Original article Pathogenic effects of Rift Valley fever virus NSs gene are alleviated in cultured cells by expressed antiviral short hairpin RNAs

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1 Antivirl Therpy 2012; 17: (doi: /IMP2073) Originl rticle Pthogenic effects of Rift Vlley fever virus NSs gene re llevited in cultured cells by expressed ntivirl short hirpin RNAs Tristn Scott 1, Jnusz T Pwesk 2, Ptrick Arbuthnot 1, Mrc S Weinberg 1 * 1 Antivirl Gene Therpy Reserch Unit, School of Pthology, Helth Sciences Fculty, University of the Witwtersrnd, Johnnesburg, Guteng, South Afric 2 Specil Pthogens Unit, Ntionl Institute for Communicble Diseses of the Ntionl Helth Lbortory Service, Sndringhm, Guteng, South Afric *Corresponding uthor e-mil: mrc.weinberg@wits.c.z Bckground: Rift Vlley fever virus (RVFV), member of the Bunyviride fmily, my cuse severe heptitis, encephlitis nd hemorrhgic fever in humns. There re currently no vilble licensed vccines or therpies to tret the virl infection in humns. RNA interference (RNAi)-bsed virl gene silencing offers promising pproch to inhibiting repliction of this highly pthogenic virus. The smll (S) segment of the RVFV triprtite genome crries the genetic determintes for pthogenicity during infection. This segment encodes the nonstructurl S (NSs) nd essentil nucleocpsid (N) genes. Methods: To dvnce RNAi-bsed inhibition of RVFV repliction, we designed severl Pol III short hirpin RNA (shrna) expression cssettes ginst the NSs nd N genes, including multimerized plsmid vector tht included four shrna expression cssettes. Results: Effective trget silencing ws demonstrted using full- nd prtil-length trget reporter ssys, nd confirmed by western blot nlysis of exogenous N nd NSs expression. Smll RNA northern blots showed detectble RNAi guide strnd formtion from single nd multimerized shrna constructs. Using cell culture model of RVFV repliction, shrnas trgeting the N gene decresed intrcellulr nucleocpsid protein concentrtion nd virl repliction. The shrnas directed ginst the NSs gene reduced NSs protein concentrtions nd llevited NSsmedited cytotoxicity, which my be cused by host trnscription suppression. Conclusions: These dt re the first demonstrtion tht RNAi ctivtors hve potentil therpeutic benefit for countering RVFV infection. Introduction Rift Vlley fever virus (RVFV) is zoonotic mosquitoborne member of the Phlebovirus genus of the Bunyviride fmily [1]. It my cuse severe nd lrge infection outbreks in humns nd livestock, which occur most commonly in Afric [2,3]. Outbreks of RVFV outside Afric, in Yemen nd Sudi Arbi, were reported in 2002 [4 7]. The bility of RVFV to replicte in wide rnge of mosquito vectors [8,9], nd recent dissemintion of rthropod-borne viruses [10,11] hve rised concerns tht RVFV my spred to nonendemic regions of the world. The virus is lso listed s potentil biowepon [12]. Humns become infected with RVFV s result of contct with blood or tissues of infected nimls or from mosquito bites. Infected individuls usully develop mild to modertely severe febrile illness. Severe complictions, including oculr disese, encephlitis nd ftl hemorrhgic disese, occur in smll proportion of ptients. The genome of RVFV comprises triprtite singlestrnded RNA mde up of negtive-sense lrge (L), medium (M) nd mbisense smll (S) segments [13 15]. The S segment encodes non-structurl (NSs) nd nucleocpsid (N) proteins nd hs been shown to be the genetic determinnt for pthogenicity in Bunyviride [16,17]. The NSs protein is involved in inhibiting host innte virl defences. NSs chieves this by four mechnisms: blocking host messenger RNA (mrna) trnscription by sequestering p44 nd XPB to inhibit mturtion of trnscription fctor II H (TFIIH); promoting the degrdtion of the TFIIH subunit p62 in the nucleus 2012 Interntionl Medicl Press (print) (online) 643

2 T Scott et l. of infected cells [18]; binding of the trnscription fctor Yin Yng 1 (YY1) on the interferon-b promoter to suppress its ctivtion through the SAP30 complex [19 22]; nd, in the cytoplsm, promoting the down-regultion of double-strnded RNA-dependent protein kinse erly in infection, which prevents phosphoryltion of eukryotic initition fctor 2 [23,24]. Removl of NSs results in ttenution of RVFV, which hs been demonstrted in nturlly occurring ttenuted strin of RVFV nd in recombinnt RVFV lcking fully functionl NSs [25,26]. The S segment-encoded N protein is essentil for virion cpsid formtion nd plys other roles in the virus life cycle. These include interction with RVFV surfce glycoproteins nd involvement in trnscription nd repliction [27]. There re currently no vilble RVFV therpies or vccines for use in humns. Development of improved therpies to combt the infection is therefore priority. The sequence-specific nd potent gene silencing tht my be chieved by hrnessing the RNA interference (RNAi) pthwy is promising pproch to inhibiting pthogenic viruses [28] nd my be hrnessed ginst RVFV. Both synthetic nd expressed RNAi ctivtors hve been used to silence virl gene expression nd their therpeutic potentil hs been demonstrted in vrious chronic nd cute infections [29]. Recent investigtion into the fesibility of RNAibsed therpies ginst hemorrhgic fevers cused by Ebol, Mrburg nd Lss viruses demonstrted tht synthetic nd expressed RNAi effectors inhibit virl repliction in cell culture nd non-humn primtes [30 33]. However, existing dt on using RNAi to counter hemorrhgic fever viruses re limited. A recent study demonstrted the efficcy of smll interfering RNA (sirna) trgeted to inhibit Hzr virus, virus tht belongs to the Crimen-Congo hemorrhgic fever serogroup [34]. This study underscores