Combinations of siRNAs against La Autoantigen with NS5B or … · 2016. 9. 20. · Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition

Research ArticleCombinations of siRNAs against La Autoantigen with NS5B orhVAP-A Have Additive Effect on Inhibition of HCV Replication

Anirban Mandal1 Krishna Kumar Ganta1 and Binay Chaubey12

1Centre for Advance Studies Department of Botany University of Calcutta 35 Ballygunge Circular Road Kolkata 700019 India2Laboratory of Recombinant Vaccines Intercollegiate Faculty of Biotechnology UG and MUG Abrahama 58 Street80-307 Gdansk Poland

Correspondence should be addressed to Binay Chaubey bchaubeyhotmailcom

Received 28 March 2016 Revised 23 May 2016 Accepted 30 May 2016

Academic Editor Piero Luigi Almasio

Copyright copy 2016 Anirban Mandal et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hepatitis C virus is major cause of chronic liver diseases such as chronic hepatitis liver cirrhosis and hepatocellular carcinomaPresently available direct-acting antiviral drugs have improved success rate however high cost limits their utilization especiallyin developing countries like India In the present study we evaluated anti-HCV potential of several siRNAs targeted against theHCV RNA-dependent RNA polymerase NS5B and cellular factors La autoantigen PSMA7 and human VAMP-associated proteinto intercept different steps of viral life cycle The target genes were downregulated individually as well as in combinations and theirimpact on viral replication was evaluated Individual downregulation of La autoantigen PSMA7 hVAP-A and NS5B resulted ininhibition of HCV replication by about 672 507 39 and 52 respectively However antiviral effect was more pronouncedwhen multiple genes were downregulated simultaneously Combinations of siRNAs against La autoantigen with NS5B or hVAP-Aresulted in greater inhibition in HCV replication Our findings indicate that siRNA is a potential therapeutic tool for inhibitingHCV replication and simultaneously targeting multiple viral steps with the combination of siRNAs is more effective than silencinga single target

1 Introduction

HCV is an enveloped single-stranded positive sense RNAvirus belonging to Flaviviridae family that causes acute andchronic hepatitis in humans Persistent HCV infection oftenleads to cirrhosis and hepatocellular carcinoma [1] Currently130ndash150 million people are chronically infected with HCVand sim05 million people die every year due to HCV relatedliver diseases [2] World Health Organization (WHO) hasrecognized hepatitis C as a global health problem [3] Till2011 the mainstay of the HCV therapy has been a combina-tion of pegylated interferon alfa (PEG-IFN 120572) in combinationwith ribavirin [4 5]The addition of telaprevir and boceprevir(NS34Aprotease inhibitors) to this arsenal improved successrate but rapid development of drug resistance severe sideeffects and low spectrum activity proved to be barriers totreatment and therefore it is no longer used after interferon-free regimens became widely available [6ndash9] HCV treatment

has been further improved with the recent introduction ofHCV NS5B inhibitor sofosbuvir and NS5A inhibitor ledi-pasvir [10] In early 2016 second-generation NS34A proteaseinhibitor grazoprevir and second-generation NS5A inhibitorelbasvir were approved for the treatment of genotypes 1and 4 infected patients [11] However due to the poor rep-licative fidelity of HCV frequent emergences of drug-resist-ant strains are inevitable Furthermore high cost and geno-type dependency of these drugs often lead to treatment non-compliance [12 13] This necessitates a continuing effort todesign and develop additional agents to combat HCV

RNA interference (RNAi) is a gene silencing phenom-enon duringwhich small dsRNAmolecules induce sequence-specific degradation of homologous endogenous mRNA [1415] In general HCV is highly susceptible to RNAi becauseHCV genome is a (+) single-stranded RNA which functionsas both messenger RNA and template for replication in thecytoplasm of hepatocyte [16] Silencing of HCV RNA could

Hindawi Publishing CorporationHepatitis Research and TreatmentVolume 2016 Article ID 9671031 11 pageshttpdxdoiorg10115520169671031

2 Hepatitis Research and Treatment

stop its replication and propagation Therefore utilization ofthis technology holds tremendous potential as a therapeutictool against HCV Previous reports have shown that silencingof cellular or viral determinants could inhibit HCV entryand replication [17ndash23] siRNAs targeted against the core andNS4B regions specifically decreased viral load in a dose-dependent manner [24] siRNAs against NS5B and NS3regions inhibit HCV replication and protein expressionwithout any IFN response [25] Alternatively many studieshave also been focused on the cellular factors that directlyor indirectly interact with the different regions of the viralgenome and play a vital role in viral life cycle [26ndash29]However monotherapy with siRNA has led to the emergenceof viral escape mutants through point mutation within thesiRNA target regions [30 31] Therefore combinations ofsiRNAs comprising multiple viral andor cellular targetshave also been evaluated Few recent reports showed thatcombinations of siRNAs targeted against different cellular orviral factors have synergistic anti-HCV effects [32ndash36] Mostof these studies were restricted to either viral entry or the viralgenome However different stages in HCV life cycle suchas viral entry viral genome translation replication complexformation and replication and virion assembly and releaserepresent a broader spectrum of molecular events that mayserve as potential targets against HCVTherefore we proposecocktail of siRNAs targeting different viral and cellular factorswhichmay intercept various crucial steps in the viral life cycleand may enforce a strong synergistic antiviral effect

In the present study gene specific siRNAs were designedagainst cellular factors La autoantigen PSMA7 hVAP-A andviral factor HCV NS5B and screened for HCV inhibitionindividually as well as in combinationsThese cellular factorsand viral protein NS5B play crucial roles in HCV life cycle[37ndash45] Downregulation of viral replications was assessed bysemiquantitative RT-PCR western blotting and immunos-taining

2 Materials and Methods

21 Cell Culture Huh-75 cells (a kind gift from Dr V NPandey New Jersey Medical School USA) a highly permis-sive cell line for HCV replication were grown in DulbeccorsquosModified Eaglersquos Medium (Gibco USA) supplemented with10 FBS (Gibco USA) penicillin 100UmL and strepto-mycin 100 120583gmL (HiMedia India) 01mM nonessentialamino acids (Gibco USA) and 2mM glutamax-I (GibcoUSA) Cells were maintained in a humidified incubator at37∘C in 5 CO

2

22 HCV Production in Cell Culture For HCV culture pFL-J6JFH plasmid [24] (a kind gift from Dr V N Pandey NewJersey Medical School USA) encoding full-length chimericHCV genotype 2a was linearized with XbaI and subjectedto in vitro transcription using MEGAscript Kit (AmbionUSA) following manufacturerrsquos protocol Huh-75 cells weretransfected with 10 120583g transcript using Lipofectamine 2000transfection reagent (Invitrogen USA) using manufacturerrsquosprotocol and 6 h after transfection media were removedcells were washed with PBS and incubated in fresh media

The culture supernatant was collected after 72 h and passedthrough 045 120583m filter and stored at minus80∘C in small aliquots

23 Viral Load Quantification Viral RNAs were isolatedfrom cell culture supernatants using Trizol LS reagent(Ambion USA) and quantified by real-time quantitative RT-PCR using primer pair of HCV 51015840UTR forward 51015840-CCT-AATAGGGGCGACACTCCG-31015840 reverse 51015840-CCACAA-GGCCTTTCGCAACC-31015840 Assays were performed usingSybr green real-time PCRmaster mix reagent kit (InvitrogenUSA) and mastercycler ep realplex2 (Eppendorf Germany)according to the manufacturerrsquos instructions

