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Journal of Virology, January 2007, p. 669-676, Vol. 81, No. 2
0022-538X/07/$08.00+0     doi:10.1128/JVI.01496-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Small Interfering RNA Targeted to Hepatitis C Virus 5' Nontranslated Region Exerts Potent Antiviral Effect{triangledown}

Tatsuo Kanda,1 Robert Steele,1 Ranjit Ray,2,3 and Ratna B. Ray1,2,3*

Departments of Pathology,1 Internal Medicine,2 Liver Center,Saint Louis University, St. Louis, Missouri 631103

Received 13 July 2006/ Accepted 20 October 2006


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ABSTRACT
 
Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5' nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.


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INTRODUCTION
 
Chronic HCV infection affects at least 170 million people worldwide and is the most common cause of cirrhosis and hepatocellular carcinoma in the United States (22, 46). Despite intensive clinical efforts, a limitation for the combination of interferon (IFN) or pegylated IFN and ribavirin therapy exists (14). There is an important need to develop new therapeutic options for treatment of chronic HCV infection. HCV belongs to the family Flaviviridae, and its genome is a positive-strand 9.6-kb RNA. HCV has a 5' nontranslated region (NTR), a long open reading frame, and a 3' NTR. An internal ribosome entry site (IRES), containing the 5' NTR and part of the core coding region, forms a secondary structure and supports translation initiation of an HCV genome in a cap-independent manner (13, 41). The HCV genome encodes a single precursor polyprotein that is processed by host signal peptidases and HCV proteases into structural (core, envelope E1, and E2/p7), and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. HCV replication was overcome, in part, by the development of selectable, dicistronic, subgenomic (SR), or full-length (FL) replicons derived from HCV genotype 1 or 2a in Huh-7 cells or their derivatives (reviewed in reference 1). Recently, different groups of investigators have reported the generation of infectious virus by transfecting full-length HCV genomic RNA from genotypes 1a and 2a into cells of human hepatocyte origin (6, 11, 17, 29, 54, 59, 61).

MicroRNA (miRNA) and small interfering RNA (siRNA) are small RNAs of 18 to 25 nucleotides that play important roles in the regulation of gene expression. Although, both miRNA and siRNA utilize the RNA-induced silencing complex for gene silencing, their mechanisms for inhibiting protein synthesis are not the same. The siRNAs shut down gene expression at the posttranscriptional level through mRNA degradation (9, 30, 33). In mammals, exposure to double-stranded RNAs greater than 30 base pairs in length induces a generalized antiviral interferon response that globally represses mRNA translation (27, 51). However, introduction of siRNA into mammalian cells leads to mRNA degradation with exquisite sequence specificity without activating an interferon response. Thus, siRNA is a promising vehicle for induction of intracellular immunity. Unlike classical antisense techniques, siRNA taps into existing gene-silencing pathways. Resistance of particular RNAs to RNA interference (RNAi)-mediated degradation has also been observed in cases where accessibility of the target sequence was restricted. For viral RNAs, resistance can also be related to the intracellular location and/or nucleocapsid association of genomic-RNA molecules (53). RNAi effectors can be delivered to cells using two different approaches: (i) chemically synthesized siRNAs can be delivered as a drug or (ii) a gene therapy approach can be used, in which DNA encoding shRNA expression cassettes is delivered into cells and is then processed into active siRNAs by the host cell (5). Delivery of DNA expression vectors is possible either by integration into the genome or in self-replicating episomal form, which could allow constitutive expression of the shRNA cassette. shRNAs transfected into cells are initially transcribed in the nucleus and are thought to be exported into the cytoplasm with the aid of exportin 5, like miRNAs (32). The loops of shRNA are trimmed by Dicer in cytoplasm to generate siRNA complexes for mRNA degradation, although it is possible that translational repression might be involved in gene silencing by vector-based shRNA (37). Therefore, RNA silencing provides a new platform that may effectively treat HCV infection, in addition to traditional antiviral therapies.

We have shown that siRNA targeted to NS5A knocks down NS5A expression and impairs NS5A-mediated interleukin-8 activation (44). Other investigators have also reported the inhibition of HCV replication by targeting core, NS3, or NS5B sequences (19, 39, 45, 57, 58). Therefore, RNAi can inhibit HCV replication, and we have demonstrated the proof of concept. However, HCVs are error prone in replication and produce mutated progeny molecules or quasispecies. Some of these natural mutations help viruses to escape immune surveillance or reduce inhibition by antiviral drugs, and they may prevent recognition by siRNAs. To overcome these obstacles, we need to target multiple sites of viral RNA sequences that are conserved and normally invariant between different HCV strains or simultaneously target several viral sequences. In the present study, we have shown that vectors encoding shRNAs targeted to the 5' NTR of HCV efficiently act upon replicon systems and cell-culture-grown HCV, inhibiting virus genome replication and suggesting a potential clinical application of this novel approach.