the potentil ppliction for RNAi-bsed pproches to inhibit members of the Bunyviride fmily known to be humn pthogens. A potentil impediment to the development of gene silencing-bsed therpy for Bunyviride is tht RNAi my be disrupted during infection with these viruses. Although direct inhibition of RNAi by RVFV hs not been shown, tomto spotted wilt virus [35,36] nd L Crosse virus (LACV) [35,36] re cpble of suppressing RNAi. The bility of RVFV NSs to inhibit globl trnscription, nd therefore the expression of multiple proteins required for the RNAi pthwy, is of concern for the development of RNAi-bsed RVFV tretment. In this study pnel of short hirpin RNA (shrna) expression cssettes ws designed to trget the NSs nd N genes of RVFV. These gene silencers were shown to suppress virl gene expression successfully. The shrnas trgeting NSs reduced the pthogenic effects of NSs by lleviting host trnscription suppression nd cellulr toxicity. shrnas ginst N inhibited virl repliction in cell culture, demonstrting tht RNAi hs potentil for therpy of infections cused by Bunyviride. Methods NSs nd N reporters nd expression plsmids The expression nd reporter plsmids encoding NSs nd N were generted (GenBnk ccession numbers ZH548, EU312138; ZH501, EU312137; RVF 825/79 [Zimbbwe], EU312131; RVF Ar 21229, EU312115; RVF Tmbul [Egypt], EU312110; RVF B1143 [Keny], EU312119; RVF CAR 1662, EU312122; RVF 52\99\1 [South Afric], EU312127; Smithburn [vccine], EU312129; nd Lunyo [Ugnd], EU312121). PCR primers were designed to mplify these sequences. The primers contined either XhoI (forwrd) or NotI (reverse) restriction sites (underlined) nd sequences were s follows: NSs forwrd primer: 5 -GATCCTCGAGTATCATG- GATTACTTTCCTGT-3, N forwrd primer: 5 -GATC- CTCGAGATGGACAACTATCAAGAGCT-3, NSs reverse primer: 5 -GATCGCGGCCGCTAATCAACCT- CAACAAATCC-3 nd N reverse primer: 5 -GATCGCG- GCCGCTTAGGCTGCTGTCTTGTAAGC-3. NSs from 10 strins of RVFV nd N from 2 strins of the virus where mplified by PCR. The full-length NSs nd N PCR products were digested with XhoI nd NotI (Ferments, Glen Bernie, MD, USA) then inserted t equivlent sites downstrem of the Renill luciferse open reding frme in psicheck -2 (Promeg, Fitchburg, WI, USA). The plsmids expressing NSs nd N from the cytomeglovirus (CMV) Pol II trnscriptionl regultory element were propgted in the mmmlin expression vector pci-neo (Promeg) using similr procedures. Plsmids expressing mutnt versions of NSs, PP1, PP2 nd C13, were kindly donted by Michele Bouloy [22]. pci-gfp ws generted s previously described [37]. To generte the 3 FLAG-tgged constructs, NSs nd N were mplified by PCR using the sme NSs forwrd nd N forwrd primers described bove. Sequences of reverse primers contining n in-frme 3 FLAG sequence nd NotI site were s follows: NSs 501, PP1 nd PP2 3 FLAG reverse: 5 -gtcgcggccgcctcttgtctcgt- CATCCTTGTAGTCGATGTCATGATCTTTATAAT- CACCGTCATGGTCTTTGTAGTCAGCCCGGGATC- TATCAACCTCAACAAATCC-3 nd N FLAG reverse: 5 -gtcgcggccgcctcttgtctcgt- CATCCTTGTAGTCGATGTCATGATCTTTATAAT- CACCGTCATGGTCTTTGTAGTCAGCCCGG- GATCTGGCTGCTGTCTTGTAAGCC-3. The PCR products were cloned into pci-neo s described bove. The miniml trgets consisted of only the NSs nd N shrna trget sequences nd were generted from complementry oligonucleotides tht contined XhoI Interntionl Medicl Press

3 Trgeting RVFV NSs using expressed RNAi nd NotI sequence overhngs. The oligonucleotides were phosphorylted with polynucleotide kinse (Promeg), nneled nd inserted t XhoI nd NotI sites of the psicheck -2 plsmid. The sense strnd oligonucleotides were s follows: positive NSs TCGA- GATATCGTCC TAGTCACGAGGTTCG-3, positive NSs3 5 -TCGAGATATCGATGGTCCTCCCAGGA- TAC-3, positive NSs4 5 -TCGAGATATCGTTGT- GTCAGTGGAGTACT-3, positive N1 5 -TCGAGA- TATCTCAAG CAGTGGACCGCAATGA-3 nd negtive NSs2 5 -GGCCGCCGAACCTCGTGAC- TAGGACGATAT-3, negtive NSs3 5 -GGCCGCG- TATCCTGGGAGGACCATCGATATC-3, negtive NSs4 5 -GGCCGCAGTACTCCACTGACACAACGA- TATC-3 nd negtive N1 5 -GGCCGCTCATTGCG GTCCACTGCTTGAGATATC-3. The sequences of ll expression nd reporter constructs were confirmed (Inqb Biotech, Pretori, South Afric). Single nd multimeric short hirpin RNA expression cssettes All U6 Pol III promoter-driven shrnas were generted by PCR using universl U6 Pol III forwrd primer (5 -GATCGGGCCCATCGACAAGGTCGGGCAG- GAAGAGGCCCT-3 ) with pproprite reverse primer. The plsmid, ptz-u6+1 contining U6 Pol III promoter ws used s templte [38]. The PCR products were ligted to ptz-57r/t using the InsTAClone PCR cloning kit (Ferments), ccording to the mnufcturer s instructions. The sequences of the four shnss reverse primer sequences were s follows: shnss1 5 -AAAAAAGGCTTACACAGGATGATAGTCTCTT- GAACTATCATCCTGTGTAAGCCGGTGTTTCGTC- CTTTCCACAA-3, 5 -AAAAAAGTCCT AGTCACGAGGTTCGTCTCTTGAACGAAC- CTCGTGACTAGGACGGTGTTTCGTCCTTTC- CACAA-3, shnss3 5 -AAAAAGATGGTCCTCC- CAGGATACTCTCTTGAAGCATCCTGGGAG- GACCATCGGTGTTTCGTCCTTTCCACAA-3 nd shnss4 5 -AAAAAAGTTGTGTCAGTGGAGTAC- TACTCTTGATAGTACTCCACTGACACAACGGT- GTTTCGTCCTTTCCACAA 3. The sequences of the four shn reverse primers were s follows: shn1 5 -AAAAAAGTCAAGCAGTGGACCGCAATCT CTTGGACTACAGTCCACTGCTTGACGGT- GTTTCGTCCTTTCCACAA-3, shn2 5 -AAAAAA- GCAGCCAATGAATGCAGCATCTCTTGAATAC- CGCATTCATTGGCTGCGGTGTTTCGTCCTTTC- CACAA-3, shn3 5 -AAAAAAGTTATAAGCCAT- GAGAAGAGTCTCTTGAACCCTCCTCATG- GCTTATAACGGTGTTTCGTCCTTTCCACAA-3 nd shn4 5 -AAAAAAGCTCTCTGTATCTGCT- GCAGTCTCTTGAACCACAACAGATACAGAGA- GCGGTGTTTCGTCCTTTCCACAA-3. The H1 Pol III promoter driven shrnas were mplified using H1 Pol III forwrd primer (5 -GATCGAATTCACTAGT- GAACGCTGACGTCATCAA-3 ), which contined EcoRI (bold) nd SpeI (underlined) restriction sites. Genomic DNA from HEK293 cells ws used s templte. The sequences