24 RNA Isolation and RT-PCR Total RNA was extractedfrom cells using Trizol reagent (Ambion USA) followingmanufacturerrsquos guidelines RNA was then subjected to cDNAsynthesis using RevertAid reverse transcriptase (ThermoFisher Scientific USA) following manufacturerrsquos protocolin a thermocycler (Eppendorf Germany) In brief cDNAwas synthesized using 2120583g of total RNA in 20120583L reactionvolume using gene specific reverse primers and further 2120583Lof cDNA was used for PCR amplification of specific genesRespective primer sequences were La autoantigen forward51015840-GGATAGACTTCGTCAGAGGAGCA-31015840 reverse 51015840-CTGGTCTCCAGCACCATTTTCTG-31015840 hVAP-A forward51015840-GTGCTCCATCTGATTTACCCCA-31015840 reverse 51015840-TTC-CACAGGCTTGCTCAGTATT-31015840 PSMA7 forward 51015840-GCCATCACCGTCTTCTCGCC-31015840 reverse 51015840-CGTTGT-CATCCAAAGCACAG-31015840 HCV NS5B forward 51015840-ACA-TCAAGTCCGTGTGGAAGG-31015840 reverse 51015840-GCTCCC-ATTACCGCCTGAGG-31015840 and 120573-actin forward 51015840-GCG-GGAAATCGTGCGTGACATT-31015840 reverse 51015840-GATGGA-GTTGAAGGTAGTTTCGTG-31015840 The PCR products wereresolved by electrophoresis in 15 (wv) agarose gels andimages were captured by a Chemidoc XRS system (Bio-RadUSA) The band intensities were quantified using QuantityOne software

25 Design and Synthesis of siRNAs siRNAs were designedusing IDT online designing tool and were synthesized fromIDT USA Sequences of siRNAs were as follows La autoanti-gen (GenBank accession number NM 003142) sense 51015840-GGAACAAAGAAGUGACUU-31015840 antisense 51015840-ACUUCC-CAAGUCACUUCU-31015840 hVAP-A (GenBank accession num-ber NM 1944342) sense 51015840-GGUUUAGACAGGUUC-AAUUAGCUCA-31015840 antisense 51015840-UGAGCAAAUUGA-ACCUGUCUAAACCCG-31015840 PSMA7 (GenBank accessionnumberNM 0027923) sense 51015840-CCUCUUCCAAGUGGA-GUA-31015840 antisense 51015840-CUGCGCGUACUCCACUUG-31015840HCV NS5B (HCV genotype 2a strain) sense 51015840-CCCUCU-AUGACAUUACACA-31015840 antisense 51015840-UGUGUAAUG-UCAUAGAGGG-31015840 scrambled negative control sense 51015840-CUUCCUCUCUUUCUCUCCCUUGUGA-31015840 antisense51015840-UCACAAGGGAGAGAAAGAGAGGAAGGA-31015840

26 siRNA Transfection and HCV Infection Huh-75 cells(2 times 105 cellswell) grown overnight in 12-well plates weretransiently transfected with 50 nM for single and 100 nMfor combinations of siRNAs using Lipofectamine RNAiMAX

Hepatitis Research and Treatment 3

transfection reagent (Invitrogen USA) as per manufacturerrsquosprotocol After 48 h of transfection cells were infected withan equal amount of HCV (MOI 1 01) and incubated for 4 hCells were once again transfected with siRNAs to maintainthe gene silencing effect and incubated for another 48 hTotal RNA or protein was isolated and subjected to RT-PCR or western blotting respectively Scrambled siRNAnoncomplementary to any known sequence in the humanand HCV genome was included as a negative control

27 Western Blotting For western blotting cells were lysedwith RIPA buffer (50mM Tris-Cl 5mM EDTA 150mMNaCl 1 Triton X-100 1 sodium deoxycholate and 1sodium dodecyl sulfate) supplemented with 1mM proteaseinhibitor PMSF and then centrifuged at 13000 rpm for10min at 4∘C Supernatants were collected and total proteinwas quantified by Bradfordmethod Equal amount of protein(20120583g) from the control and transfected cells was subjectedto SDS-PAGE and then transferred to a PVDF membrane(GE healthcare USA) After blocking with 5 BSA blotswere incubated overnight at 4∘Cwith primary antibodies spe-cific for La autoantigen (ab75927 Abcam UK) HCV NS5B(sc-34040 Santa Cruz Biotechnology USA) and 120573-actin(NB100-56874 Novus Biologicals USA) Membranes wereincubated with corresponding secondary antibodies conju-gated with horseradish peroxidase (sc-2005 sc-2354 and sc-2004 Santa Cruz Biotechnology USA)The blots were devel-oped using ECL chemiluminescence detection kit (Bio-RadUSA) by a Chemidoc XRS system (Bio-Rad USA) and bandintensities were quantified using Quantity One software [46]

28 Indirect Immunofluorescence Huh-75 cells (5 times 104 cellswell) grown on 17mm poly-D-lysine coated glass coverslips(MP Biomedicals USA) were fixed with 4 paraformalde-hyde for 20min at room temperature Cells were washedtwice with PBS and then permeabilized with 02 TritonndashX100 for 10min followed by blocking with 3 BSA in PBSAfter blocking cells were incubated overnight at 4∘C withprimary antibody specific for HCV NS5B (sc-34040 SantaCruz Biotechnology USA) Cells were then incubated withFITC conjugated secondary antibody (sc-2356 Santa CruzBiotechnology USA) for one hour at room temperature andthen mounted the coverslips on glass slides with mountingmedia (50mM Tris pH 8 150mM NaCl and 1mgmL p-phenylenediamine in 50 glycerol) containing 400 nMDAPIand observed under a confocal laser scanning microscope(Olympus Fluoview FV1000) [47]

29 Cellular Viability Assay The cytotoxicity of siRNA-Lipo-fectamine RNAiMAX complex was assessed byMTT [3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide] as-say [48] Huh-75 cells grown overnight in 96-well plate(20000 cellswell) were transfected with individual or com-bination of siRNAs and incubated for 72 h After incubation20120583L of 5mgmL MTT reagent (HiMedia LaboratoriesIndia) was added to each well and incubated for 4 h at 37∘CThe formazan crystals were dissolved in acidified isopropanolsolution and absorbance was taken in a microplate reader

(iMark Microplate Reader Bio-Rad USA) at 595 nm with areference wavelength of 650 nm

210 Statistical Analysis Data are presented asmeanplusmn stand-ard deviation (119899 = 3) and one-way ANOVA test was per-formed using SPSS software (version 22000) 119875 value lt 005versus control was considered statistically significant

3 Result

31 Downregulation of Cellular and Viral Genes SeverelyInhibits HCV Replication HCV NS5B an RNA-dependentRNA polymerase synthesizes viral genome through an inter-mediate negative RNA strandHence it is an important targetto inhibit the viral replication Among the host cell factorsLa autoantigen an RNA-binding protein plays an importantrole in viral translation and replication and protects viralRNA from rapid degradation [37ndash39] Expression of Laautoantigen is induced by HCV infection and it activatestelomerase activity in Huh-75 cells Therefore it could beimportant to suppress La autoantigen protein not only forthe inhibition of HCV but also for reducing the oncogenicpotential of hepatocytes infected with HCV [49] PSMA7 120572-subunits of 20S proteasome regulates HCV-IRES (internalribosome entry site) mediated cap-independent viral trans-lation a phenomenon essential for HCV replication Fur-thermore inhibition of the 20S proteasome by proteasomeinhibitor exerts a dose-dependent inhibition on HCV-IRESactivity [40] Another host cell factor hVAP-A a SNARE-likevesicle transport protein is present in the ER and ERGolgiintermediate compartment (ERGIC) and plays a general rolein vesicle traffic between the ER andGolgi compartment [50]It makes a bridge between viral proteins NS5B and NS5A bymaking its N-terminus interact with NS5B and C-terminusinteract with NS5A and mediates the formation of HCVreplication complex on a lipid raft [41] We downregulatedthese viral and host genes using single and combinatorialRNAi-based approach in Huh-75 cells infected with HCVand evaluated its effect on viral replication