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MATERIALS AND METHODS
 
Cells and virus. Huh-7 and Huh-7.5 (2) cells were maintained at 37°C in an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (Cambrex, Walkersville, MD) containing 10% fetal bovine serum, 200 U/ml of penicillin G, and 200 µg/ml of streptomycin. IHH cells were maintained in SABM medium (40). Huh-7 cells (2) harboring an SR or an FL replicon (31, 38) of HCV genotype 1b were used in this study. Cell-culture-grown HCV (genotypes 1a and 2a) was also used (17).

shRNA expression constructs. The GenBank database was searched for unique sequences within HCV to exclude identity with known cellular genes. Three sites, HCV-53 (5'-AACUACUGUCUUCACGCAGAA-3') and HCV-274 (5'-AAAGGCCUUGUGGUACU GCCU-3') in the 5' NTR and HCV-375 (5'-AAACGUAACACCAACCGUCGC-3') in the core of the common sequences of the HCV 1a strain H77 (GenBank accession number AF009606) (24), the 1b strain Con1 (GenBank accession number AJ238799) (31), and the 2a strain JFH1 (GenBank accession number AB047639) (20) were chosen as the targets for shRNAs (Fig. 1A and B).


Figure 1
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  		FIG. 1. Target structures of HCV-specific siRNAs for shRNA expression. (A) Modified schematic representation of domain II and subdomains IIId and IIIe of the HCV IRES, including the adjacent coding sequences. Sequence homology of the target sequences is indicated by dashes. (B) Predicted secondary structure of the IRES from HCV genotype 1a (strain H77). An IRES model of HCV-N, AF139594 (genotype 1b) (13), was modified using the RNA secondary-structure prediction program (http://www.genebee.msu.su/services/rna2_reduced.html). shRNA targets are indicated by arrows.

shRNA oligonucleotides were designed to contain a sense strand of 19-nucleotide sequences (from the HCV genome or scrambled sequence [not matched with the HCV genome or the host genome]), followed by a short spacer (TTCAAGAGA), the reverse complement of the sense strand, and five thymidines as an RNA polymerase III transcriptional stop signal. Oligonucleotides were annealed with the reverse strand and cloned into the BamHI and HindIII sites of pRNAT-H1.3/Hygro (GenScript Corporation, Piscataway, NJ). The resultant plasmid (psh-53, psh-274, or psh-375) was used in the experiments. The plasmids psh-53, psh-274, and psh-375 were named to correspond with their respective targets, HCV-53, HCV-274, and HCV-375. The constructs contained the 3' end of the sense strand and the 5' end of the antisense strand, connected by a 7-nucleotide loop sequence. Scrambled siRNA (control) cloned into the same vector was used as a negative control in all of the experiments.

Reporter assay. The plasmid pSV40-HCV IRES-luc carries, in a bicistronic fashion, the Renilla reniformis luciferase (Rluc) gene, the entire HCV core gene under translational control of the HCV 5' NTR, and the firefly luciferase (Fluc) gene, followed by the 3' NTR of HCV genotype 1a (25). Approximately 1 x 105 Huh-7 cells per well were placed in a six-well plate 24 h prior to transfection. Using Lipofectamine (Invitrogen, San Diego, CA), psh-53, -274, or -375 (0.4 µg) was transfected into the cells, along with pSV40-HCV IRES-luc (0.6 µg). Seventy-two hours posttransfection, the cells were harvested using reporter lysis buffer (Promega, Madison, WI), and Fluc and Rluc activities were determined with an Optocomp II luminometer (MGM Instruments, Hamden, CT). Activity was normalized with respect to the protein concentrations of the cell lysates.

Treatment of cells harboring the HCV genome with shRNAs. Vector carrying shRNA was introduced into Huh-7 cells harboring SR or FL replicon or infected with HCV JFH1 using Lipofectamine, as described above. Cells were harvested at 48 h or 72 h posttransfection for RNA and protein analyses. We have also established Huh-7.5 cells stably expressing psh-53, psh-274, or psh-375. These cells were infected with HCV JFH1, and the virus genome copy number was determined by real-time reverse transcription (RT)-PCR as described previously (17).