of the H1 Pol III shrna reverse primers were s follows: 5 -AAAAAAGTCCTAGT- CACGAGGTTCGTCTCTTGAACGAACCTCGT- GACTA GGACGGATCCGAGTGGTCTCATAC-3 (73 nucleotides), shnss3 5 -AAAAAAGATGGTCCTCC- CAGGATACTCTCTTGAAGCATCCTGGGAGGAC- CATCGGATCCGAGTGGTCTCATAC-3, shnss4 5 -AA AAAAGTTGTGTCAGTGGAGTACTACTCTT- GATAGTACTCCACTGACACAACGGATCCGAGTG- GTCTCATAC-3 nd shn1 5 -AAAAAAGTCAAGC AGTGGACCGCAATCTCTTGGACTACAGTCCACT- GCTTGACGGATCCGAGTGGTCTCATAC-3. The PCR products were inserted into ptz-57r/t in the sme mnner s tht of the U6 Pol III shrnas. All the shrnas-expressing plsmids (shnss1, 2, 3 nd 4 nd shn1, 2, 3 nd 4) were confirmed by sequencing. To generte multimers, n shrna ws initilly digested with ScI nd SpeI enzymes then ligted into shrna cssette tht ws digested with ScI nd XbI (Ferments). The sme process ws repeted using cssettes contining two shrnas to generte the cssettes contining four shrnas. Cell culture nd trnsfections Cells of the humn embryonic kidney cell line, HEK293, were mintined s previously described [39]. The humn heptom cell line, Huh7, ws mintined in Egle s miniml essentil medi (BioWhittker, Wlkersville, MD, USA) supplemented with 10% fetl clf serum, 50 IU/ml penicillin/50 mg/ml streptomycin mix, 1% non-essentil mino cids (Sigm, St. Louis, MO, USA), nd 1% l-glutmine (Sigm) nd incubted t 37 C with 5% CO 2. Trnsfections were crried out using Lipofectmine2000 (Invitrogen, Crlsbd, CA, USA) ccording to the mnufcturer s instructions. The medi ws chnged 24 h post-trnsfection, nd nlysis crried out 24 h therefter. Equivlent trnsfection efficiencies were verified by fluorescence microscopy by cotrnsfecting plsmid tht constitutively produces enhnced green fluorescent protein (pci-egfp) [37]. To evlute the effect of the shrna-encoding plsmids on trget reporter ssys, trnsfection ws performed in HEK293 cells s previously described [39] except t rtio of 1:10 (trget to shrna). To determine the effect the CMV Pol II expressed plsmids would hve on reporter ssy, HEK293 cells were seeded s described bove, nd trnsfected with the CMV Pol II expression plsmids nd 100 ng of prl- CMV. The shrna-encoding constructs were subsequently introduced t the sme rtio s indicted bove. Antivirl Therpy

4 T Scott et l. To ssess the effect of shrna-encoding plsmids on the llevition of interferon-b suppression, HEK293 cells were seeded s described bove nd trnsfected with 2.5 ng of the CMV Pol II expression plsmids with pifd (-125) lucter (pif-luc) contining the interferon-b promoter (positions -125 to 72) fused to firefly luciferse gene (kindly donted by Stephen Goodbourn [40]), nd 200 ng of the shrna encoding plsmids. At 24 h posttrnsfection, cells were treted with Poly (I:C) for 16 h. To evlute the effect the CMV Pol II expressed plsmids nd shrna-encoding plsmid would hve on cell vibility, HEK293 cells were seeded 24 h prior to trnsfection t 40% confluence in 96-well culture dish. HEK293 cells were trnsfected with the CMV Pol II expressed trget plsmids nd the shrna-encoding plsmids t the sme rtio s bove nd 10 ng of pci-gfp. For the western blot nlysis, HEK293 cells were seeded 24 h prior to trnsfection t 50% confluence in 6-well culture dishes. The HEK293 cells were trnsfected with the CMV Pol II expressed 3 FLAG trget plsmids nd the shrna encoding constructs rtio of 1:6 (shrna to trget). To ssess the effect the shrna-encoding plsmids would hve on 1981 SA isolte of RVFV in n infection chllenge ssy, Huh7 cells were seeded t 50% confluence 24 h prior to trnsfection in 24-well culture dishes. The medi ws chnged 30 min prior to trnsfections to remove penicillin-streptomycin. Huh7 cells were trnsfected with 900 ng of the shrna-encoding plsmids nd the medi contining penicillin-streptomycin replced 24 h post-trnsfection. Renill nd dul-luciferse ssys The dul-luciferse reporter ssys were crried out using the Promeg Dul-Luciferse Reporter ssy s per the mnufcturer s instructions (Promeg) nd Verits dul-injection luminometer (Turner Biosystems, Sunnyvle, CA, USA). Knockdown ws determined s the rtio of Renill luciferse to tht of firefly luciferse, normlizing the rtio of the shrnas to the mocktreted cells. The experiment ws performed in triplicte with the dt expressed s men ±sd. Protein extrction nd western blot nlysis Whole cell protein ws extrcted using RIPA buffer (25 mm Tris-HCl ph 7.6, 150 mm NCl, 1% NP-40, 1% sodium deoxycholte, 0.1% SDS) ccording to the mnufcturer s instructions (ThermoScientific, Wlthm, MA, USA). Lystes were stored t -20 C until redy to be used. Lystes were thwed nd centrifuged t 13,000 g for 10 min t 4 C. Superntnts were collected nd Pierce BSA ssy ws performed on the smples s per the mnufcturer s instructions (Thermo- Scientific). Lystes were seprted by 12% SDS-PAGE nd trnsferred onto polyvinylidene fluoride membrne (Immunoblot-P, Millipore, Billeric, MA, USA). For immunoblotting, membrnes were first incubted with mouse nti-flag or mouse nti--tubulin (Sigm) then with secondry polyclonl rbbit nti-mouse ntibody coupled to horserdish peroxidse (Dko, Glostrup, Denmrk). Primry nd secondry ntibodies were diluted t 1:1000. The chemiluminescent signl ws detected using Syngene G:BOX Chemi Gel imging equipments (Syngene, Frederick, MD, USA). Cytotoxicity ssys To determine cytotoxicty, n MTT cell vibility ssy (Sigm) ws performed s per the mnufcturer s instructions. The plte ws red on BioRd microplte reder (Model 680; BioRd Lbortories Inc., Hercules, CA, USA). Northern blot nlysis Northern blot nlyses were performed s previously described [39] using 30 mg of totl RNA per smple tht ws resolved on ure denturing 15% polycrylmide gel nd blotted onto nylon membrne. Moleculr weights were