We found that siRNA targeted against La autoantigenmRNA reduced its expression by 965 in HCV-infectedHuh-75 cells and consequently HCV replication was alsoreduced by 672 (Figures 1(a) and 1(b)) indicating a directimpact of downregulation of La autoantigen on viral replica-tion Similarly there was 39 reduction in viral replicationupon 501 downregulation of hVAP-A (Figures 1(c) and1(d)) and 65 downregulation of PSMA7 resulted in 507lower level of HCV level (Figures 1(e) and 1(f)) Theseobservations clearly indicate a reduction in HCV replicationin response to downregulation of selected cellular genesinvolved in viral life cycle Similarly HCV propagation wasalso inhibited by 52 compared to control upon treatmentwith siRNA targeted against NS5B region of HCV genome(Figures 1(g) and 1(h))

32 Effects of Combinatorial Gene Silencing on HCV Replica-tion In order to determine the effect of simultaneous down-regulation of multiple genes on HCV replication Huh-75


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Figure 1 Downregulation of La autoantigen hVAP-A PSMA7 andNS5B leads to reduction in viral replication overnight grownHuh-75 cellswere transfected with La autoantigen hVAP-A and PSMA7 siRNAs for 48 h Cells were then infected with the virus and again transfectedwith same siRNAs After incubation total RNA was isolated and subjected to RT-PCR NS5B siRNA was transfected once only after cellswere infected (a) (c) (e) and (g) represent RT-PCR results for target genes and NS5B gene upon treatment with La autoantigen hVAP-APSMA7 and NS5B siRNAs respectively Densitometric analysis of (a) (c) (e) and (g) images is expressed in percentage in (b) (d) (f) and(h) respectively The data represent mean plusmn standard deviation lowast119875 value lt 005 versus control considered statistically significant


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Figure 2 Simultaneous downregulation of multiple genes more effectively inhibits HCV replication Huh-75 cells in a similar experimentalsetup as mentioned in Figure 1 were transfected with following combinations of siRNAs La autoantigen + NS5B La autoantigen + hVAP-ALa autoantigen + PSMA7 NS5B + hVAP-A NS5B + PSMA7 and PSMA7 + hVAP-A Downregulation of viral replication were determined byRT-PCR as shown in (a) (b) represents densitometry analysis of HCVNS5B downregulationThe data represent mean plusmn standard deviationlowast

119875 value lt 005 versus control was considered statistically significant

Table 1 Effect of siRNAs used individually and in combination on HCV replication Only combination of La autoantigen with hVAP-A orHCV NS5B shows syndergistic effect on HCV inhibition Other combinations of siRNAs could not improve the overall level of inhibition ofHCV

Target gene (siRNAposition)

Target genesilencing ()

Reduction of HCV aftersingle gene silencing ()

Reduction of HCV after multiple gene silencing ()(+) La autoantigen (+) hVAP-A (+) PSMA7 (+) NS5B

La autoantigen(1064ndash1081) 965 672 mdash mdash mdash mdash

hVAP-A (5929ndash5953) 501 390 822uarr mdash mdash mdashPSMA7(199ndash217) 650 507 699uarr 490darr mdash mdash

HCV NS5B(531ndash549) 520 520 856uarr 533darr 438darr mdash

cells were transfected with the following combinations ofsiRNAs La autoantigen + NS5B La autoantigen + hVAP-ALa autoantigen + PSMA7 NS5B + PSMA7 NS5B + hVAP-Aand PSMA7 + hVAP-A Viral replication was reduced by856 822 699 438 533 and 490 with combi-nations of La autoantigen + NS5B La autoantigen + hVAP-ALa autoantigen + PSMA7 NS5B + PSMA7 NS5B + hVAP-Aand PSMA7 + hVAP-A siRNAs respectively (Figure 2) Thecombination of La autoantigen + hVAP-A siRNAs reducedthe HCV replication by 822 while it was 672 and 39respectively when the geneswere downregulated individually(Figure 1) Similarly the combination of La autoantigen +NS5B siRNAs also inhibited the HCV replication by 856whereas the individual downregulation of La autoantigenand NS5B genes demonstrated 672 and 52 inhibition ofviral replication respectively (Figure 1)These results indicatesynergistic anti-HCV effects due to simultaneous downreg-ulation of La autoantigen + hVAP-A or La autoantigen +NS5B on viral replicationHowever similar synergistic effectson viral replication were not observed after simultaneous

downregulation of La autoantigen + PSMA7 NS5B + PSMA7NS5B+hVAP-A or PSMA7+hVAP-A siRNAsThis indicatesthat multiplexing any combination of siRNAs is not sufficientto have a synergistic antiviral effect (Table 1) Their roles inviral biology are crucial to be effective antiviral targets

33 Combinatorial Effect of La Autoantigen with NS5B siRNAon HCV Protein Expression We further checked the inhib-itory effect of downregulation of La autoantigen and HCVNS5B on the expression of the viral protein by western blotanalysis We found that combination of La autoantigen +NS5B siRNA demonstrated 95 reduction in NS5B proteinlevels compared with 73 and 545 respectively whenthe genes were downregulated individually (Figures 3(a) and3(b)) Similar results were obtained from immunostaining ofNS5B protein As shown in Figure 3(c) downregulation of theviral protein NS5B was observed and it was found that cellstreated with combination of siRNAs showed a cumulativeeffect on HCV inhibition compared to individual siRNAstreated cells Cytotoxicity for all the siRNAs was measured at


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Figure 3 Analysis of NS5B protein expression after transfecting NS5B and La autoantigen siRNAs HCV-infected Huh-75 cells were treatedwith siRNAs against La autoantigen and NS5B as mentioned above After 48 h total cellular protein was isolated and subjected to westernblot analysis (a) indicates downregulation of La autoantigen (upper gel) and NS5B (middle gel) and 120573-actin (lower gel) (b) representsdensitometric analysis of La autoantigen andHCVNS5B expressed in percentage Immunofluorescence ofNS5B from single and combinationof siRNAs transfected cells as represented in (c) where row (A) indicates cells in bright field and row (B) is the merging of DAPI and FITCstained cells

50 nM (for single siRNAs) and 100 nM (for combinations ofsiRNAs) and no significant effect was observed on cell growthand viability upon treatment with test or control siRNAs(Figure 4)

4 Discussion

The HCV life cycle is broadly divided into following majorsteps entry and uncoating translation replication assemblyand release of virion particles as depicted in Figure 5 [51 52]Each of these steps involves interactions with the host cellfactors which are crucial to establish infection and maintainthe viral life cycle [53 54] Interventions of these interactions

can severely impair the viral life cycle Cellular factors hVAP-A La autoantigen and PSMA7 have multiple roles duringdistinct stages of HCV life cycle and are potential targets forhost targeting antivirals (HTA) as depicted in the schematicmodel of HCV life cycle in Figure 5 [37ndash43 55 56]

During uncoating the internalized viral nucleocapsidfuses with endosomalmembranes and release its genome intothe cytosol [57] Alteration in cholesterol composition in theendosomal compartment can inhibit this step hVAP-A withoxysterol-binding protein (OSBP) is involved in cholesterolhomeostasis which in turn helps endocytic release of virioninto the cytosol [42 43]Therefore downregulation of hVAP-A inhibits the fusion of clathrin coated virion with endo-somal membranes thereby inhibiting then invasion step In


