Journal of Medical Virology 76:511–519 (2005)

Small Interfering RNA Effectively Inhibits ProteinExpression and Negative Strand RNA SynthesisFrom a Full-Length Hepatitis C Virus Clone

Ramesh Prabhu,1 Padmaja Vittal,1 Qinyan Yin,1 Erik Flemington,1 Robert Garry,2

William H. Robichaux,3 and Srikanta Dash1*1Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana2Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana3Department of Pathology, Thibodaux Regional Medical Center, Thibodaux, Louisiana

Hepatitis C virus (HCV) infection is usually treatedwith the combination of interferon and ribavirin,but only a small fraction of patients develop asustained remission. There is need for the devel-opment of specific molecular approaches for thetreatment of chronic HCV infection. We proposethat RNA interference is highly effective antiviralstrategy that offers great potential for the treat-ment of HCV infection. Three plasmid constructsexpressing small interfering RNAs (siRNAs) tar-geted to sequences encoding the structural gene(E2) and non-structural genes (NS3, NS5B) ofHCV1a genome were prepared. Antiviral proper-ties of siRNAs against the HCV1a strain werestudied in a transient replication model that in-volved the use of a transcription plasmid con-taining the full-length HCV genome and anadenovirus expressing T7 RNA polymerase. Wefound that siRNAs targeted to the E2, NS3 andNS5B regions of the HCV genome efficientlyinhibited expression of the HCV core and NS5Aprotein measured by Western blot analysis andimmunocytochemical staining. Intracytoplasmicimmunization of siRNAs in HCV-transfected cellsefficiently degraded genomic positive strand HCVRNA, as shown by ribonuclease protection assay(RPA). All three siRNAs efficiently inhibited syn-thesis of replicative negative strand HCV RNA inthe transfected cells. A control siRNA plasmidagainst a Epstein–Barr virus latency gene did notinhibit protein expression and negative strandHCV RNA. These results suggest that RNAi is aneffective and alternative approach that canbe used to inhibit HCV expression and replication.J. Med. Virol. 76:511–519, 2005.� 2005 Wiley-Liss, Inc.

KEY WORDS: hepatitis C virus replication;negative strand HCV RNA;small interfering RNA; T7

RNA polymerase; ribonucle-ase protection assay

INTRODUCTION

Hepatitis C virus (HCV) was a major cause of post-transfusion hepatitis in the United States, whichfrequently leads to chronic liver diseases [Choo et al.,1989; Alter, 1997]. There are an estimated four millionpeople infected with HCV in the United States with anoverall incidence in the world population of approxi-mately 3%. Long-term persistence of chronic virusinfection leads to the development of liver cirrhosisand hepatocellular carcinoma [Jeffers, 2000]. Cur-rently, standard therapy for chronic HCV infection, acombination of IFN-alpha and ribavirin, is ineffective inthemajority of chronic hepatitis patients [Hoofnagle andDiBisceglie, 1997;McHutchison et al., 1998;Moradpourand Blum, 1999]. Even with the newer therapeutic re-gimens (pegylated interferons plus ribavirin), completeclearance of HCV infection has not been accomplished.Therefore, development of a more specific antiviralstrategy would be beneficial to those patients not res-ponding to standard pharmacologic therapy.

HCV, a positive stranded RNA virus, belongs to theFlaviviridae family [Miller and Purcell, 1990]. TheHCVgenome is approximately 9,600 nucleotides in length.The organization of HCV genome begins with a highlyconserved 50 untranslated region (50UTR) followed by a

Grant sponsor: NIH; Grant number: CA89121; Grant sponsor:Tulane Cancer Center.

*Correspondence to: Srikanta Dash, Department of Pathologyand Laboratory Medicine, Tulane University Health SciencesCenter, 1430 Tulane Avenue, Sl-79, New Orleans, LA 70112.E-mail: sdash@tulane.edu

Accepted 23 March 2005

DOI 10.1002/jmv.20391

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2005 WILEY-LISS, INC.

single large open reading frame (ORF), and a 30 un-translated region (30UTR) [Houghton et al., 1991; Majorand Feinstone, 1997; Reed and Rice, 2000]. The 50 UTRof HCV genome functions as an internal ribosome entrysite (IRES) for translation of the viral genome [Brownet al., 1992; Bukh et al., 1992]. The HCV polyprotein ispost-translationally cleaved into at least 10 differentstructural and non-structural proteins by the combinedaction of cellular and viral enzymes [Hijikata et al.,1991;Grakoui et al., 1993; Lin et al., 1994]. Viral pro-teins produced in HCV-infected cells include: core,envelope proteins E1 and E2, p7, and non-structuralproteins, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.The structural proteins are important in viral assembly,while non-structural proteins are important in replica-tion of positive and negative strand HCV RNA ininfected cells. The 30 UTR of the HCV genome consistsof three distinct regions: a variable region, a poly (U-UC)sequence of variable length and a highly conservedstretch of 98 nucleotides, which represents the authen-tic 30 end of the viral genome [Kolykhalov et al., 1996;Tanaka et al., 1996]. The highly conserved 50 UTRand 30