determined by loding Decde mrker (Ambion, Crlsbd, CA, USA). DNA probes complementry to the ntisense strnds of the shrnas were hybridized to the blot in order to detect the shrna processing. Virus chllenge At 24 h post-trnsfection, the cells were infected with 1981 SA isolte of RVFV using 50% tissue culture infective dose of ml -1. A totl of 200 ml of superntnt ws removed dily for 3 dys. An ELISA ws performed on the superntnts, s previously described [41]. Briefly, it involved the use of rbbit nti-rvfv N-detecting ntibody (dilution 1:3000) with secondry horserdish peroxidse conjugted nti-rbbit immunoglobulin G (dilution 1:8000). Smples were red t n opticl density of 405 nm using BioRd microplte reder (Model 680; BioRd Lbortories Inc). Sttisticl nlysis Sttisticl clcultions were determined using the GrphPd Prism softwre pckge (GrphPd Softwre, Inc., L Joll, CA, USA). Sttisticl difference ws considered significnt when P<0.05 nd ws determined using n unpired Student s t-test. Results Design nd efficcy of short hirpin RNA expression cssettes trgeting NSs nd N in cell culture A set of shrna expression cssettes ws designed to trget conserved sites within the NSs nd N genes. Conservtion ws ssessed from lignments of multiple Interntionl Medicl Press

5 Trgeting RVFV NSs using expressed RNAi virl sequences vilble from GenBnk. Four shrnas trgeting ech of the NSs (shnss1,, shnss3 nd shnss4) nd N (shn1, shn2, shn3 nd shn4) open reding frmes were tested. The shrnas ginst NSs nd N were initilly expressed from the U6 Pol III promoter nd comprised duplex stem of pproximtely 21 bse pirs with 5-nucleotide terminl loop. Silencing efficcy of shrna-expressing plsmids ws initilly ssessed using dul luciferse reporter cotrnsfection ssy. Full-length NSs or N trget sequences were inserted downstrem of the Renill luciferse open reding frme in the psicheck plsmid (Figure 1A). The rtio of Renill to Firefly luciferse ctivity in trnsfected cells ws then used s n indictor of silencing efficcy. The shrnas trgeting NSs nd N sequences of RVFV strins ZH548 nd ZH501 demonstrted >80% knockdown (Figure 1B nd 1C). The shrnas were lso tested ginst NSs sequences of eight other strins of RVFV (Additionl file 1). With the exception of ineffective trgeting of sequences of RVFV CAR1992 nd Lunyo strins by shnss 1, ll other trget sites of the other shrnas were knocked down effectively. Mismtches between the intended guide of shnss1 nd virl cognte re likely to ccount for the diminished silencing efficcy (Additionl file 1). U6 Pol III promoters trnscribe shrnas t high rtes nd my cuse toxicity s result of interference with the nturl RNAi pthwy [42]. To overcome this concern, the most effective shrnas (, shnss3, shnss4 nd shn1) were incorported into weker H1 Pol III expression cssettes. Efficcy of trget knockdown, s ssessed using the dul luciferse reporter ssy, ws retined in the H1 expression cssettes (Figure 1D), which were then used in further investigtions. RNAi ctivtors trgeting multiple RVFV sites To ssess whether silencing efficcy would be improved by simultneous trgeting of NSs nd N, multimeric expression cssettes were generted in which four H1 Pol III shrnas were inserted in hed-to-til ssembly in single plsmid vector (Additionl file 2). Positions of the shrna expression cssettes within the tndem rrngement were vried in eight constructs nd multimeric cssettes were nmed ccordingly. Assessment of efficcy of the multimers ws determined fter cotrnsfection with reporter plsmids contining miniml trgets comprising individul shrna guide cogntes. Although efficcy of knockdown vried between the multimers, the multimer chieved knockdown of ll four miniml trgets (Figure 2A nd Additionl file 2). Expression nd processing of shrnas trgeting RVFV To nlyse expression nd processing of shrnas, RNA ws extrcted from trnsfected cells nd subjected to smll RNA northern blot hybridiztion. Probes complementry to the virl ntisense component of the shrnas detected precursor unprocessed shrna (pproximtely 60 nucleotides) nd the 21-nucleotide intended guide sequence (Figure 2B). Anlysis reveled tht processing of the multimeric expression cssettes ws fr less efficient thn tht of individul expression cssettes. Processed products of shnss4 nd shn1 were detectble, but t low concentrtions, when expressed from multimer cssettes. These dt indicte tht shr- NAs re less efficiently processed from multimer cssettes. The ws tested ginst fulllength NSs nd N trgets in dul-luciferse ssy nd showed similr levels of knockdown ginst the NSs but reduced bility to knockdown N (Figure 3A). This observtion, tken together with the miniml trget dul luciferse reporter ssy dt, indicted tht the multimeric shrnas re expressed nd processed but t reduced levels compred to individul shrnas, This reduced expression is likely to ffect the bility of shn1 to knockdown full-length N trget. shrnas reduce RVFV protein expression in trnsfected cells Western blot nlysis ws crried out to ssess effects of gene silencing on NSs nd N proteins in trnsfected cells. Sequences encoding these proteins were fused to 3 FLAG tg nd plced under control of CMV Pol II promoter to form NSs-3 FLAG nd N-3 FLAG plsmids. Cotrnsfection of plsmids expressing nd shn1 with NSs-3 FLAG nd N-3 FLAG diminished the concentrtion of the tgged proteins (Figure 3B). Cotrnsfection of the multimer hd comprble knockdown of the NSs protein to tht of, but the multimer hd no effect on the levels of N protein. This is most likely to be cused by reduced expression of shn from the multimer, which ws evident from the northern blot nlysis (Figure 2B). As result of the bility of NSs to inhibit globl