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Figure 4 Cytotoxicity assay of single and different combinationsof siRNAs Huh-75 cells grown for 24 h in a 96-well plate weretransiently transfected with single or different combination ofsiRNAs and incubated for 72 h Cell viabilitywas then determined byMTT assay Absorbance was taken at 595 nm by a microplate readerand readings were plotted as percentage The data represent mean plusmnstandard deviation lowast119875 value lt 005 versus control was consideredstatistically significant

the present study upon 501 downregulation of hVAP-AHCV was also inhibited by 39 (Figures 1(c) and 1(d))However upon more efficient downregulation of hVAP-Asim60 reduction in HCV replication has also been observed[58] Short hairpin against hVAP-A expressed through anadenoviral vector effectively inhibits the HCV replication[59] Variations in the level of downregulation of target geneand HCV could be attributed to the silencing efficiency ofsiRNA as well as different HCV strains

Following entry into the cytosol the viral RNA becomeshighly susceptible to siRNA-mediated degradation A num-ber of groups have shown that NS5B is an important factorfor HCV replication and an attractive antiviral drug target[60 61] We designed siRNA targeting the active site ofHCVNS5B a highly conserved region among different HCVgenotypes As expected upon downregulation of NS5B genesignificant decrease in viral RNA and protein expression wasobserved (Figures 1(g) 1(h) and 3) Several groups havedirected siRNAs against different regions of viral genomeincluding NS5B and demonstrated different levels of inhi-bition of viral replication and protein expression [62 63]DuringHCV life cycle several cellular factors bind to the viralRNA at different regions in order to help with translationand replication or to protect viral RNA [26 37ndash39 64] Thecellular factor La autoantigen binds to the U-rich region ofHCV 31015840UTR and shield viral RNA from degradation [39]Therefore lower expression of La autoantigenmakes the viralRNA vulnerable to cellular nucleases

HCV uses host translationmachinery and several cellularfactors to synthesize polyprotein precursor La autoantigenalso binds to the initiatorAUGcodon of 51015840UTRand facilitatesefficient HCV-IRES mediated translation [38] In our studywhen La autoantigen was downregulated by 965 conse-quently reduction of 672 in HCV level was also observed(Figures 1(a) and 1(b)) In few studies La autoantigen was

used as a therapeutic target for successful inhibition ofHCV although the extent of inhibition of target gene andHCV varied among different studies due to different siRNAtarget regions experimental setup or efficiency of siRNAdelivery [58 59] PSMA7 has been implicated for its role inIRES mediated translation of HCV [40] We downregulatedPSMA7 by 65 and as a result of this viral replicationwas also reduced by 507 (Figures 1(e) and 1(f)) About50 inhibition of HCV replication has also been observedwhen PSMA7 expression was downregulated by the siRNAdelivered through retroviral vector [36]

HCVmakes a replication complexwithin cholesterol-richlipid raft membrane using viral nonstructural proteins NS3NS4A NS4B NS5A and NS5B and cellular protein hVAP-A The cellular factor makes a bridge between viral proteinsNS5B and NS5A [41] Therefore due to downregulation ofhVAP-A the association of HCV nonstructural proteins onlipid raftmembrane is severely disturbed which consequentlyhampers viral replication Several cellular factors and HCVnonstructural proteins are involved during viral replication[65 66] La autoantigen and PSMA7 have been involved atthis stage and hence downregulation of these genes severelyaffected the viral replication (Figures 1(a) 1(b) 1(e) and 1(f))Therefore these cellular proteins are the important anti-HCVtarget The overall schematic model of HCV life cycle andsiRNA-mediated inhibition at different steps has been shownin Figure 5

It is also important to confirm that silencing of thesecellular genes has no lethal effects on the host La autoantigeninfluences RNApolymerase III transcription in vitro [67] butdepletion does not hamper the transcription rate synthesizedby class III RNA polymerase although reduction in ribonu-cleoprotein (RNP) formation has been observed [68] hVAP-A is predominantly involved in vesicle trafficking betweenmembrane compartments including endoplasmic reticulumand Golgi complex [69 70] and reduction of hVAP-A mayaffect the vesicle transport functions of cells siRNA-mediatedinhibition of PSMA7 reduced vesicular stomatitis viral repli-cation through enhanced production of virus-induced type IIFN [71] still it may be speculated that downregulation mayhamper the intracellular proteolysis However MTT assay oncells transfected with all the siRNAs individually or in com-bination showed no significant changes in cellular viabilitycompared with the nontransfected controls (Figure 4)

When multiple genes were downregulated and morethan one viral step was challenged inhibition of HCV wasmore pronounced (Table 1) Viral replication was reduced by856 and 822 particularly with combinations of siRNAsagainst La autoantigen with NS5B or hVAP-A respectively ascompared to other combinations of siRNAs (Figure 2) Theseobservations confirmed that downregulation of La autoanti-gen alongwithNS5B or hVAP-A facilitates inhibition ofHCVreplication cooperatively These results were also confirmedby western blotting and immunostaining (Figure 3(c)) Thecombination of La autoantigen with NS5B simultaneouslytargets the viral RNA translation and replication steps Sim-ilarly when La autoantigen and hVAP-Awere simultaneouslydownregulated viral uncoating translation replication com-plex formation replication and viral RNAwere concurrently


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LDLR

Figure 5 Schematic model of HCV life cycle and siRNA-mediated inhibition of different steps After receptor-mediated entry hostprotein hVAP-A interacts in the uncoating and replication complex formation step within the membranous web network region Thereforedownregulating hVAP-A with siRNA severely affects the viral replication siRNA against La autoantigen and PSMA7 simultaneously hinderviral translation and replication step La autoantigen also binds to the 31015840UTR of viral RNA and protects it from cellular nuclease hencedownregulation of La autoantigenmakes the viral RNA susceptible to nuclease NS5B siRNAwas directed against the NS5B conserved regionof viral RNA

affected A few recent studies exhibited the effect of combi-natorial RNAi on HCV replication Enhanced reduction inHCV entrywas noted after downregulation of entry receptorsCD81 along with LDLR or SR-BI [33] In another study dualsiRNAs directed against HCV 51015840UTR rapidly inhibit viralreplication and also minimized escape mutants [34] Adeno-associated viral (AAV) vector expressed multiple short hair-pin oligos against HCV NS5B and HCV 51015840UTR has shownlong lasting HCV inhibition in a nonhuman primate model[35] siRNAs against HCV envelope protein E2 along withentry receptor LDLR or CD81 showed an enhanced reductionin HCV entry [32] However these studies were targeted toeither viral entry or the viral genome Virus is often ableto escape the single inhibitory blocks created by antiviralsand capable of perpetuating its life cycle Therefore targetingmultiple steps at the same time is more realistic approachin order to inhibit HCV more effectively and prevent escapemutants It is pertinent to mention that designing of siRNAand selection of viral and cellar targets are the key to thesuccess of siRNA based inhibition of HCV Application ofsiRNA has tremendous possibilities because it not just allowsdownregulating host and viral factors simultaneously but alsoallows tremendous flexibility in designing of siRNAs withdynamic changes in the viral sequences

5 Conclusion

Currently approved anti-HCV drugs are mainly genotypedependent and because of error prone replication of HCVemergence of quasi-species are inevitable Targeting con-served regions among different genotypes can reduce emer-gence of resistant strains and also inhibit virus in a pan-genotypic manner Targeting host cell factor is a novelapproach particularly for patients who fail to respond tocurrent direct antiviral agents (DAA) treatment or developdrug resistance As siRNAs work at mRNA level the hostDNA remains unaffected and emergence of mutational resis-tance to the host or the virus does not arise thereforecreating a possibility of pan-genotypic anti-HCV treatmentSeveral studies showed that siRNAs targeted against differentregions (stem-loops II III and IV) of 51015840UTR or cellularfactor CD81 inhibit HCV infection in genotype-independentmanner [72ndash74] Therefore siRNA has tremendous potentialas a therapeutic tool against HCV We showed simultaneousinhibition of La autoantigen with NS5B or hVAP-A is moreeffective than silencing a single gene to inhibit the viralreplication Therefore a cocktail of siRNAs directed againstdifferent crucial steps in viral life cycle is likely to be a moreeffective approach in the treatment of chronic hepatitis C


