFH1 virus cell culture system. Transfection of the siRNA #331 14 into HCVinfected Huh7.5.1 cells resulted in substantial decrease of intracellular HCV RNA, while a control siRNA showed no effect (Fig. 6a) . Similarly, infection of AxshRNA-HCV into Huh7.5.1/ HCV-JFH1 cells specifically suppressed expression of HCV RNA (Fig. 6b) . 

Relative luciferase activity 

The effects of the shRNA expression on the expression of the viral structural proteins in vivo were investigated using conditional HCV cDNA-transgenic mice, CN2-29. 28 Adenoviruses, AxshRNA-HCV, AxshRNA-Control or AxCAw1 were injected into CN2-29 mice in combination with AxCANCre, an adenovirus expressing Cre DNA recombinase. The mice were killed on the fourth day after the injection, and the hepatic expression of the HCV core protein was measured. The expressed amounts of the core protein were 143.0 Ϯ 56.2 pg/mg and 108.5 Ϯ 42.4 pg/mg in AxCAw1 and AxshRNA-Control-infected mice, respectively, and the expressed amount was significantly lower in mice injected with AxshRNA-HCV (28.7 Ϯ 7.0 pg/mg, P < 0.05, Fig. 7a) . Similarly, the induced expression of HCV core protein was not detectable by immunohistochemistry in AxshRNA-HCV infected liver tissue (Fig. 7c) . Staining of a host cellular protein, albumin, was not obviously different between the liver infected with AxCAw1, AxshRNA-HCV and AxshRNA-Control (Fig. 7d) . The expression levels of two ISG, IFN-beta and Mx1, in the liver tissue were not significantly different between individuals with and without injection of the adenovirus vectors (Fig. 7b) . These results indicate specific shRNA silencing of HCV structural protein expression in the liver.

The requirements to achieve a high efficiency using RNAi are: (i) selection of target sequences that are the most susceptible to RNAi; (ii) persistence of siRNA activity; and (iii) efficient in vivo delivery of siRNA to cells. We have used an shRNA sequence that was derived from a highly efficient siRNA (siRNA331), and constructed a DNA-based shRNA expression cassette that showed competitive effects with the synthetic siRNA (Fig. 2 ). 14 The shRNA-expression cassette does not only allow extended half-life of the RNAi, but also enables use of gene-delivery vectors, such as virus vectors. As shown in the results, a retrovirus vector expressing shRNA-HCV could stably transduce cells to express HCVdirected shRNA, and the cells acquired protection against HCV subgenomic replication (Fig. 3 ). An adenovirus vector expressing shRNA-HCV resulted in suppression of HCV subgenomic and protein expression by around three logs to almost background levels (Fig. 4) . Consistent results were obtained by using an HCV cell culture (Fig. 6 ). More importantly, we have demonstrated in-vivo effects on viral protein expression in the liver using a conditional transgenic mouse model (Fig. 7) . These results suggest that efficient delivery of siRNA could be effective against HCV infection in vivo. An obstacle to applying siRNA technology to treat virus infections is that viruses are prone to mutate during their replication. 32 HCV continuously produces mutated viral strains to escape immune defense mechanisms. Even in a single patient, the circulating HCV population comprises a large number of closely related HCV sequence variants called quasispecies. Therefore, siRNA targeting the protein-coding sequence of the HCV genome, which have been reported by others, [15] [16] [17] [18] [19] may vary considerably among different HCV genotypes, and even among strains of the same genotype. 33 Our shRNA sequence targeted the 5′-UTR of HCV RNA, which is the most conserved region among various HCV isolates. 33 In addition, the structural constraints on the 5′-UTR, in terms of its requirement to direct internal ribosome entry and translation of viral proteins, might not permit the evolution of escape mutations. Our preliminary results have shown that the siRNA-HCV suppressed replication of an HCV genotype 2a replicon 34 to the same extent as the HCV 1b replicon.