trnscription nd therefore protein expression [18,19,22,23], bis in the western blot nlysis needed to be excluded. To chieve this, the mutnt forms of NSs, PP1 nd PP2, which hve ltered nucler locliztion proline motifs, were cotrnsfected with the shrna constructs. Similr reductions in the mutnt NSs proteins were observed (Additionl file 3). To exclude functionl effects cused by ddition of the FLAG tg, inhibition of reporter gene expression cused by trnscriptionl suppression ws mesured fter cotrnsfection of cells with NSs- 3 FLAG or wild-type NSs. NSs-3 FLAG nd wild-type NSs showed similr reporter suppression (Additionl file 3), which indictes tht the function of the tgged protein is retined. These dt support the notion tht the shrnas reduce levels of the NSs nd N proteins by n RNAi-bsed mechnism tht is independent of globl suppression of cellulr trnscription. Antivirl Therpy

6 T Scott et l. Figure 1. Knockdown of RVFV NSs nd N trget sites by different shrnas A NSs or N shrna trgets NSs N 798 bp 738 bp psicheck hrluc hfluc B C Men normlized rtio of hrluc:hfluc shnss1 psicheck NSs shnss3 ZH548 ZH501 shnss4 shhiv Men normlized rtio of hrluc:hfluc shn1 psicheck N shn2 shn3 ZH548 ZH501 shn4 shhiv D Men normlized rtio of hrluc:hfluc (H1) (H1) shnss3 (H1) shnss4 (H1) (H1) shn1 (H1) psicheck NSs (ZH548) psicheck N (ZH548) (A) The Rift Vlley fever virus (RVFV) non-structurl S (NSs) nd nucleocpsid (N) genes were inserted into psicheck (Promeg, Fitchburg, WI, USA) dul-luciferse reporter vector downstrem of Renill luciferse (hrluc). The control firefly luciferse (hfluc) cssette, present on the sme vector, is lso illustrted. Both cssettes re under the control of constitutively ctive trnscription regultory elements: herpes simplex virus thymidine kinse nd Simin virus 40 promoters. The short hirpin RNAs (shrnas) were cotrnsfected into HEK293 cells with the (B) NSs nd (C) N from two strins of RVFV, ZH548 nd ZH501, inserted into the psicheck vectors. Men normlized rtios of hrluc:hfluc were determined reltive to mock (plsmid U6+1). An shrna tht trgets HIV (shhiv) ws included s nonspecific control. Mens were obtined from experiments performed in triplicte, with stndrd devitions indicted by error brs. (D) Trgeting of RVFV NSs nd N using H1 Pol III (H1) promoter expressed shrnas. The men of normlized rtios of hrluc:hfluc ctivity ws determined when HEK293 cells were cotrnsfected with plsmids expressing H1 Pol III driven shrnas nd psicheck reporter vectors contining full-length RVFV ZH548 NSs nd N sequences. The mens were obtined from experiments performed in triplicte, with stndrd devitions indicted by error brs. P<0.05, unpired Student s t-test, mde reltive to the mock-trnsfected control. bp, bse pirs Interntionl Medicl Press

7 Trgeting RVFV NSs using expressed RNAi RVFV repliction in cultured cells is inhibited by shrnas trgeting NSs nd N To determine the effects of nti-rvfv shrnas on virl repliction, Huh7 cells were trnsfected with the shrna constructs. At 24 h post-trnsfection, cells were infected with the 1981 SA strin of RVFV. Therefter, superntnts were collected dily for 3 dys nd ELISA mesurement of the N protein [41] ws used to quntify virl repliction. No effect on virl repliction cused by shnss sequences ws observed (Figure 4). These dt re not surprising s previous studies hve shown tht NSs is not required for RVFV repliction in cultured cells [26]. Importntly, reduction in repliction ws effected by shn1. Compred Figure 2. Expression nd processing of shrnas from single nd multimer constructs A B Mrker Men normlized rtio of hrluc:hfluc (H1) (U6) Probe: psicheck NSs2 psicheck NSs3 psicheck NSs4 psicheck N1 (H1) shnss3 (U6) shnss3 (H1) shnss4 (U6) shnss4 (H1) shn1 (U6) shn1 Probe: shnss3 Probe: shnss4 Probe: shn1 60 nt ~60 nt 40 nt 20 nt ~21 nt Probe: U6 snrna (H1) (H1) shnss3 (H1) (H1) shnss3 (H1) shnss4 (H1) shn1 (H1) shnss4 (H1) shn1 (A) The men normlized rtios of Renill:Firefly luciferse (hrluc:hfluc) ctivity determined on extrcts of HEK293 cells tht hd been cotrnsfected with dulluciferse psicheck (Promeg, Fitchburg, WI, USA) reporter vector contining miniml trgets nd short hirpin RNA (shrna) constructs. The single shrnas driven off U6 Pol III (U6) nd H1 Pol III (H1) promoters were included s positive controls. A control shrna trgeting HBV () ws lso included. The mens, reltive to the mock-trnsfected control, were obtined from experiments performed in triplicte, with stndrd devitions indicted by error brs. (B) Smll RNA northern blot nlysis of totl RNA extrcted from Huh7 cells, trnsfected with the indicted H1 Pol III driven shrna constructs. Probes for the, shnss3, shnss4 nd shn1 were used to detect the precursor shrnas nd processed products. The pproximtely 60 bse pir hirpin duplex nd the pproximtely 21 bse pir smll interfering RNA products re nnotted. U6 smll nucler RNA (snrna) ws detected to confirm equl loding. N, nucleocpsid; NSs, non-structurl S; nt, nucleotide. Antivirl Therpy

8 T Scott et l. Figure 3. Reduction in NSs nd N expression by shrnas A Men normlized rtio of hrluc:hfluc shn1 psicheck NSs psicheck N B NSs-3xFLAG N-3xFLAG FLAG α-tubulin shn1 shn1 (A) The men normlized rtios of Renill:Firefly luciferse in HEK293 cells cotrnsfected with the full-length non-structurl S (NSs) nd nucleocpsid (N) trget psicheck (Promeg, Fitchburg, WI, USA) constructs nd short hirpin RNA (shrna) expression vectors. An shrna tht trgets HBV () ws included to demonstrte the specificity of the Rift Vlley fever virus (RVFV)-trgeted shrnas. The mens were obtined from experiments performed in triplicte, with stndrd devitions indicted by the error brs. (B) Western blot nlyses of HEK293 cells cotrnsfected with NSs-3 FLAG or N-3 FLAG cytomeglovirus expressing vectors of the ZH501 strin of RVFV nd either mock,, shn1 or. Anti-FLAG ntibodies were used to detect the FLAG constructs. Detection of -tubulin ws used s loding control. The vlues bove the lnes re the normlized densities of FLAG to -tubulin, which were determined reltive to the mock-trnsfected control (H1 Pol III plsmid). P<0.05, unpired Student s t-test, mde reltive to the mock-trnsfected control. to controls, shn1 demonstrted 29.4% inhibition of cpsid production on dy 2 nd 15.5% reduction on dy 3 (Figure 4). The N protein is required for RVFV repliction nd knockdown of this trget resulted in decrese in the ntigenic mrker of repliction. Reduced expression nd processing of shn1, when generted from the tetrmeric cssette, is likely to result in ttenuted silencing of virl repliction. Collectively these dt indicte tht RNAi expression cssettes my be used to diminish repliction of RVFV. Although NSs-trgeting sequences were not effective ginst the virus in cultured cells, they my well hve utility in vivo, where NSs is importnt for propgtion of RVFV. Short hirpin RNA reduces the pthogenic effects of NSs The bility of NSs to inhibit host mrna expression is unique nd highly pthogenic property of the protein [19]. Attenution of this effect by silencing NSs expression is therefore potentilly of significnt therpeutic benefit. Inhibition of trnscription by NSs, which is more significnt thn prtilly inctive NSs mutnts (PP1 nd PP2), hs been confirmed here using Renill reporter ssy (Figure 5A) nd by others [22]. The dose-relted nture of the effect ws lso demonstrted (Additionl files 3 nd 4). To ssess RNAi-medited llevition of globl cellulr trnscription inhibition by NSs, shnss-expressing vectors were cotrnsfected with Interntionl Medicl Press

9 Trgeting RVFV NSs using expressed RNAi luciferse reporter plsmid nd sequences encoding NSs. Renill luciferse ctivity incresed in the presence of sequences, which indictes tht suppression of trnscription is lessened (Figure 5B). Importntly the llevition ws mintined in the presence of incresing mounts of trnsfected NSs (Additionl files 3 nd 4). This effect ws specific to silencing of wild-type NSs, s cotrnsfection with plsmids encoding NSs mutnts (C13, PP1 nd PP2) or N protein did not ffect reporter ctivity (Figure 5C). The,3,4,-N1 multimeric expression cssettes hd similr effects to those of the single (Figure 5B nd 5C), which is in ccordnce with previous results tht demonstrted tht silencing efficcy ws not enhnced by multimeriztion. The NSs-induced host mrna suppression results in phenotypic chnges tht re consequence of the protein s cytotoxicity. To mesure the possible reversl of this phenomenon by RNAi-medited silencing of NSs, HEK293 cells were cotrnsfected with plsmids constitutively expressing NSs, GFP reporter construct nd shrna expression cssettes. Monolyer formtion ws disrupted in NSs-expressing cells, nd morphology of individul cells confirmed the cytotoxic effect, which ws bsent in control cells expressing NSs mutnts (Additionl files 4 nd 5). Inhibition of trnscription by NSs is lso supported by the visibly-reduced expression of GFP (Figure 5A nd Additionl file 5). An MTT cell vibility ssy, which mesures mitochondril ctivity, showed tht vibility of NSs-expressing cells ws reduced when compred to cells tht were trnsfected with NSs mutnts (C13 nd PP1) or n empty vector (Additionl file 5). When the or the construct ws cotrnsfected with NSs, cell vibility incresed reltive to mock-treted control (Figure 5D). No significnt increse in vibility ws observed when C13, PP1 or n empty vector were cotrnsfected with the shrna constructs. The NSs protein suppresses the interferon pthwy by blocking trnscription nd by direct interction with the interferon-b promoter [20,22]. To ssess whether nti-nss shrnas could llevite the suppression of the interferon-b promoter, HEK293 cells were trnsfected with plsmid expressing NSs or C13 nd reporter construct expressing firefly luciferse driven off n interferon-b promoter. Poly (I:C) ws used to ctivte the interferon-b promoter nd luciferse ctivity ws mesured. The multimeric construct, nd not the mock-treted control, ws cpble of brogting the suppressive effects of NSs on interferon-b promoter ctivity (Figure 5E). The extent of RNAimedited derepression of NSs on poly (I:C)-induced interferon-b promoter ctivity ws comprble to the levels of interferon-b ctivtion in the cells expressing the mutnt NSs, C13. Tken together, these dt indicte tht RNAi-medited silencing of NSs ttenutes the pthogenic effects of this protein. Figure 4. RVFV chllenge ssy Reltive virl output Discussion RVFV SA Time post-infection, h shn1 Virus only No virus The short hirpin RNA (shrna) constructs were trnsfected into Huh7 cells 24 h prior to infection with Rift Vlley fever virus (RVFV; isolte 1981 SA). The multiplicity of infection ws t 50% tissue culture infective dose of Virl prticles from the culture superntnt were hrvested every 24 h for 72 h nd N ws detected by indirect ELISA. Wells contining only virus nd n shrna ginst HBV () were included s negtive controls. Mens were obtined from mesurements performed in duplicte. Hrnessing the RNAi pthwy to chieve powerful nd specific silencing of virl genes hs promising therpeutic ppliction. Both expressed nd synthetic RNAi ctivtors inhibit the repliction of wide vriety of viruses [43]. Preclinicl proof-of-concept nd lso erly clinicl trils hve demonstrted fesibility of RNAi-bsed tretment of HIV-1, HBV, HCV, respirtory syncytil virus nd the hemorrhgic Ebol virus. The fulminte nture nd high repliction rte of hemorrhgic fever viruses poses significnt chllenges for dvncing RNAi-bsed therpy. Ebol is inhibited by sirnas dministered 1 h fter infection of guine pigs nd 30 min post-infection of non-humn primtes [32,33]. In clinicl setting, ptients with RVFV infection re most likely to require therpeutic intervention upon presenttion with symptoms. Typiclly this coincides with the pek of the viremi. Circulting virl prticle counts my rech concentrtions s high s infectious prticles/ml in humns [26,32], which my overwhelm the efficcy of therpeutic gene silencing. Interestingly, non-ftl infections re ssocited with lower pek viremi [26,44,45] nd indicte tht host immune responses my be cpble of Antivirl Therpy