Ethical Approval

The authors state that present study did not involve anyhuman or animal experimental investigations

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the Department of Biotech-nology New Delhi India for financial support vide Sanctionno BTPR9284Med29612007

References

[1] T Kanda O Yokosuka and M Omata ldquoHepatitis C virus andhepatocellular carcinomardquo Biology vol 2 no 1 pp 304ndash3162013

[2] World Health Organization ldquoHepatitis Crdquo Fact Sheet Number164 World Health Organization Geneva Switzerland 2015httpwwwwhointmediacentrefactsheetsfs164en

[3] World Health OrganizationHepatitis C World Health Organi-zation Geneva Switzerland 2003 httpwwwwhointcsrdis-easehepatitisHepcpdf

[4] J J Feld and JHHoofnagle ldquoMechanismof action of interferonand ribavirin in treatment of hepatitis Crdquo Nature vol 436 no7053 pp 967ndash972 2005

[5] R T Chung M Gale Jr S J Polyak S M Lemon T JLiang and J H Hoofnagle ldquoMechanisms of action of interferonand ribavirin in chronic hepatitis C summary of a workshoprdquoHepatology vol 47 no 1 pp 306ndash320 2008

[6] K J Wilby N Partovi J-A E Ford E D Greanya and E MYoshida ldquoReview of boceprevir and telaprevir for the treatmentof chronic hepatitis Crdquo Canadian Journal of Gastroenterologyvol 26 no 4 pp 205ndash210 2012

[7] A A Butt and F Kanwal ldquoBoceprevir and telaprevir in themanagement of hepatitis C virus-infected patientsrdquo ClinicalInfectious Diseases vol 54 no 1 pp 96ndash104 2012

[8] HIVandHepatitiscom ldquoVertex to Discontinue Sale of Telapre-vir (Incivek) for Hepatitis Crdquo San Francisco Cailf USA 2014httpwwwhivandhepatitiscomhcv-treatmentapproved-hcv-drugs4808-vertex-to-discontinue-sale-of-telaprevir-incivek-for-hepatitis-c

[9] HIVandHepatitiscom Merck Plans to Discontinue Boceprevirfor Hepatitis C by December 2015 HIVandHepatitiscom SanFrancisco Calif USA 2015 httpwwwhivandhepatitiscomhcv-treatmentapproved-hcv-drugs5021-merck-plans-to-dis-continue-boceprevir-for-hepatitis-c-by-december-2015

[10] N Afdhal K R Reddy D R Nelson et al ldquoLedipasvir andsofosbuvir for previously treated HCV genotype 1 infectionrdquoTheNew England Journal of Medicine vol 370 no 16 pp 1483ndash1493 2014

[11] M P Manns J M Vierling B R Bacon et al ldquoThe com-bination of MK-5172 peginterferon and ribavirin is effectivein treatment-naive patients with hepatitis c virus genotype 1infection without cirrhosisrdquoGastroenterology vol 147 no 2 pp366ndash376e6 2014

[12] G Diana and H Gregory ldquoLedipasvirsofosbuvir (Harvoni)improving options for hepatitis C virus infectionrdquo P amp T vol40 no 4 pp 256ndash276 2015

[13] FDA News Release ldquoFDA approves Zepatier for treatment ofchronic hepatitis C genotypes 1 and 4rdquo US Food and DrugAdministration Silver Spring Md USA 2016 httpwwwfdagovNewsEventsNewsroomPressAnnouncementsucm-483828htm

[14] A Reynolds D Leake Q Boese S ScaringeW SMarshall andA Khvorova ldquoRational siRNA design for RNA interferencerdquoNature Biotechnology vol 22 no 3 pp 326ndash330 2004

[15] G J Hannon ldquoRNA interferencerdquoNature vol 418 no 6894 pp244ndash251 2002

[16] M Motavaf S Safari and S M Alavian ldquoTherapeutic potentialof RNA interference a new molecular approach to antiviraltreatment for hepatitis Crdquo Journal of Viral Hepatitis vol 19 no11 pp 757ndash765 2012

[17] J A Wilson S Jayasena A Khvorova et al ldquoRNA interferenceblocks gene expression and RNA synthesis from hepatitis Creplicons propagated in human liver cellsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 5 pp 2783ndash2788 2003

[18] J-S Moon S-H Lee E-J Kim et al ldquoInhibition of hepatitisC virus in mice by a small interfering RNA targeting a highlyconserved sequence in viral IRES pseudoknotrdquo PLoS ONE vol11 no 1 Article ID e0146710 2016

[19] T Kanda R Steele R Ray and R B Ray ldquoSmall interferingRNA targeted to hepatitis C virus 5rsquo nontranslated region exertspotent antiviral effectrdquo Journal of Virology vol 81 no 2 pp669ndash676 2007

[20] S Khaliq S Jahan B Ijaz W Ahmad S Asad and S HassanldquoInhibition of hepatitis C virus genotype 3a by siRNAs targetingenvelope genesrdquo Archives of Virology vol 156 no 3 pp 433ndash442 2011

[21] H Ding Y Liu Z-Q Bian et al ldquoStably silencing of CD81expression by small interfering RNAs targeting 31015840-NTR inhibitsHCV infectionrdquo Hepatitis Monthly vol 8 no 4 pp 267ndash2742008

[22] G Randall M Panis J D Cooper et al ldquoCellular cofactorsaffecting hepatitis C virus infection and replicationrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 31 pp 12884ndash12889 2007

[23] S Jahan B Samreen S Khaliq et al ldquoHCV entry receptors aspotential targets for siRNA-based inhibition of HCVrdquo GeneticVaccines and Therapy vol 9 article 15 2011

[24] G Randall A Grakoui and C M Rice ldquoClearance of repli-cating hepatitis C virus replicon RNAs in cell culture by smallinterfering RNAsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 1 pp 235ndash240 2003

[25] Y Takigawa M Nagano-Fujii L Deng et al ldquoSuppression ofhepatitis C virus replicon by RNA interference directed againstthe NS3 and NS5B regions of the viral genomerdquo Microbiologyand Immunology vol 48 no 8 pp 591ndash598 2004

[26] D Harris Z Zhang B Chaubey and V N Pandey ldquoIdentifi-cation of cellular factors associated with the 31015840-nontranslatedregion of the hepatitis C virus genomerdquoMolecular and CellularProteomics vol 5 no 6 pp 1006ndash1018 2006

[27] D M Jones P Domingues P Targett-Adams and J McLauch-lan ldquoComparison of U2OS and Huh-7 cells for identifying hostfactors that affect hepatitis C virus RNA replicationrdquo Journal ofGeneral Virology vol 91 no 9 pp 2238ndash2248 2010


[28] T I Ng H Mo T Pilot-Matias et al ldquoIdentification of hostgenes involved in hepatitis C virus replication by small interfer-ing RNA technologyrdquo Hepatology vol 45 no 6 pp 1413ndash14212007

[29] A W Tai Y Benita L F Peng et al ldquoA functional genomicscreen identifies cellular cofactors of hepatitis C virus replica-tionrdquo Cell Host amp Microbe vol 5 no 3 pp 298ndash307 2009

[30] M Konishi C H Wu M Kaito et al ldquosiRNA-resistance intreated HCV replicon cells is correlated with the developmentof specific HCV mutationsrdquo Journal of Viral Hepatitis vol 13no 11 pp 756ndash761 2006