Electroporation of cultured cells with vectors carrying shRNA and selection. Vector carrying shRNA was transfected into cells harboring SR or FL replicon cell lines by electroporation. Briefly, subconfluent cells were detached by trypsin treatment, collected by centrifugation (500 x g; 10 min), and washed two times in RNase-free phosphate-buffered saline. Plasmid vectors (3.0 µg) were mixed at 0.4 ml of 2.0 x 106 cells in a 4-mm-gap-width cuvette (Bio-Rad, Hercules, CA) and pulsed using a Bio-Rad GenePulser X cell (electroporation conditions, 270 V and 950 µF). The pulsed cells were left to recover for 10 min at room temperature and then seeded into two 100-mm-diameter culture dishes. Twenty-four hours later and every 3 or 4 days during selection, the medium was replaced with fresh Dulbecco's modified Eagle's medium supplemented with 800 µg of G418 for 3 weeks in SR- or FL replicon-harboring cells. One dish from each transfection was used for a colony formation assay. G418-resistant colonies were fixed with 3.7% formaldehyde and stained with 1% crystal violet. In the other dish, G418-resistant colonies were pooled, expanded, and used for RNA and/or protein analyses and serial treatments with vector carrying shRNA. For screening of colonies resistant to vector carrying shRNA, two subsequent serial treatments were performed by electroporation of surviving G418-resistant colonies with the same vector carrying shRNA described above.

RNA purification, RT-PCR, and real-time RT-PCR. Total RNA was isolated from the cells using a Purescript RNA isolation kit (Gentra Systems, Minneapolis, MN). To analyze the development of escape mutations like HIV-1 (3), the corresponding region of the HCV replicon was analyzed using the Superscript one-step RT-PCR with a platinum Taq kit (Invitrogen). PCR primers for the HCV 5' NTR and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were previously described (17). The PCR products were subjected to electrophoresis on a 1.8% agarose gel. HCV-specific RNA was detected by real-time PCR as the increase in fluorescence of SYBR Green I on an ABI PRISM 7700 (Applied Biosystems, Foster City, CA). The GAPDH housekeeping gene was used as a control for normalization. Each real-time PCR assay was performed in triplicate.

Western blot analysis for HCV protein. Cells were harvested using sodium dodecyl sulfate sample buffer. Proteins were subjected to electrophoresis on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was probed with a monoclonal antibody to NS5A (Biodesign International, Saco, ME) or actin (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were visualized using an enhanced-chemiluminescence detection kit (Amersham Pharmacia, Piscataway, NJ) and scanned by an image analyzer to quantify the density of the protein bands using Image Quant software (Amersham Molecular Dynamics, Sunnyvale, CA).

Immunofluorescence study. For intracellular-immunofluorescence studies, infected hepatocytes were fixed with 3.7% formaldehyde at day 3 postinfection and incubated at room temperature for 1 h with an NS4-specific fluorescein isothiocyanate-conjugated monoclonal antibody (Biodesign International, Saco, ME). Nuclear staining was performed with TO-PRO3-iodide (Molecular Probes). Finally, the washed cells were mounted for confocal microscopy (Bio-Rad 1024), and focus-forming units (FFU) per ml were counted as described previously (17).


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RESULTS
 
shRNA directed against HCV 5' NTRs inhibits IRES-mediated translation. We previously reported that the introduction of siRNA targeted to NS5A of HCV genotype 1a (H77) inhibits HCV protein expression (44). Interestingly, these siRNAs failed to inhibit HCV NS5A expression from genotype 1b. Close analysis indicated three mismatched nucleotides in the middle of the siRNA sequences. Therefore, we plan to identify the sequences that are common among different HCV genotypes. The 5' NTR is one of the most conservative regions in the HCV genome and among the genotypes that differ from each other by 31% to 33% (43, 47, 56, 60). The core is the most conservative sequence among the HCV proteins. The 5' NTR and core are important regions, as IRES functions to translate HCV proteins (41). Although it is well known that the 3' NTR is also conserved among different HCV strains, it was reported that the effects of siRNA against the 5' NTR were almost greater than those of the 3' NTR siRNAs (16, 25). Therefore, in the present study, we designed siRNAs targeting the common regions of the IRESs (Fig. 1A) of both genotypes 1a and 1b, as well as 2a, sequences and evaluated their antiviral activities.

The ternary interaction of the IRES, the 40S ribosomal subunit, and eukaryotic initiation factor 3 are essential for translation initiation (21, 23, 36, 48). The IRES sequence may be conserved among HCV and related flaviviruses and pestiviruses. The proposed secondary structure of the HCV IRES is thought to contain four major domains (I to IV) (4, 13). Targets of psh-53 and psh-274 were located in domains II and III, respectively (Fig. 1B). Domain III contains subdomains IIIa to IIIf. The target of psh-274 was located in the portion from subdomain IIId to IIIe. It is interesting that hairpins IIId and IIIe, comprising nucleotides 253 to 302, are known to be essential for binding to the 40S subunit (34). Initially, we examined the effects of shRNAs on HCV IRES-mediated translation using a luciferase reporter assay. Huh-7 cells were transfected with pSV40-HCV IRES-luc and different shRNA constructs (psh-53, psh-274, or psh-375). Inhibition of luciferase activity at different levels was observed from all three constructs, although the highest activity was noted with psh-274 (Fig. 2). We and others showed earlier that suppression of Fluc activity correlates with that of Rluc activity, indicating that sequences located downstream and upstream of the target site are degraded (18, 28).


Figure 2
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  		FIG. 2. Inhibition of HCV IRES-mediated translation by shRNAs. The bicistronic plasmid pSV40-HCV IRES-luc carries the Rluc gene; the HCV IRES, including the full-length core; and the Fluc gene under the control of the simian virus 40 (SV40) promoter, with a polyadenylation signal (pA). Huh-7 cells (105) were cotransfected with 0.6 µg of pSV40-HCV IRES-luc vector and 0.4 µg of psh-53, -274, or -375 or control DNA. At 72 h posttransfection, cells were lysed and the luciferase activity was determined. The mean firefly luciferase activity from control cells was set at 100% and used to normalize the firefly luciferase activities of transfected cells. The resulting relative luciferase values are shown. The error bars represent standard errors of the mean from three different experiments.

shRNA inhibits replication of cell-culture-grown HCV. We next analyzed whether the shRNAs could inhibit replication of cell-culture-grown HCV. Recently, we established persistently infected Huh-7.5 cells with HCV genotype 2a (JFH1). shRNAs were introduced into these cells. A significant level of suppression of HCV genotype 2a replication by psh-274 was observed compared to other shRNAs (Fig. 3A and B). Although HCV-53 and HCV-274 had perfect sequence homology with an infectious molecular clone of HCV genotype 2a, psh-274 displayed better inhibitory activity. psh-375 displayed the weakest inhibitory activity against JFH1 replication. The sequence of JFH1 displayed two mismatches at nucleotide positions 4 and 6 compared with HCV-375 (Fig. 1A), which may be the reason for lower activity (Fig. 3A and B). This could be due to the off-target effects, as reported earlier (15). Densitometric scanning displayed ~95% inhibition of HCV genome replication upon treatment with psh-274 (Fig. 3B). We also examined whether HCV growth in Huh-7.5 cells could be inhibited by stably expressing psh-53 or psh-274. Analysis by real-time RT-PCR (Fig. 3C) suggested that psh-274 inhibited HCV genotype 2a replication (88.5%) more strongly than inhibition by psh-53 (68.6%). We used psh-53 and psh-274 for subsequent study to identify a common sequence present in other HCV genotypes.


Figure 3
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  		FIG. 3. Antiviral activities of shRNAs against cell-culture-grown HCV genotype 2a (JFH1) in Huh-7 cells. (A) Semiquantitative RT-PCR analysis of HCV RNA after 72 h of transfection with vector expressing shRNA in Huh-7.5 cells harboring HCV JFH1 growth. The results with HCV 5' NTR and GAPDH RNA are shown. (B) The HCV 5' NTR/GAPDH ratios from three independent experiments were measured using Scion Image. (C) Real-time PCR analysis of HCV RNA extracted after 72 h of virus infection from Huh-7.5 cells stably expressing control, psh-53, or psh-274. The error bars represent the means from three independent experiments.