10 T Scott et l. Figure 5. Allevition of the trnscriptionl suppression nd ssocited toxicities induced by NSs when trgeting NSs with shrnas A 15 B 7.5 hrluc, hrluc, NS 0 NSs 548 (WT) NSs C13 NSs PP1 NSs PP2 Empty vector 0.0 NSs 548 (WT) C Normlized hrluc b shn NSs 548 (WT) NSs PP1 NSs PP2 NSs C13 N 548 (WT) Empty vector (A) The men Renill luciferse (hrluc) ctivity ws determined when HEK293 cells were cotrnsfected with cytomeglovirus (CMV) plsmids expressing NSs nd hrluc. A truncted NSs, C13, prtilly ctive NSs mutnts, PP1 nd PP2, nd n empty vector were included s negtive controls. The mens were obtined from experiments performed in triplicte, with stndrd devitions indicted. (B) The men hrluc ctivity ws determined fter cotrnsfection of HEK293 cells with CMV plsmids expressing NSs nd Renill luciferse with n, nd n shrna trget to HBV () or mock-treted cells. The mens were obtined from experiments performed in triplicte, with stndrd devitions indicted. (C) The men hrluc ctivity ws determined when HEK293 cells were cotrnsfected with CMV plsmids expressing NSs nd hrluc with, shn1,, nd. Other CMV-expressed trgets were included. These represented truncted NSs, C13, prtilly ctive NSs mutnts, PP1 nd PP2, N nd n empty vector. The mens were obtined from experiments performed in triplicte, with stndrd devitions indicted. (D) An MTT ssy ws performed on HEK293 cells cotrnsfected with CMV plsmid expressing NSs, C13, PP1 nd n empty vector with, nd. Mens were obtined from experiments performed in triplicte, with stndrd devitions indicted by error brs. (E) HEK293 cells were cotrnsfected with CMV plsmid expressing NSs or C13 nd the pif-luc ( plsmid expressing Firefly luciferse driven off n IFN-b promoter) vector with. Cells were treted with Poly (I:C) 16 h prior to Firefly luciferse mesurement. The dt re shown s fold-ctivtion of luciferse reltive to no-poly (I:C) controls. Mens were obtined from experiments performed in triplicte, with stndrd devitions indicted. P<0.05, unpired Student s t-test; b P<0.05, unpired Student s t-test, mde reltive to the mock-trnsfected control. WT, wild-type Interntionl Medicl Press

11 Trgeting RVFV NSs using expressed RNAi Figure 5. Continued D b E Mitochondril dehydrogense ctivity (normlized) NSs 548 (WT) NSs C13 NSs PP1 Empty vector IFN-β promoter ctivity (fold ctivtion reltive to no poly I:C control) NSs 548 (WT) NS NSs C13 eliminting lower-level virus infection. Therpy for n infection my therefore need only to diminish circulting virl prticle numbers rther thn eliminting the virus completely, s is required for tretment of slowlyreplicting viruses such s HIV or HBV. RNAi-bsed therpies ginst cute infections my therefore be useful to reduce virl lods below threshold to chieve virl clernce. An extensive chrcteriztion of RNAi efficcy fter infection with highly pthogenic viruses will be required to determine the vlue of RNAi-bsed therpies in clinicl setting. In this study, we provide support for the potentil therpeutic utility of expressed shrnas tht counter RVFV. RNAi ctivtors trgeting NSs- nd N-encoding RNA proteins llevite RVFV pthogenic effects nd lso inhibit virl repliction. The shrnas designed to trget NSs efficiently silenced virl trgets in reporter gene construct, knocked down virl NSs nd llevited pthogenic effects cused by this protein. Trgeting NSs in cell culture model of virl infection did not inhibit virl repliction, which is consistent with previous studies demonstrting tht NSs is dispensble for repliction in vitro [46]. However, it is well-described tht the removl of NSs ttenutes virl pthogenesis in vivo. This is evident in ttenuted vccine mutnts nd in the nturlly occurring RVFV strin, C13, which lck functionl NSs [25,26]. We lso demonstrted tht the mutnt version of NSs, PP1, did not exhibit cytotoxicity. PP1 mutnts re retined in the cytoplsm, suggesting tht the nucler locliztion of NSs is required for cytotoxic side effects, which is likely owing to the effects of NSs on globl cellulr trnscription suppression [19]. An lterntive mechnism of NSs cytotoxicity is through its interction with trnscription fctor YY1 on pericentromeric DNA sequences resulting in segregtion nd cohesion defects [20,22,47]. Assessment of whether these specific effects of NSs re llevited by the shrnas would be worthy of investigtion. Importntly, the toxicity of NSs is suspected to ply role in tertogenic disorders nd bnormlly high bortion rtes in nimls [47,48], nd sustined NSs inhibition my thus be of potentil vlue in livestock frming where RVFV outbreks re common. A potentilly interesting question remins whether RFVF NSs is cpble of directly suppressing the expression or function of fctors ssocited with the RNAi pthwy. Erlier it ws reported tht LACV inhibits exogenous sirna-medited post-trnscriptionl silencing [36]. However, these effects my be indirect since LACV NSs, like RVFV NSs, hs been recently shown to be n interferon ntgonist [49]. Not surprisingly, LACV NSs lso functions in similr wy to RVFV NSs by blocking RNA polymerse II ctivity [49]. In our study we show tht shrnas trgeted to NSs re functionlly cpble of reducing NSs protein levels, derepressing NSs-medited inhibition of trnscription, nd lleviting the cellulr toxicities of NSs. While we do not rule out pleiotropic effect for NSs on the RNAi pthwy, our dt show tht robust RNAi pthwy, t the level of ctive Dicer nd Argonute, cn function in the presence of NSs. Antivirl Therpy

12 T Scott et l. Combintoril RNAi is the use of two or more RNAi effectors ginst given trget within virus [50]. We ttempted this strtegy becuse RNA viruses re prone to muttionl escpe, nd the simultneous trgeting of multiple sequences is likely to prevent the formtion of RNAi-resistnt virl qusispecies [51]. Moreover, combintoril RNAi pproch dds to the ccumultive suppressive effects on virl repliction, gined by trgeting multiple proteins within the virl life cycle. Our multimeric expression cssette, consisting of four tndem H1-driven shrnas, showed reduced sirna guide strnd production for ech embedded shrna (Figure 2B). While we cnnot discount the contribution of sequence-specific positionl effects on expression nd processing of ech shrna, since shrna precursors were lso reduced, it is likely tht tndem rrys of H1 Pol III promoters resulted in wekened expression for ech promoter, s observed by severl others [51 54]. Interestingly, the three nti-nss shrnas pper to combine to effect comprble knockdown to tht observed with the individul shrna ginst NSs (Figures 3B nd 5B E). Combined guide strnds t low concentrtion my hve n dditive effect nd potentite equivocl suppression of highly-bundnt single guide sequence. This might suggest tht improvement in our current multimeric design is possible by incresing the number of shrnas trgeted to N. Alterntively, incresed potency my be chieved by rising the dose of ech expressed shrna by replcing H1 with stronger Pol III promoter. While cre must be tken to ensure the nturl RNAi pthwy is not ffected, this ltter pproch my ironiclly prove to be sfer in the context of multimeric design. This study dds to the growing body of knowledge which supports the use of RNAi-bsed gene silencing s fesible pproch for therpy for virl infections, nd in prticulr to combt RVFV. Nevertheless, there re significnt obstcles tht remin before RNAi-bsed RVFV therpy is relized. These include chieving rpid nd highly effective virl trget gene silencing, sustined silencing for n dequte durtion to chieve therpeutic effect, efficient delivery to trget tissues nd ensuring tht unintended off-trget effects re voided. Mny of these concerns re similr to those fcing the development of RNAi-bsed tretment of other virl infections. Despite these hurdles, the wider topic of developing RNAi-bsed ntivirls is progressing rpidly. Achieving successful RNAi-bsed RVFV tretment will benefit from technologicl dvnces tht re mde in the broder field. Acknowledgements We would like to cknowledge the ssistnce of Petrus Jnsen vn Vuren from the Specil Pthogens Unit of the NICD-NHLS nd Sheen Symn for ssistnce with northern blot nlysis. Funding is cknowledged from the Poliomyelitis Reserch Foundtion (PRF), Ntionl Reserch Foundtion (NRF), Medicl Reserch Council (MRC) the Germn Acdemic Exchnge Service (DAAD) nd the Stell nd Pul Loewenstein Chritble nd Eductionl Trust. Disclosure sttement The uthors declre no competing interests. Additionl files Additionl file 1: Supplementry figure illustrting knockdown of eight of strins of RVFV NSs nd sequence of the CAR1662 nd Lunyo strins ligned with the 548 wild-type sequence cn be found t 11-OA-2128_Scott_Add_file1.pdf Additionl file 2: Supplementry figure illustrting the determintion of expression nd processing of shrnas from single nd multimer constructs cn be found t 11-OA-2128_Scott_Add_file2.pdf Additionl file 3: Supplementry figure illustrting tht knockdown of NSs is independent of its trnscriptive suppressive effects cn be found t OA-2128_Scott_Add_file3.pdf Additionl file 4: Supplementry methods cn be found t AVT-11-OA-2128_Scott_Add_file_4.pdf Additionl file 5: Supplementry figure illustrting the cytotoxic effects of NSs cn be found t OA-2128_Scott_Add_file_5.pdf References 1. Bishop DH, Clisher CH, Csls J, et l. Bunyviride. Intervirology 1980; 14: Peters CJ, Liu CT, Anderson GW, Morrill JC, Jhrling PB. Pthogenesis of virl hemorrhgic fevers: Rift Vlley fever nd Lss fever contrsted. Rev Infect Dis 1989; 11 Suppl 4:S743 S Gerdes GH. Rift vlley fever. Vet Clin North Am Food Anim Prct 2002; 18: Miller BR, Godsey MS, Crbtree MB, et l. Isoltion nd genetic chrcteriztion of Rift Vlley fever virus from Aedes vexns rbiensis, Kingdom of Sudi Arbi. Emerg Infect Dis 2002; 8: Jupp PG, Kemp A, Grobbelr A, et l. The 2000 epidemic of Rift Vlley fever in Sudi Arbi: mosquito vector studies. Med Vet Entomol 2002; 16: Interntionl Medicl Press

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J Biol Chem 1994; 269: Jnsen vn Vuren P, Pwesk JT. Lbortory sfe detection of nucleocpsid protein of Rift Vlley fever virus in humn nd niml specimens by sndwich ELISA. J Virol Methods 2009; 157: Grimm D, Wng L, Lee JS, et l. Argonute proteins re key determinnts of RNAi efficcy, toxicity, nd persistence in the dult mouse liver. J Clin Invest 2010; 120: Arbuthnot P. Hrnessing RNA interference for the tretment of virl infections. Drug News Perspect 2010; 23: Bird BH, Bwiec DA, Ksizek TG, Shoemker TR, Nichol ST. Highly sensitive nd brodly rective quntittive reverse trnscription-pcr ssy for highthroughput detection of Rift Vlley fever virus. J Clin Microbiol 2007; 45: Antivirl Therpy