[31] J A Wilson and C D Richardson ldquoHepatitis C virus repliconsescape RNA interference induced by a short interfering RNAdirected against the NS5b coding regionrdquo Journal of Virologyvol 79 no 11 pp 7050ndash7058 2005

[32] S Jahan S Khaliq B Samreen et al ldquoEffect of combined siRNAof HCV E2 gene and HCV receptors against HCVrdquo VirologyJournal vol 8 article 295 2011

[33] S Jahan B Samreen S Khaliq et al ldquoHCV entry receptors aspotential targets for siRNA-based inhibition of HCVrdquo GeneticVaccines andTherapy vol 9 no 1 article 15 2011

[34] P K Chandra A K Kundu S Hazari et al ldquoInhibition ofhepatitis C virus replication by intracellular delivery of multiplesiRNAs by nanosomesrdquo Molecular Therapy vol 20 no 9 pp1724ndash1736 2012

[35] D A Suhy S-C Kao T Mao et al ldquoSafe long-term hepaticexpression of anti-HCVshRNA in anonhumanprimatemodelrdquoMolecular Therapy vol 20 no 9 pp 1737ndash1749 2012

[36] M Korf D Jarczak C Beger M P Manns and M KrugerldquoInhibition of hepatitis C virus translation and subgenomic rep-lication by siRNAs directed against highly conserved HCVsequence and cellular HCV cofactorsrdquo Journal of Hepatologyvol 43 no 2 pp 225ndash234 2005

[37] A M Domitrovich K W Diebel N Ali S Sarker and ASiddiqui ldquoRole of La autoantigen and polypyrimidine tract-binding protein in HCV replicationrdquo Virology vol 335 no 1pp 72ndash86 2005

[38] N Ali and A Siddiqui ldquoThe La antigen binds 51015840 noncodingregion of the hepatitis c virus RNA in the context of theinitiator AUG codon and stimulates internal ribosome entrysite-mediated translationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 94 no 6 pp2249ndash2254 1997

[39] K Spangberg L Wiklund and S Schwartz ldquoBinding of theLa autoantigen to the hepatitis C virus 31015840 untranslated regionprotects the RNA from rapid degradation in vitrordquo Journal ofGeneral Virology vol 82 no 1 pp 113ndash120 2001

[40] M Kruger C Beger P J Welch J R Barber M P Manns andF Wong-Staal ldquoInvolvement of proteasome 120572-subunit PSMA7in hepatitis C virus internal ribosome entry site-mediatedtranslationrdquo Molecular and Cellular Biology vol 21 no 24 pp8357ndash8364 2001

[41] L Gao H Aizaki J-W He and M M C Lai ldquoInteractionsbetween viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replicationcomplex on lipid raftrdquo Journal of Virology vol 78 no 7 pp3480ndash3488 2004

[42] P Metz A Reuter S Bender and R Bartenschlager ldquoInter-feron-stimulated genes and their role in controlling hepatitis Cvirusrdquo Journal of Hepatology vol 59 no 6 pp 1331ndash1341 2013

[43] S Amini-Bavil-Olyaee Y J Choi J H Lee et al ldquoThe antiviraleffector IFITM3 disrupts intracellular cholesterol homeostasis

to block viral entryrdquo Cell Host and Microbe vol 13 no 4 pp452ndash464 2013

[44] W Zhong A S Uss E Ferrari J Y N Lau and Z HongldquoDe novo initiation of RNA synthesis by hepatitis C virus non-structural protein 5B polymeraserdquo Journal of Virology vol 74no 4 pp 2017ndash2022 2000

[45] V Lohmann A Roos F Korner J O Koch and R Barten-schlager ldquoBiochemical and structural analysis of the NS5BRNA-dependent RNA polymerase of the hepatitis C virusrdquoJournal of Viral Hepatitis vol 7 no 3 pp 167ndash174 2000

[46] S Gallagher S E Winston S A Fuller and J G R HurrellldquoImmunoblotting and immunodetectionrdquo in Current ProtocolsinMolecular Biology FMAusubel Ed chapter 10 unit 108 pp1081ndash10828 John Wiley amp Sons New York NY USA 2008

[47] J G Donaldson ldquoImmunofluorescence stainingrdquo in CurrentProtocols in Cell Biology K S Morgan Ed chapter 4 unit 43pp 431ndash436 John Wiley amp Sons New York NY USA 1998

[48] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[49] T Shirasaki M Honda H Mizuno et al ldquoLa protein requiredfor internal ribosome entry site-directed translation is a poten-tial therapiutic target for hepaatitis C virus replicationrdquo TheJournal of Infectious Diseases vol 202 no 1 pp 75ndash85 2010

[50] M L Weir A Klip and W S Trimble ldquoIdentification of ahuman homologue of the vesicle-associated membrane protein(VAMP)-associated protein of 33 kDa (VAP-33) a broadlyexpressed protein that binds toVAMPrdquoBiochemical Journal vol333 no 2 pp 247ndash251 1998

[51] T Suzuki K Ishii H Aizaki and T Wakita ldquoHepatitis C virallife cyclerdquo Advanced Drug Delivery Reviews vol 59 no 12 pp1200ndash1212 2007

[52] D Moradpour F Penin and C M Rice ldquoReplication ofhepatitis C virusrdquoNature ReviewsMicrobiology vol 5 no 6 pp453ndash463 2007

[53] J Dubuisson and F-L Cosset ldquoVirology and cell biology of thehepatitis C virus life cyclemdashan updaterdquo Journal of Hepatologyvol 61 no 1 supplement pp S3ndashS13 2014

[54] T K H Scheel and C M Rice ldquoUnderstanding the hepatitisC virus life cycle paves the way for highly effective therapiesrdquoNature Medicine vol 19 no 7 pp 837ndash849 2013

[55] M B Zeisel J Lupberger I Fofana and T F Baumert ldquoHost-targeting agents for prevention and treatment of chronic hepati-tis Cmdashperspectives and challengesrdquo Journal of Hepatology vol58 no 2 pp 375ndash384 2013

[56] J M Baugh J A Garcia-Rivera and P A Gallay ldquoHost-tar-geting agents in the treatment of hepatitis C a beginning andan endrdquo Antiviral Research vol 100 no 2 pp 555ndash561 2013

[57] J G OrsquoLeary and G L Davis ldquoHepatitis C virus replication andpotential targets for direct-acting agentsrdquoTherapeutic Advancesin Gastroenterology vol 3 no 1 pp 43ndash53 2010

[58] Q Xue H Ding M Liu et al ldquoInhibition of hepatitis C virusreplication and expression by small interfering RNA targetinghost cellular genesrdquoArchives of Virology vol 152 no 5 pp 955ndash962 2007

[59] J Zhang O Yamada T Sakamoto et al ldquoDown-regulation ofviral replication by adenoviral-mediated expression of siRNAagainst cellular cofactors for hepatitis C virusrdquo Virology vol320 no 1 pp 135ndash143 2004


[60] Y Waheed A Bhatti and M Ashraf ldquoRNA dependent RNApolymerase of HCV a potential target for the development ofantiviral drugsrdquo Infection Genetics and Evolution vol 14 no 1pp 247ndash257 2013

[61] L Trejo-Avila R Elizondo-Gonzalez K D C Trujillo-MurilloP Zapata-Benavides C Rodrıguez-Padilla and A M Rivas-Estilla ldquoAntiviral therapy inhibition of hepatitis C virus expres-sion by RNA interference directed against the NS5B region ofthe viral genomerdquo Annals of Hepatology vol 6 no 3 pp 174ndash180 2007

[62] U Ali Ashfaq M Ansar M T Sarwar T Javed S Rehmanand S Riazuddin ldquoPost-transcriptional inhibition of hepatitisC virus replication through small interference RNArdquo VirologyJournal vol 8 no 1 article 112 2011