Although the siRNA techniques rely on a high degree of specificity, several studies report siRNA-induced non-specific effect that may result from induction of ISG responses. 18, 31 These effects may be mediated by activation of double-strand RNA-dependent protein kinase, toll-like receptor 3, 35 or possibly by a recently identified RNA helicase, RIG-I. 36 It remains to be determined whether these effects are generally induced by every siRNA construct. Sledz et al. have reported that transfection of two siRNA induced cellular interferon responses, 37 while Bridge et al. report that shRNA-expressing plasmids induced an interferon response but transfection of synthetic siRNA did not. 31 Speculatively, these effects on the interferon system might be construct dependent. Our shRNA-expression plasmids and adenoviruses did not activate ISG responses in vitro (Fig. 5a,b) or in vivo (Fig. 7b) . We have preliminarily detected phosphorylated PKR (P-PKR) by western blotting, and found no apparent increase of P-PKR (data not shown). These results indicate that these target sequences and structures are of sufficient specificity to silence the target gene without eliciting non-specific interferon responses. Beside the canonical action of siRNA, a sequence-specific cleavage of target mRNA, the siRNA could act as a micro-RNA that suppresses translational initiation of mRNA, 38 or it could mediate transcriptional gene silencing. 39 Regarding our in-vivo experiments, it was difficult to differentially analyze the effect of siRNA at individual sites of action because post-translational effect of siRNA concomitantly destabilizes target mRNA, which leads to apparent decrease of mRNA transcripts. (c) Immunofluorescence microscopy of HCV core protein in the liver tissue. Liver sections of mice were stained using rabbit anticore polyclonal antibody and normal rabbit IgG as a negative control. The upper photographs were obtained at 400¥ magnification, and the lower photographs were at 1000¥. (d) Immunofluorescence microscopy of albumin in liver. Liver sections from the mice were fixed and stained using rabbit antialbumin antibody and normal rabbit IgG as a negative control. Efficiency and safety of gene transfer methods are the key determinants of the clinical success of gene therapy and an unresolved problem. There are several reports of delivery of siRNA or siRNA-expression vectors to cells in vivo; 12, 40, 41 however, gene delivery methods that are safe enough to apply to clinical therapeutics are currently under development. Adenovirus vectors are one of the most commonly used carriers for human gene therapies. [42] [43] [44] Our present results demonstrate that the adenoviral delivery of shRNA is effective in blocking HCV replication in vitro and virus protein expression in vivo. Adenovirus vectors have several advantages of efficient delivery of transgene both in vitro and in vivo and natural hepatotropism when administered in vivo. The AxshRNA-HCV specifically blocked expression of HCV structural proteins in a conditional transgenic mouse expressing those proteins. The current adenovirus vectors may cause inflammatory reactions in the target organ, 45 however, and produce neutralizing antibodies which make repeated administration difficult. These problems may be overcome by the improved constructs of virus vectors with attenuated immunogenicity or by the development of non-viral carriers for gene delivery. 46 In conclusion, our results demonstrate the effectiveness and feasibility of the siRNA expression system. The efficiency of adenovirus expressing shRNA that target HCV suggests that delivery and expression of siRNA in hepatocytes may eliminate the virus and that this RNA-targeting approach might provide a potentially effective future therapeutic option for HCV infection.

Epidemiology of hepatitis C

Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Modulation of HIV-1 replication by RNA interference

Short interfering RNA confers intracellular antiviral immunity in human cells

Inhibition of influenza virus production in virus-infected mice by RNA interference

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA

Inhibition of HBV replication by siRNA in a stable HBV-producing cell line

RNA interference in adult mice

Inhibition of hepatitis B virus expression and replication by RNA interference

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

Interference of hepatitis C virus RNA replication by short interfering RNAs

Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs

Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs

Letter to the editor: small interfering RNA-mediated inhibition of hepatitis C virus replication in the human hepatoma cell line Huh-7

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

Effect of alpha interferon on the hepatitis C virus replicon

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon-alpha

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

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

Sequence-dependent and independent inhibition specific for mutant ataxin-3 by small interfering RNA

Regulation of hepatitis C virus replication by interferon regulatory factor-1

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Robust hepatitis C virus infection in vitro

Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA)

Mechanisms of antiviral action of interferon

Induction of an interferon response by RNAi vectors in mammalian cells

Silencing viruses with RNA

Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

Recognition of double-stranded RNA and activation of NF-kB by Toll-like receptor 3

The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

Activation of the interferon system by short-interfering RNAs

siRNAs can function as miRNAs

siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code

siRNA-mediated gene silencing in vitro and in vivo

Caspase 8 small interfering RNA prevents acute liver failure in mice

Transfer of a foreign gene into the brain using adenovirus vectors

Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors

A model system for in vivo gene transfer into the central nervous system using an adenoviral vector

Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo

Helper-dependent adenovirus vectors devoid of all viral genes cause less myocardial inflammation compared with first-generation adenovirus vectors

This study was supported by grants from Japan Society for the Promotion of Science, 15590629 and 16590580, and partly supported by a grant from the Viral Hepatitis Research Foundation of Japan.