UTR sequences are critical for virus replication [Friebeet al., 2001; Friebe and Bartenschlager, 2002; Yi andLemon, 2003].The availability of infectious clones representing the

major genotypes (1a and 1b) facilitated experimentalstudies on hepatitis C [Kolykhalov et al., 1997; Yanagiet al., 1997, 1998]. The development of sub-genomicHCV replicons in Huh-7 cells has provided a reliablein vitro model to test novel therapeutic approaches[Lohmann et al., 1999; Blight et al., 2000]. HCV re-plication has been inhibited by the use of ribozyme/anti-sense oligonucleotides, recombinant antibodies, pro-tease, helicase and polymerase inhibitors, and smallinterfering RNA (siRNA) [Wakita andWands, 1994; Altet al., 1995; Leiber et al., 1996; Welch et al., 1996;Oketani et al., 1999;Macejak et al., 2000; Sullivan et al.,2002;Kapadia etal., 2003;Randall et al., 2003;Senetal.,2003; Seo et al., 2003;Wilson et al., 2003]. Among these,the recently described RNAi-based antiviral strategyappears to be the most promising in inhibiting viralgene expression and replication. This approachhas beenwidely used to inhibit expression of a number of candi-date genes [Elbashir et al., 2001; Gitlin et al., 2002;Hannon, 2002]. RNAi is a common mechanism for post-transcriptional gene silencing, a phenomenon recentlyobserved in a variety of eukaryotic organisms. Theprocess of gene silencing in eukaryotic cells occurs viasmall double stranded RNA (dsRNA) fragments. ThesesiRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC), which in turn medi-ates the cleavage of its target mRNA [Hannon, 2002].Several recent studies have reported that this non-specific response can be generated in mammalian cellsby the use of synthetic (19–23)-nucleotide dsRNA.These dsRNA oligos are efficiently taken up by the cells,and the anti-sense strand of siRNAprevents a gene fromproducing the functional protein by specific degradationof the target mRNA. However, the half-life of synthetic

RNA duplexes is too short and in some circumstancesdoes not stay long enough for complete elimination ofvirus replication in an infected cell. To overcome theselimitations, vector-based intracellulardelivery of siRNAoffers an alternative strategy [Brummelkamp et al.,2002]. This approach allows continuous production ofsiRNAwithin the cells, and permits long-term silencingof viral gene expression. Therefore, we propose thatplasmid-based vector offers an alternative strategy forintracellular delivery of siRNA for complete inhibition ofviral gene expression and replication.

Recently, some investigators have shown that anRNAi-based approach can be utilized to inhibit replica-tion of a number of viruses, including HIV-1, cyto-megalovirus, gammaherpes virus, human rhinovirus,hepatitis delta virus, hepatitis B virus [Coburn andCullen,2002;Jacqueetal., 2002;Leeetal., 2002;Lietal.,2002; Lindenbach and Rice, 2002; Chang and Taylor,2003; Giladi et al., 2003; Jia and Sun, 2003; Klein et al.,2003; Konishi et al., 2003; Philipps et al., 2004;Wiebusch et al., 2004]. Other laboratories also havedemonstrated that siRNA inhibits the replication of sub-genomic HCV RNA [Kapadia et al., 2003; Randall et al.,2003; Seo et al., 2003;Wilson et al., 2003]. Here we haveshown that the efficient intracellular delivery of siRNAcould be achieved in a hepatic cell line by using aplasmid-based expression vector. Using the vector-mediated delivery approach, the antiviral effects ofsiRNA were examined using replication model estab-lished in our laboratory. It is demonstrated that vector-mediated intracytoplasmic delivery of siRNA targetedto E2, NS3, and NS5B efficiently inhibits expression ofcore and NS5A protein from a chimpanzee infectiousclone HCV1a. Intracellular production of siRNA fromplasmid-based vector effectively degraded genomicpositive strand HCV RNA transcripts and abolishednegative strand RNA synthesis within 48–72 hr aftertransfection.