[63] B Chang CH Lee J H Lee and S-W Lee ldquoComparative anal-ysis of intracellular inhibition of hepatitis C virus replication bysmall interfering RNAsrdquo Biotechnology Letters vol 32 no 9 pp1231ndash1237 2010

[64] A Upadhyay U Dixit D Manvar N Chaturvedi and V NPandey ldquoAffinity capture and identification of host cell factorsassociated with hepatitis C virus (+) strand subgenomic RNArdquoMolecular and Cellular Proteomics vol 12 no 6 pp 1539ndash15522013

[65] S-C Chan S-Y Lo J-W Liou et al ldquoVisualization of the struc-tures of the hepatitis C virus replication complexrdquo Biochemicaland Biophysical Research Communications vol 404 no 1 pp574ndash578 2011

[66] D Moradpour M J Evans R Gosert et al ldquoInsertion ofgreen fluorescent protein into nonstructural protein 5A allowsdirect visualization of functional hepatitis C virus replicationcomplexesrdquo Journal of Virology vol 78 no 14 pp 7400ndash74092004

[67] E Gottlieb and J A Steitz ldquoThe RNA binding protein La influ-ences both the accuracy and the efficiency of RNA polymeraseIII transcription in vitrordquo EMBO Journal vol 8 no 3 pp 841ndash850 1989

[68] S Weser M Bachmann K H Seifart and W MeibssnerldquoTranscription efficiency of human polymerase III genes invitro does not depend on the RNP-forming autoantigen LardquoNucleic Acids Research vol 28 no 20 pp 3935ndash3942 2000

[69] J P Wyles and N D Ridgway ldquoVAMP-associated protein-A regulates partitioning of oxysterol-binding protein-relatedprotein-9 between the endoplasmic reticulum and Golgi appa-ratusrdquo Experimental Cell Research vol 297 no 2 pp 533ndash5472004

[70] J P Wyles C R McMaster and N D Ridgway ldquoVesicle-associated membrane protein-associated protein-A (VAP-A)interacts with the oxysterol-binding protein to modify exportfrom the endoplasmic reticulumrdquo Journal of Biological Chem-istry vol 277 no 33 pp 29908ndash29918 2002

[71] Y Jia T Song C Wei et al ldquoNegative regulation of MAVS-mediated innate immune response by PSMA7rdquo The Journal ofImmunology vol 183 no 7 pp 4241ndash4248 2009

[72] R Prabhu R F Garry and S Dash ldquoSmall interfering RNAtargeted to stem-loop II of the 51015840 untranslated region effectivelyinhibits expression of sixHCVgenotypesrdquoVirology Journal vol3 article 100 2006

[73] TWatanabeM SudohMMiyagishi et al ldquoIntracellular-diceddsRNA has enhanced efficacy for silencing HCV RNA andovercomes variation in the viral genotyperdquo Gene Therapy vol13 no 11 pp 883ndash892 2006

[74] I Fofana F Xiao CThumann et al ldquoA novel monoclonal anti-CD81 antibody produced by genetic immunization efficientlyinhibits hepatitis C virus cell-cell transmissionrdquo PLoS ONE vol8 no 5 Article ID e64221 2013


stop its replication and propagation Therefore utilization ofthis technology holds tremendous potential as a therapeutictool against HCV Previous reports have shown that silencingof cellular or viral determinants could inhibit HCV entryand replication [17ndash23] siRNAs targeted against the core andNS4B regions specifically decreased viral load in a dose-dependent manner [24] siRNAs against NS5B and NS3regions inhibit HCV replication and protein expressionwithout any IFN response [25] Alternatively many studieshave also been focused on the cellular factors that directlyor indirectly interact with the different regions of the viralgenome and play a vital role in viral life cycle [26ndash29]However monotherapy with siRNA has led to the emergenceof viral escape mutants through point mutation within thesiRNA target regions [30 31] Therefore combinations ofsiRNAs comprising multiple viral andor cellular targetshave also been evaluated Few recent reports showed thatcombinations of siRNAs targeted against different cellular orviral factors have synergistic anti-HCV effects [32ndash36] Mostof these studies were restricted to either viral entry or the viralgenome However different stages in HCV life cycle suchas viral entry viral genome translation replication complexformation and replication and virion assembly and releaserepresent a broader spectrum of molecular events that mayserve as potential targets against HCVTherefore we proposecocktail of siRNAs targeting different viral and cellular factorswhichmay intercept various crucial steps in the viral life cycleand may enforce a strong synergistic antiviral effect

In the present study gene specific siRNAs were designedagainst cellular factors La autoantigen PSMA7 hVAP-A andviral factor HCV NS5B and screened for HCV inhibitionindividually as well as in combinationsThese cellular factorsand viral protein NS5B play crucial roles in HCV life cycle[37ndash45] Downregulation of viral replications was assessed bysemiquantitative RT-PCR western blotting and immunos-taining

2 Materials and Methods

21 Cell Culture Huh-75 cells (a kind gift from Dr V NPandey New Jersey Medical School USA) a highly permis-sive cell line for HCV replication were grown in DulbeccorsquosModified Eaglersquos Medium (Gibco USA) supplemented with10 FBS (Gibco USA) penicillin 100UmL and strepto-mycin 100 120583gmL (HiMedia India) 01mM nonessentialamino acids (Gibco USA) and 2mM glutamax-I (GibcoUSA) Cells were maintained in a humidified incubator at37∘C in 5 CO

2

22 HCV Production in Cell Culture For HCV culture pFL-J6JFH plasmid [24] (a kind gift from Dr V N Pandey NewJersey Medical School USA) encoding full-length chimericHCV genotype 2a was linearized with XbaI and subjectedto in vitro transcription using MEGAscript Kit (AmbionUSA) following manufacturerrsquos protocol Huh-75 cells weretransfected with 10 120583g transcript using Lipofectamine 2000transfection reagent (Invitrogen USA) using manufacturerrsquosprotocol and 6 h after transfection media were removedcells were washed with PBS and incubated in fresh media

The culture supernatant was collected after 72 h and passedthrough 045 120583m filter and stored at minus80∘C in small aliquots

23 Viral Load Quantification Viral RNAs were isolatedfrom cell culture supernatants using Trizol LS reagent(Ambion USA) and quantified by real-time quantitative RT-PCR using primer pair of HCV 51015840UTR forward 51015840-CCT-AATAGGGGCGACACTCCG-31015840 reverse 51015840-CCACAA-GGCCTTTCGCAACC-31015840 Assays were performed usingSybr green real-time PCRmaster mix reagent kit (InvitrogenUSA) and mastercycler ep realplex2 (Eppendorf Germany)according to the manufacturerrsquos instructions

24 RNA Isolation and RT-PCR Total RNA was extractedfrom cells using Trizol reagent (Ambion USA) followingmanufacturerrsquos guidelines RNA was then subjected to cDNAsynthesis using RevertAid reverse transcriptase (ThermoFisher Scientific USA) following manufacturerrsquos protocolin a thermocycler (Eppendorf Germany) In brief cDNAwas synthesized using 2120583g of total RNA in 20120583L reactionvolume using gene specific reverse primers and further 2120583Lof cDNA was used for PCR amplification of specific genesRespective primer sequences were La autoantigen forward51015840-GGATAGACTTCGTCAGAGGAGCA-31015840 reverse 51015840-CTGGTCTCCAGCACCATTTTCTG-31015840 hVAP-A forward51015840-GTGCTCCATCTGATTTACCCCA-31015840 reverse 51015840-TTC-CACAGGCTTGCTCAGTATT-31015840 PSMA7 forward 51015840-GCCATCACCGTCTTCTCGCC-31015840 reverse 51015840-CGTTGT-CATCCAAAGCACAG-31015840 HCV NS5B forward 51015840-ACA-TCAAGTCCGTGTGGAAGG-31015840 reverse 51015840-GCTCCC-ATTACCGCCTGAGG-31015840 and 120573-actin forward 51015840-GCG-GGAAATCGTGCGTGACATT-31015840 reverse 51015840-GATGGA-GTTGAAGGTAGTTTCGTG-31015840 The PCR products wereresolved by electrophoresis in 15 (wv) agarose gels andimages were captured by a Chemidoc XRS system (Bio-RadUSA) The band intensities were quantified using QuantityOne software