We recently established in vitro growth of HCV genotype 1a (H77) in immortalized human hepatocytes (IHH) (17). To further investigate whether shRNAs could inhibit the replication of H77 virus, we transfected IHH with shRNA and then infected them with a known number of FFU of HCV H77. After 72 h of infection, cells were collected to measure either the genome copy number or FFU. Real-time RT-PCR analysis revealed that psh-274 can inhibit virus replication (80%) at the RNA level more than a scrambled shRNA-transfected control (Fig. 4A). An immunofluorescence study also revealed that psh-274 can inhibit HCV genotype 1a (H77) titers (77.8%) more than the control (Fig. 4B and C). Similarly, psh-53 was examined for inhibition of HCV H77 replication. We did not observe knockdown of HCV RNA to a significant level, which corroborated with inhibition of HCV genotype 1a IRES translation (Fig. 2). Together, these results suggested that psh-274 effectively inhibits the replication and growth of two different HCV genotypes.


Figure 4
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  		FIG. 4. Antiviral activities of shRNAs against cell-culture-grown HCV genotype 1a (H77) in IHH. (A) Real-time PCR analysis of HCV RNA following transfection of psh-274 into IHH harboring HCV H77. (B) Suppression of HCV infectivity for naïve IHH transfected with psh-274 or negative control. After 48 h of transfection, IHH were infected with cell-culture-grown HCV H77, and the virus titer was determined by immunofluorescence using antibody to the NS4 protein. (C) A representative photomicrograph of H77 virus production in IHH by focus-forming assay in the presence of the negative control (a) and a psh-274 construct (b).

shRNA inhibits replication of HCV genotype 1b. We next examined the efficacies of shRNAs for inhibition of HCV genotype 1b replication. Since we do not have cell-culture-grown genotype 1b HCV, we evaluated the role of shRNA on the SR or FL replicon of HCV strain Con1. We transiently transfected psh-53, psh-274, or control shRNA into Huh-7 cells harboring SR or FL replicon. Replication of HCV RNA was examined by semiquantitative RT-PCR analysis from total RNA. We have normalized the expression of HCV RNA with the GAPDH housekeeping gene and presented the results as a PCR product/GAPDH ratio. In the subgenomic replicon, psh-53 and psh-274 inhibited replication of HCV genotype 1b to 40% and 70%, respectively, compared to the control (Fig. 5A and B). The expression of HCV NS5A was also determined by Western blot analysis after 48 h of transfection of shRNAs. Suppression of the NS5A protein was observed at higher levels following treatment with psh-274 (Fig. 5C and D). Similarly, psh-274 suppressed HCV replication more efficiently in Huh-7 cells harboring the FL replicon (Fig. 5E and F), although treatment with psh-53 displayed significant reduction of HCV replication.


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Figure 5
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  		FIG. 5. Antiviral activities of shRNAs against genotype 1b HCV. (A) Huh-7 cells (106) harboring subgenomic replicons were transfected with 0.4 µg shRNA expression vector. Forty-eight hours after transfection, RNA was extracted for analysis. Semiquantitative RT-PCR analysis of the HCV 5' NTR and GAPDH RNA in Huh-7 cells harboring subgenomic replicon 1b is shown. (B) The HCV 5' NTR/GAPDH ratios from three independent experiments were measured using Scion Image. (C) Western blot analysis of HCV NS5A and actin in SR cells. The expression level of the NS5A protein was determined by Western blot analysis using a specific antibody. The blot was reprobed with antibody to actin for comparison of equal loads. (D) Densitometric scanning of the autoradiograms from three independent experiments after normalization against actin. (E) Semiquantitative RT-PCR analysis of the HCV 5' NTR and GAPDH RNA in Huh-7 harboring FL replicon 1b cells. (F) The HCV 5' NTR/GAPDH ratios from three independent experiments were measured using Scion Image. (G) Antiviral activities of shRNAs on the HCV genotype 1b replicon. A histogram of relative percent HCV replicon colony growth after electroporation of shRNA(s) is shown. The results are presented as the mean of three independent experiments. The number of colonies obtained from control shRNA-treated cells was arbitrarily set as 100%. The error bars represent the means from three independent experiments.

To further determine the effects of the shRNAs on the HCV replicon, we transfected shRNAs in Huh-7 cells harboring full-length or subgenomic HCV replicons and treated with G418 for recovery of resistant colonies as described earlier (58). Our results suggested that psh-274 has a stronger effect for inhibition of HCV genotype 1b replication than psh-53 (Fig. 5G and Table 1). We observed many colonies upon introduction of psh-53. We pooled these colonies and transfected them with another round of psh-53. Each time, we observed an increase in cell colony numbers. However, after the introduction of psh-274 into the cells, a reduction in colony numbers was observed, further suggesting the effectiveness of the shRNAs. To examine whether selective pressure of psh-53 generated escape mutants in the HCV genome, sequence analysis of the shRNA-directed region (300 bp) was performed. Interestingly, sequence variation was not observed compared with the parental HCV sequence. Together, these results suggested that psh-274 is more effective in suppression of HCV genotype 1b replication.


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  		TABLE 1. Relative percent colony formation after single electroporation of vectors expressing shRNA into Huh-7 cells harboring subgenomic 1b and full-length 1b replicons


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DISCUSSION
 
In the present study, we have examined the effects of vector carrying shRNA in hepatic cell lines infected with HCV genotype 1a or 2a. We observed a strong inhibition of replication of both the HCV genotypes by psh-274. Drugs against HCV, such as interferon, are influenced by HCV genotypes. Although IFN-{alpha} with or without ribavirin therapy is more effective against genotype 2 than in genotype 1 patients (8, 12), psh-274 can suppress HCV replication of both genotypes to similar extents in cell culture. To our knowledge, this is the first report that siRNA can suppress the replication of cell-culture-grown HCV of two different genotypes. Recently, Wang et al. (55) reported that HCV core is an inhibitor of RNAi and that core suppresses the function of Dicer. shRNA cannot skip the dicing step, and our study revealed that vector encoding shRNA significantly inhibits HCV replication.

HCV replicates in the cytoplasm, and the virus RNA seems to be a suitable target for RNAi. IRESs possess stem-loop structures and provide easy access for siRNAs. Our results also support the idea that the vector carrying shRNAs against the 5' NTR is effective for suppression of the replication of HCV genotypes 1a, 1b, and 2a. Transfection of cells with vectors may lead to activation of the interferon signaling pathway. However, double-stranded RNA-activated protein kinase (PKR) activation following siRNA treatment was not observed in our previous experiments (44). Vector carrying shRNA is thought to be able to inhibit HCV replication longer than simple siRNA and to be more effective. The siRNAs directed against HCV have been shown to inhibit virus genome replication, although generation of resistant mutants, mainly from HCV replicon cells, has also been reported (3, 10, 19, 26, 39, 42, 58). Our study suggested the appearance of resistant colonies following treatment with psh-53, although sequence analysis did not reveal mutations around the siRNA-targeted region. Therefore, this targeted region may not be effective for efficient attack by the siRNA for inhibition of virus replication.

A major obstacle to achieving in vivo gene silencing by RNAi technology is targeted delivery to infected cells. siRNAs are negatively charged and do not readily cross mammalian cell membranes. Effective siRNA-mediated prevention and treatment of HCV infection requires efficient nontoxic means to deliver siRNAs to the liver. Optimizing systemic delivery requires stabilization of the siRNA, targeting of the effector to the correct tissue, and facilitation of cellular uptake. For the effector to target particular cell types, different ligands (35) and antibodies (49) are incorporated into/conjugated to the effector RNAi. Soutschek et al. (50) have reported that cholesterol-conjugated siRNAs can silence an endogenous gene (apoB) in the mouse liver. These findings hold promise for the development of a new class of therapeutics that harnesses the RNAi mechanism. The viral vectors may be useful for systemic delivery of RNAi effectors. Although viral vectors can provide an excellent tissue-specific tropism and transduction efficiency needed for clinical delivery, each type of viral vector brings with it a unique set of risks and safety concerns (52). The benefits of RNAi therapeutics are compelling, and both lentivirus and adeno-associated virus vectors are being considered for clinical delivery of shRNAs (7). We plan to study an appropriate delivery system to develop anti-HCV siRNA(s) for sustained inhibition of HCV replication and for potential therapeutic applications in future studies.


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ACKNOWLEDGMENTS
 
We thank R. Bartenschlager for providing HCV replicons, M. Kruger for the HCV IRES-luciferase vector, T. Wakita for the HCV JFH1 clone, and C. M. Rice for the HCV H77 clone and Huh-7.5 cells.

This work was supported by research grants AI45144 (R.B.R.) and CA85486 (R.R.) from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: 1402 S. Grand Blvd., St. Louis, MO 63104. Phone: (314) 577-8331. Fax: (314) 771-3816. E-mail: rayrb{at}slu.edu. Back

{triangledown} Published ahead of print on 1 November 2006. Back


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Journal of Virology, January 2007, p. 669-676, Vol. 81, No. 2
0022-538X/07/$08.00+0     doi:10.1128/JVI.01496-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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