MATERIALS AND METHODS

Cell line and Transcription plasmid

Huh-7 cell line was maintained in Dulbecco’s Modi-fied Media (D-MEM; Invitrogen Life Technologies,Carlsbad, CA) containing non-essential amino acids,sodium pyruvate, and 10% fetal bovine serum (GibcoBRL, Grand Island, NY). Chimpanzee infectious clonepCV-H77C (HCV1a) was obtained from Jens Bukh,National Institute ofHealth [Lietal., 2002;Yanagi etal.,1999]. We prepared HCV transcription plasmid usingthe chimpanzee infectious clone (pCV-H77C). Therecombinant plasmid, pNIH1a-Rz, contains a T7 pro-moter, full-length cDNA of HCV genome, followed by acDNA copy of autolytic ribozyme from antigenomicstrand of hepatitis delta virus and T7 transcriptionalterminator sequences at the 30 end of HCV cDNA. Wemade a control plasmid in which the Hind III and NdeIfragment (7,862–9,160) was removed, DNA was bluntended and relegated. This recombinant plasmid is calledpNIH1a-Rz-GDD mutant. This clone will not produce

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functional NS5B protein. Therefore, the RNA tran-scripts derived from this plasmid should not replicate.Detailed description of transcription plasmid andmethods has been described previously [Myung et al.,2001; Prabhu et al., 2004].

siRNA Delivery Vectors

Three different siRNA were selected targeted to E2(nts 2,426–2,444), NS3 (nts 4,498–4,517) and NS5B(nts 8,666–8,685) regions in the HCV1a genome (Fig. 1)by using web-based OligoEngine software. As control,siRNA targeted to Epstein–Barr virus (EBV) nuclearantigen was used. A commercially available plasmidvector called pSuper-retro (Oligo-Engine) was used forintracellular delivery of siRNA. The siRNA constructswere prepared at two different steps. In a first step, apair of (sense and anti-sense orientation) 64-nts oligocontaining 19nucleotides ofHCV in sense and antisenseorientations were selected, separated by a 9-nt spacersequence. Restriction enzymes Xho1 and Bgl II wereintroduced at the 50 end of sense and antisense64 nucleotide oligos for cloning. In the second step,the sense and antisense primers were annealed byincubation at 908C for 4 min then 728C for 10 min. Theannealed oligos were then cooled slowly to 108C andligated to the pSuper-retro vector usingXho1 andBgl IIrestriction sites. The recombinant clones containingsiRNA insert were selected by restriction enzyme diges-tion. Large-scale plasmid DNA isolation was performedusing themaxi kit (Qiagen, Sciences,MD). The presenceof siRNA sequences was confirmed by DNA sequenceanalysis.

Intracellular Delivery of siRNA

Efficient delivery of siRNA andHCV-cDNA to a hepa-tic cell line, Huh-7 cells, was achieved by co-transfectionstudies. Briefly, we first infected Huh-7 cells with areplicative defective adenovirus expressing T7 RNApolymerase. After 2 hr, cells co-transfected with 10 mg ofsiRNA plasmid and 10 mg full-length transcriptionplasmid pHCV-NIH1a-Rz using FuGENE 6 Reagent(Roche Molecular Biochemical’s, Indianapolis, IN). Theinhibitory effects of siRNA onHCV expression in Huh-7cells was determined by measuring core protein, posi-

tive and negative strand HCV RNA at 48–72 hr aftertransfection. Preliminary experiments were performedusing 10 mg of HCV transcription plasmid with fourdifferent concentrations of siRNA plasmid (10 mg, 5 mg,2.5 mg, and 1.25 mg) to determine the optimum concen-tration of siRNA plasmid required for maximum inhi-bitory effect.

Western Blot Analysis

Western blot analysis for HCV core andNS5A proteinwas performed using total protein lysate from trans-fected cells according to a standard protocol in ourlaboratory [Prabhu et al., 2004]. Briefly, transfectedcells were treated with 500 ml of lysis buffer containing150 mM sodium chloride, 50 mM Tris-HCl, 1% NP-40,0.5% deoxycholate, 0.1% SDS, and protease inhibitors(Protease Inhibitor Cocktail, Roche Biochemical’s).Fifty micrograms of the total cell lysate was separatedby 10% SDS–PAGE and transferred onto nitrocellulosemembranes (Amersham, Arlington Heights, IL). Themembranes were blocked with phosphate-bufferedsaline (PBS) containing 5% non-fat dried milk and0.1% Tween-20 for 1 hr at room temperature. The mem-brane was then incubated with monoclonal antibodyagainst core (AffinityBioreagents,Denver,CO) orNS5A(a gift fromChenLiu,University ofGainesville, Florida)at 1:100 dilution for 1 hr. The membrane was washedthree times with 0.1% Tween-20 in PBS. Following thisstep, the membranes were incubated with peroxidase-labeled secondary antibody (ECL Western blottinganalysis system, Amersham Biosciences, NJ) at a dilu-tion of 1:1,000 for 1 hr. After this step, membraneswere washed three times with PBS and developed usingECL Chemiluminescence Detection Kit (AmershamBiosciences,NJ).To verify that equal amounts of proteinwere loaded onto each lane of the SDS–PAGE, themembrane was incubated with monoclonal antibody tobeta-actin (Sigma Chemical Co., Saint Louis, MO).

Immunoperoxidase Staining

The extent of inhibition of viral protein expression inthe siRNA-transfected cells was examined by immunos-taining using the same monoclonal antibodies againstcore and NS5A. For this experiment, transfected Huh-7cells were immobilized onto glass slides by cytospinsusing a standard protocol. The cells were washed withPBS pH 7.4 twice, air-dried and fixed with chilledacetone for 10 min. The cells were permeabilized bytreatment with 0.05% saponin for 10 min at roomtemperature. Blocking was performed with 3% normalgoat serum (SigmaChemical Co., St. Louis, MO) dilutedin minimum essential medium for 30 min at roomtemperature.Blocking for endogenousbiotin-avidinwasperformed using blocking reagents from the kit (Avidin/Biotin Blocking Kit, Vector Laboratories, Inc., Burlin-game, CA) and blocking for endogenous peroxidase wasdone with 0.9% H2O2 for 30 min at room temperature.The cells were incubated with a monoclonal anti-core

Fig. 1. Schematic representation of location of siRNA targets in thefull-length HCV1a genome. The ribonucleotide sequence representingthe siRNA targets for E2 (2426–2444), NS3 (4498–4517), and NS5B(8666–8685) gene are shown. Each siRNA targeted is 19 nucleotidelong. Intracellular delivery and processing of siRNA was achievedusing pSuper-retro vector. The host cell RNA polymerase transcribesthe short hairpin RNA from pSuper-retro vector, which is processedintracellularly to generate functional siRNA.

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antibody or anti-NS5A antibody (1:100 dilution) over-night at 48C. The slide was then washed three timesand incubated with anti-mouse biotin conjugated anti-body for 1 hr at room temperature. The slides werethen washed and incubated for 30 min with Eliteavidin-biotin peroxidase complex (Vector Laboratories,Burlingame, CA). The slides were reacted with diami-nobenzidine for 10min. Counterstainingwas performedwith hematoxylin for 1 min. After dehydration, theslides were mounted with paramount and observedunder light microscopy. To examine the nonspecificinhibition of viral protein expression due to the siRNAtransfection, cells were stained with b-actin antibody(Sigma Chemical Co., St. Louis, MO).

Detection of Positive Strand HCV RNA

Huh-7 cells were co-transfected with siRNA plasmidand full-length HCV 1a plasmid using two-step trans-fection procedure described earlier. Detecting the levelsof positive strand HCV RNA by ribonuclease protectionassay (RPA) assessed anti-HCV effect of each siRNAplasmid. Cells were harvested at 0, 24, 48, and 72 hrafter transfection by treatment with trypsin-EDTA.Total RNA was isolated by the GITC method. RNAextracts were treated with DNase I (Roche Molecular,Mannheim, Germany) 5 U/mg of RNA for 1 hr at 378C toremove any residual plasmid DNA templates. RPA wasperformed to detect the presence of positive strandHCVRNA in transfected Huh-7 cells (Ambion Inc., Austin,TX).TheRNAprobe targets thehighly conserved50UTRof HCV genome. The plasmid pCR II-296 was linearizedwithXba I and used to prepare an anti-sense RNAprobeusing the SP6 RNA polymerase. For RPA assays,approximately 1� 106 cpm of the labeled anti-senseprobe was added to 25 mg of RNA sample and vacuumdried. Hybridization was performed in 10 ml of thehybridization buffer after denaturing for 3 min at 958Cand followed by overnight incubation at 458C. RNasedigestion was performed in 200 ml of RNase cocktail(1:100) (Ambion Inc., Austin, TX) in a buffer consistingof 10 mM Tris, pH 7.5, 5 mMEDTA, and 0.3 M NaCl for1 hr at 378C. Reactions were stopped by the additionof 2.5 ml of 25% SDS and 10 ml of proteinase K (10 mg/ml)at 378C for 15 min. Samples were extracted withphenol/chloroform and precipitated with ethanol. Thepellet was air dried and resuspended in 15 ml of gelloading buffer. The samples were then boiled for 3 minand separated on an 8% acrylamide/8 M urea gel. Thegel was dried and exposed to X-ray film (Kodak, X-OMAT-AR; Eastman Kodak Co., Rochester, NY).

Detection of Negative Strand HCV-RNA

HCV is a positive strand RNA virus it replicates bysynthesizing full-length negative strand RNA. Thepresence of replicative negative-strand HCV RNA inthe siRNA transfected cells was examined by RPAmethod using a sense riboprobe targeted to the 50UTR.The plasmid pCR II-296 was linearized with Hind III

and used to prepare a sense RNA probe using the T7RNA polymerase. Transfected cells were harvested atdifferent time intervals and totalRNA isolatedusing theGITC method. Approximately 25 mg of RNA extractswere digested with DNase I and then processed for RPAanalysis using the procedure described earlier. Huh-7cells transfectedwith aNS5Bmutant clonewere used asa control for negative strand HCV RNA detection to besure that it is not synthesized by T7 RNA polymerase.Since this is a DNA-based replication model, theefficiency of our DNase I digestion protocol was examin-ed by testing RNA extracts collected at 0 time aftertransfection.

RESULTS

siRNA Inhibits Viral Protein Expression

Three different siRNA targets were selected using thenucleotide sequence of HCV 1a chimpanzee infectiousclone (pCV-H77C). The location and nucleotide se-quences representing the RNAi target sites are shownin Figure 1. We used the pSuper-retro vector whichdirects the synthesis of siRNAs in Huh-7 cells aftertransfection. siRNAare transcribed intracellularly fromthe plasmidDNAby the host cell RNApolymerase usingthe polymerase III-H1-RNA gene promoter. The siRNAproduced from this vector is a small RNA transcriptlacking a polyadenosine tail, and has a well-definedstart of transcription and a termination signal consist-ing of five consecutive thymidines. The gene-specificinsert was designed so that it specifies a 19-nucleotidesequence derived from the HCV sequence, separated bya short spacer from the reverse complement of the same19-nucleotide sequence and five thymidines as atermination signal. The resulting transcript is predictedto fold back on itself to form a 19-base pair stem-loopstructure. The stem-loop structure transcript generatedfrom the pSuper-retro vector is cleaved inside the cell togenerate a functional siRNA. The specific inhibitoryeffect of siRNA against HCV was determined in a tran-sient transfection experiment using a chimpanzeeinfectious clone for HCV1a. We first examined effect ofthree siRNA targets by co-transfecting pSuper-retro-siRNA plasmids along with full-length transcriptionplasmid into Huh-7 cells. The success of siRNA effectagainst HCV was examining by measuring translationof viral core and NS5A proteins by Western blot anal-ysis. Results of this experiment are shown in Figure 2demonstrating that all three siRNAs effectively inhib-ited HCV protein expression. The inhibition of core orNS5A protein expression was not altered in cells trans-fected with control siRNA plasmid against EBV latencyantigen, EBNA1. Protein extracts derived from thesiRNA-transfected cellswere tested for the expression ofbeta-actin and found to be similar. The extent of inhibi-tion of HCV protein expression by siRNA in the trans-fected cells was determined by immuno-cytochemicalstaining. This method allows to measure directly, thenumber of HCV positive cells abolished core and NS5Aprotein expression due to siRNA. We used the same

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monoclonal antibodies (core and NS5A) used for Wes-tern analysis. siRNA transfected cells were harvested at48 hr and subjected to immunocytochemical staining.The results of this experiment are shown in Figure 3,

which indicate that intracytoplasmic delivery of siRNAwas a very efficient and inhibited core as well as NS5Aprotein in themajority of the cells expressingHCV. Thiseffect appears to be specific since the expression of coreand NS5A protein was not altered with an unrelatedsiRNA directed against EBNA1. Levels of beta-actinprotein expression in Huh-7 cells transfected with all ofthe siRNA plasmids were similar.

siRNA Mediated Degradationof Positive Strand HCV RNA

RNAi is the process of sequence specific gene silencingby dsRNA. This RNAi can be accomplished in culturedcells using the pSuper-retro vector, which generatefunctional siRNA of 19 base pairs size. This techno-logy selectively silences gene expression by degradingtarget mRNA. To investigate the therapeutic potentialof siRNAs, we examined the levels of positive strandHCV RNA in the siRNA-transfected cells were exam-ined by RPA. Huh-7 cells were co-transfected with full-length HCV transcription plasmid and individualpSuper-retro plasmid carrying siRNA targets. Theability of vector derived siRNA to degrade specificallypositive strand HCV RNA in the transfected cells wasdeterminedusingmultiple controls. Preliminary experi-ments were performed to determine the optimal time ofefficacy and the optimumamounts of plasmidDNA to betransfected for maximum inhibition of HCV in thissystem. It was found that use of 10 mg of HCV plasmidand 10 mg of pSuper-retro plasmid produced maximumeffect. To examine optimum time requirement for themaximum inhibitory effect of siRNA constructs againstHCV1a by measuring the levels of positive strand HCVRNA at 0, 24, 48, and 72 hr after transfection. Results ofthese analysesare summarized inFigure4A, suggestingthat siRNA mediated cleavage occurs in a time depen-dant manner. Maximum effect was seen in between 48and 72 hr after transfection. There was no significantdifference in levels of intracellular positive strand HCVRNA between 48 and 72 hr after transfection. All three

Fig. 2. Western blot analysis shows that all three siRNA constructsinhibit core, NS5A protein expression. Huh-7 cells were co-transfectedwith siRNA carrying pSuper-retro plasmid and full-length HCV1ausing the two-step transfection procedure. After 48hr, transfected cellswere isolated by the treatment with trypsin-EDTA. Cells were washedonce with PBS and protein lysates were prepared and electrophoresedon 10% SDS–PAGE gels. The proteins were transferred to nitrocellu-lose membranes, blocked and immunoreacted with a primary antibody(core and NS5A) at 1:100 dilution. After this step, the membrane waswashed and incubated with peroxidase labeled secondary antibody(1:1,000). In the final step, the membrane was developed by ECL-chemiluminescence method. Panel A shows the expression of HCVcore protein in the siRNA transfected cells and controls.PanelB showsthe level of b-actin protein in the same sets of transfected cells.PanelCshows the expression of HCV NS5A protein in the siRNA transfectedcells and controls. Panel D shows the level of b-actin protein in thesame sets of transfected cells. All the three siRNAs constructsspecifically inhibit HCV core and NS5A protein.

Fig. 3. Immunocytochemical staining shows that all three siRNAconstructs abolished the expression of core and NS5A protein. Huh-7cells were co-transfected with siRNA carrying pSuper-retro plasmidand full-length HCV1a using the two-step transfection procedure.After 48 hr, transfected cells were isolated by the treatment withtrypsin-EDTA. Cells were washed with PBS and immobilized onto aglass slide by cytospin method. For each sample, one set of slides was

stained for core and the other one stained for NS5A protein using amousemonoclonal antibody. Immunostaining forHCVcoreprotein andNS5A were performed using a standard protocol. Top panel A showsthe expression of core protein in the siRNA-transfected cells and con-trols. Bottom panel B shows the expression of NS5A protein in thesIRNA-transfected cells and controls. All three siRNA constructs abo-lishes core and NS5A protein in the majority of cells expressing HCV.

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siRNA targets were equally effective in the degrada-tion of genomic HCV RNA transcripts in Huh-7 cells(Fig. 4B). The inhibitory effect of siRNA is sequencespecific since HCV RNA levels was not altered in thecells transfected with siRNA for EBNA1. The levels ofGAPDH mRNA in the nucleic extracts of all experi-mental sampleswere comparable. These results suggestthat intracellular immunization with siRNA targeted toE2, NS3, and NS5B using pSuper-retro vector was veryeffective in the degradation of positive strandHCVRNAtranscribed in Huh-7 cells.

siRNA Inhibits NegativeStrand HCV RNA Synthesis

HCV is a positive strand RNA virus, which replicatesby synthesizing negative-strand RNA. Therefore, detec-tion of negative strand HCV RNA in this DNA-based

model is used to demonstrate virus replication. Speci-ficity of negative strand HCV RNA detection in thismodel was determined by performing experimentsby using NS5B deleted mutant transcription plasmid.This plasmid generate NS5B deleted HCV RNA tran-script. The full-length and NS5B deleted transcriptionplasmid constructs used in this study are illustrated inFigure 5A. Technically, Huh-7 cells transfectedwith theNS5A mutant plasmid should not replicate and nonegative strand HCV RNA can be found. We transfect-ed full-length and mutant transcription plasmid intoHuh-7 cells using a two-step transfection procedure[Prabhu et al., 2004]. Total RNA isolated from the trans-fected cells at 0, 72 hr after transfection and examinedfor the presence of negative strand HCV RNA by RPA.Results of this experiments are shown in Figure 5B.Upper panel shows that positive strand HCV RNAtranscripts were present in cells transfected with full-length as well as mutant plasmid. Lower panel showsnegative strand HCV RNA was detectable only in thefull-length transfected cells but no RNA was present incells transfected with the mutant clone. The results ofthese experiments indicate that negative strand HCVappearing in the transfected cells is due to replication offull-length genomic RNA transcripts. We then deter-mined whether transfection of plasmid that producesdifferent siRNAs could have abolished the replica-tion process and inhibit negative strand HCV RNA inHuh-7 cells. Result of RPA for negative strand HCVRNA detection in the siRNA transfected is shown inFigure 5C. It was determined that all three siRNAclones effectively inhibited the synthesis of negativestrand HCV RNA. The formation of negative strandHCV RNA was not inhibited in the case of an unrelated(EBNA1) siRNA nor in the vector control. As a loadingcontrol, RPA for GAPDH mRNA was performed andfound that they are equally present in all the samples(Fig. 5C). These results suggest that siRNA effectivelyinhibits negative strand HCV RNA, and virus replica-tion. In summary, we examined the antiviral effects ofsiRNA using a replication model that utilizes anunmodified, native full-length chimpanzee infectiousclone. The results of this study are in agreementwith allthe previous studies which suggest that siRNA is themost effective nucleic acid-based antiviral approachthat can be utilized to efficiently degrade HCV genomein the infected cells.

DISCUSSION

RNA interference represents a novel antiviral strat-egy with potential efficacy in the treatment of viraldiseases. A study was carried out to whether siRNAtargeted to an HCV1a infectious clone can inhibit geneexpression using a DNA-based transient transfectionmodel. The experimental model utilized in this studyinvolves a transcription plasmid containing the unmo-dified full-length HCV genome, and an adenoviruscarrying the gene for the T7 RNA polymerase. Highlevel expression of protein andmRNA froman infectious

Fig. 4. Ribonuclease protection assay (RPA) showingall three siRNAconstructs equally degraded positive strand HCV RNA in thetransfected Huh-7 cells. Panel A shows siRNA transfection resultedin the decrease HCV RNA levels in a time dependent manner. Huh-7cells were co-transfected with siRNA carrying pSuper-retro plasmidand full-length HCV1a using the two-step transfection procedure.After 0, 24, 48, and 72 hr, transfected cells were isolated by thetreatmentwith trypsin-EDTA.TotalRNAwas isolated and subjected toRPA for positive strand HCV RNA. The protected band intensity wasmeasured by Image gauge software and percentage of inhibition wasdetermined by comparing with untreated sample. Maximum antiviraleffect was seen between 48 and 72 hr after transfection. Panel Brepresentative RPA assay showing the levels of positive strand HCVRNA at 48 hr after siRNA plasmid transfection. Unrelated siRNAplasmid (ENBA1) did not inhibit positive strand HCV RNA levels.siRNA transfection procedure did not alter theGAPDHmRNA levels inall the samples and levels are comparable.

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HCV full-length cDNA clonewas studied by using a two-step transfection procedure.Wehave reported that highlevel expression ofHCVproteins, genomic andnegative-strand RNA can be detected in this system for HCV1aand HCV1b clone [Myung et al., 2001; Prabhu et al.,2004]. In this study, we examined whether siRNA couldinhibit specifically viral protein production and specifi-cally destroy positive-strand RNA in this transientexpression model using three different siRNAs targetedto E2, NS3, and NS5B regions of HCV1a (pCV-H77C)clone. A mammalian expression vector that directs thesynthesis of fully processed siRNA-like transcripts intransfectedHuh-7 cellswasused.HostRNApolymerasetranscribes siRNA from this plasmid using the poly-merase III-H1-RNA gene promoter. The transfectedplasmid probably enters the nucleus where RNA istranscribed from this plasmid by cellular RNA poly-merase. The primary transcript generated from thisplasmid is a short hairpin RNA with 19-base pair stemloop structure, which then enters the cytoplasm. Thistranscript is cleaved inside cytoplasm to generatefunctional siRNA, which is incorporated into the RISCalong with HCV mRNA. The success of RNAi antiviralstrategy against full-length HCV1a was studied bymeasuring core protein expression as well as positiveand negative-strand HCV RNA in co-transfection ex-periments. It was shown that all three siRNAs target-ed to either the E2, NS3, or NS5B regions of the HCVgenome effectively inhibited core protein expression.This inhibitory effect was not seen when an unrelatedsiRNA was expressed. The specific inhibition of HCVgene expressionwas due to degradation of genomicHCVRNA. This process was not due to non-specific or toxiceffects of siRNA, since GAPDHmRNA levels were com-parable. It was also determined that negative strandHCV RNA synthesis was inhibited by siRNAs targetedtoE2,NS3, andNS5Bgenes.The inhibitory effectwe seein our system does not appear to be due to activation ofPKR pathways, excluding the possibility of activation ofthe interferon system [Bridge et al., 2003; Sledz et al.,2003].

Several laboratories have demonstrated that RNAieffectively inhibits replication and expression of HCVusing replicon cell lines. Two different strategies wereused to deliver siRNA into cells: (i) vector-mediateddelivery of small hairpin RNA; (ii) direct delivery ofsiRNA. Studies performed by Kapadia et al. [2003] havetargeted NS3 and NS5B regions of replicon (HCV1b)using siRNAs synthesized in vitro. Studies reported byRandall et al. [2003] have used siRNA assembled usingchemically synthesizedRNAoligos to target the coreandNS4B regions of HCV sub-genomic replicons (HCV1b).Both studies have reported that siRNA delivery speci-fically inhibits HCV sub-genomic RNA replication andexpression. Wilson et al. [2003] have used DNA-basedmammalian vectors to produce siRNA intracellularly.They have shown that siRNAs targeted to the highlyconserved IRESofHCV, or toNS3, or toNS5Bsequenceseffectively inhibit HCV replication and expression.Studies performed by Sen et al. [2003] showed that

Fig. 5. All three siRNA constructs inhibit synthesis of negativestrandHCVRNA.Panel A shows the full-length HCV1a transcriptionplasmid and control NS5B-deleted mutant transcription plasmid usedin our experiments.PanelBRPAshowing the detection of positive andnegative strands HCV RNA. Huh-7 cells were transfected separatelywith full-length or mutant plasmid using the two-step transfectionprocedure. After 48 hr, transfected cells were isolated by the treatmentwith trypsin-EDTA. Total RNA was isolated and subjected to RPA forpositive- and negative-strand HCV RNA. Upper panel shows thedetection of positive strand HCV RNA. Both full-length and mutantclone transcribed HCV RNA. Lower panel shows the detection ofnegative strand HCV RNA in the transfected cells. Negative strandHCV RNA synthesis was detectable only in full-length transfectedHuh-7 cells. Panel C All three siRNA constructs inhibit negativestrand HCV RNA synthesis. Huh-7 cells were co-transfected withsiRNA carrying pSuper-retro and full-lengthHCV1a plasmid using thetwo-step transfection procedure. After 48 hr, transfected cells wereisolated by the treatment with trypsin-EDTA. Total RNA was isolatedand subjected to RPA for negative strand HCV RNA. All three siRNAconstructs inhibit negative strand HCV RNA in the transfected cells.The levels of negative strand HCV RNA was not affected by unrelatedsiRNA (ENBA1). The intracellular GAPDH mRNA levels in theexperimental and control samples were comparable.

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siRNA targeted to NS5A also inhibit expression of coreprotein. In this report, we examined the siRNA medi-ated antiviral effect against HCV using replicationmodel that was developed in our laboratory. Our modelis different than a replicon-basedmodel since it involvesunmodified chimpanzee infectious clones for HCV1a. Itwas demonstrated that RNAi-based antiviral strategycould be used to degrade full-length HCV genometranscripts in a transient transfection model. It wasalso shown that vector-mediated delivery of siRNA wasvery effective and degraded most of the genomic HCVRNA within 48–72 hr inhibited viral protein synthesisand abolished negative strand HCV RNA synthesis.Antiviral effect of siRNAs delivered by pSuper-retrovector was observed as early as 48 hr after transfection.In summary, results of this study in concert with thoselisted above suggest that RNAi efficiently inhibits HCV.This study also provides an experimental evidence thatshort term (48–72 hr) intracellular immunization withsiRNAcan efficiently degradeHCVgenome in the trans-fected cells.There are several reports demonstrating the applic-

ability of the RNAi-based antiviral approach againstother human viruses, including HIV-1 and hepatitisviruses [Coburn and Cullen, 2002; Jacque et al., 2002;Lee et al., 2002; Li et al., 2002; Lindenbach and Rice,2002; Chang and Taylor, 2003; Giladi et al., 2003; Jiaand Sun, 2003; Klein et al., 2003; Konishi et al., 2003;Philipps et al., 2004; Wiebusch et al., 2004]. Thesestudies indicate that the approach can be an effi-cient method for treating virus infection. We observedthat the antiviral effect of siRNA is sequence specific.For example, siRNAtargeted toHCV1amaynotproducesimilar results using another HCV1b clone (unpub-lished data). These findings are in agreementwith otherstudies in which single or couple nucleotide differencesin the siRNA design greatly diminish the antiviralefficacy.Considering this limitation, it is very importantto design siRNA against HCV using a very highly con-served sequence in order to optimize efficacy in inhibit-ing a majority of HCV strains. It is also important toidentify an optimal target of RNAi, which will showsuccessful antiviral activity against all the major HCVgenotypes. A potential problem that may arise in thistype of approach is the high mutation rate of HCV andthe generation of quasi species variants during chronicHCV infection. This problem can be overcome by design-ing siRNAs to the highly conserved region of differentHCV genotypes. The 50UTR region comprising the IRESis highly conserved among differentHCVgenotypes andmay be a good RNAi antiviral target. These studies arein progress in our laboratory. In regard to applicabilityof this antiviral approach in the treatment of chronichepatitis C, delivery of siRNA to appropriate cells ortissueswill be amajor challenge. Theuse of recombinantadenovirus and retroviruses represents an ideal way todeliver siRNA in to the liver as has been shown for thedelivery of siRNA against hepatitis B virus infection[Barton and Medzhitov, 2002; Xia et al., 2002; Giladiet al., 2003; Klein et al., 2003]. The results of our study

indicate development of viral vectors for intracellularimmunization with siRNA can be effective way to eradi-cate chronic HCV infection.

ACKNOWLEDGMENTS

This work was supported by NIH grant CA89121 andpartial support from the Tulane Cancer Center. Theauthors acknowledge Robert Purcell and Jens Bukh, attheNational Institute ofHealth for providing full-lengthchimpanzee infectious clone. The authors also acknowl-edge Donald Olivares for assistance in computer. ChenLiu, Department of Pathology, University of FloridaCollege of Medicine, Gainesville, FL, for providing theNS5A antibody. The authors thank Jeanne Frois forcritically reading this manuscript.

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