25 Design and Synthesis of siRNAs siRNAs were designedusing IDT online designing tool and were synthesized fromIDT USA Sequences of siRNAs were as follows La autoanti-gen (GenBank accession number NM 003142) sense 51015840-GGAACAAAGAAGUGACUU-31015840 antisense 51015840-ACUUCC-CAAGUCACUUCU-31015840 hVAP-A (GenBank accession num-ber NM 1944342) sense 51015840-GGUUUAGACAGGUUC-AAUUAGCUCA-31015840 antisense 51015840-UGAGCAAAUUGA-ACCUGUCUAAACCCG-31015840 PSMA7 (GenBank accessionnumberNM 0027923) sense 51015840-CCUCUUCCAAGUGGA-GUA-31015840 antisense 51015840-CUGCGCGUACUCCACUUG-31015840HCV NS5B (HCV genotype 2a strain) sense 51015840-CCCUCU-AUGACAUUACACA-31015840 antisense 51015840-UGUGUAAUG-UCAUAGAGGG-31015840 scrambled negative control sense 51015840-CUUCCUCUCUUUCUCUCCCUUGUGA-31015840 antisense51015840-UCACAAGGGAGAGAAAGAGAGGAAGGA-31015840

26 siRNA Transfection and HCV Infection Huh-75 cells(2 times 105 cellswell) grown overnight in 12-well plates weretransiently transfected with 50 nM for single and 100 nMfor combinations of siRNAs using Lipofectamine RNAiMAX








3 Result





La

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La si

RNA

(a)

La autoantigenNS5B

lowast

lowast


20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

(b)

hVAP-A

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

hVA

P-A

siRN

A

(c)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

hVAP-ANS5B


(d)

PSMA7

NS5B

Actin

Sc si

RNA

PSM

A7

siRN

A

Ctrl

Reag

ent

(e)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

PSMA7NS5B


(f)

Ctrl

Reag

ent

NS5

B siR

NA

Neg

ctrl

NS5B

Actin

(g)

lowast

020406080

100120

Targ

et g

ene e

xpre

ssio

n (

)

NS5B


(h)



NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(a)NS5B

0

20

40

60

80

100

120

HCV

RN

A ex

pres

sion

()

lowastlowast

lowast

lowast

lowastlowast

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(b)















Ctrl

Reag

ent

Sc si

RNA

La si

RNA

NS5

B siR

NA

La

NS5B

Actin

La+

NS5

B siR

NA

(a)


LaNS5B

0

20

40

60

80

100

120

Targ

et p

rote

in ex

pres

sion

()

(La + NS5B)

(b)

Ctrl

(A)

(B)


(c)



4 Discussion






lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References





















































































3 Result





La

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La si

RNA

(a)

La autoantigenNS5B

lowast

lowast


20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

(b)

hVAP-A

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

hVA

P-A

siRN

A

(c)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

hVAP-ANS5B


(d)

PSMA7

NS5B

Actin

Sc si

RNA

PSM

A7

siRN

A

Ctrl

Reag

ent

(e)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

PSMA7NS5B


(f)

Ctrl

Reag

ent

NS5

B siR

NA

Neg

ctrl

NS5B

Actin

(g)

lowast

020406080

100120

Targ

et g

ene e

xpre

ssio

n (

)

NS5B


(h)



NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(a)NS5B

0

20

40

60

80

100

120

HCV

RN

A ex

pres

sion

()

lowastlowast

lowast

lowast

lowastlowast

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(b)















Ctrl

Reag

ent

Sc si

RNA

La si

RNA

NS5

B siR

NA

La

NS5B

Actin

La+

NS5

B siR

NA

(a)


LaNS5B

0

20

40

60

80

100

120

Targ

et p

rote

in ex

pres

sion

()

(La + NS5B)

(b)

Ctrl

(A)

(B)


(c)



4 Discussion






lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References















































































La

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La si

RNA

(a)

La autoantigenNS5B

lowast

lowast


20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

(b)

hVAP-A

NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

hVA

P-A

siRN

A

(c)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

hVAP-ANS5B


(d)

PSMA7

NS5B

Actin

Sc si

RNA

PSM

A7

siRN

A

Ctrl

Reag

ent

(e)

lowast

lowast

0

20

40

60

80

100

120

Targ

et g

ene e

xpre

ssio

n (

)

PSMA7NS5B


(f)

Ctrl

Reag

ent

NS5

B siR

NA

Neg

ctrl

NS5B

Actin

(g)

lowast

020406080

100120

Targ

et g

ene e

xpre

ssio

n (

)

NS5B


(h)



NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(a)NS5B

0

20

40

60

80

100

120

HCV

RN

A ex

pres

sion

()

lowastlowast

lowast

lowast

lowastlowast

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(b)















Ctrl

Reag

ent

Sc si

RNA

La si

RNA

NS5

B siR

NA

La

NS5B

Actin

La+

NS5

B siR

NA

(a)


LaNS5B

0

20

40

60

80

100

120

Targ

et p

rote

in ex

pres

sion

()

(La + NS5B)

(b)

Ctrl

(A)

(B)


(c)



4 Discussion






lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References















































































NS5B

Actin

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(a)NS5B

0

20

40

60

80

100

120

HCV

RN

A ex

pres

sion

()

lowastlowast

lowast

lowast

lowastlowast

Ctrl

Reag

ent

Sc si

RNA

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

(b)















Ctrl

Reag

ent

Sc si

RNA

La si

RNA

NS5

B siR

NA

La

NS5B

Actin

La+

NS5

B siR

NA

(a)


LaNS5B

0

20

40

60

80

100

120

Targ

et p

rote

in ex

pres

sion

()

(La + NS5B)

(b)

Ctrl

(A)

(B)


(c)



4 Discussion






lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References















































































Ctrl

Reag

ent

Sc si

RNA

La si

RNA

NS5

B siR

NA

La

NS5B

Actin

La+

NS5

B siR

NA

(a)


LaNS5B

0

20

40

60

80

100

120

Targ

et p

rote

in ex

pres

sion

()

(La + NS5B)

(b)

Ctrl

(A)

(B)


(c)



4 Discussion






lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References
















































































lowast

020406080

100120

Cel

l via

bilit

y (

of c

ontro

l)

Con

trol

Reag

ent Sc La

La+

NS5

B

La+

hVA

P-A

La+

PSM

A7

NS5

B+

PSM

A7

NS5

B+

hVA

P-A

PSM

A7+

hVA

P-A

hVA

P-A

PSM

A7

NS5

B










La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References















































































La

HCV

CD81SR-B1

Endocytosis

Uncoating

Translation

Replication

Packaging

Release

Membranousnetwork

Attachment

Processing

Assembly

siRNA

siRNA

hVAP-A

La

PSMA7

hVAP-A

PSMA7

LaNS5B


Cldn 1

LDLR



5 Conclusion



Ethical Approval


Competing Interests


Acknowledgments


References















































































Ethical Approval


Competing Interests


Acknowledgments


References
















































































































































Documents

Combinations of siRNAs against La Autoantigen with NS5B or … · 2016. 9. 20. · Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition