Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: Stability and interactions with the core RNAi machinery

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Contents lists available at ScienceDirect

Journal of Insect Physiology

journal homepage: www.elsevier .com/ locate/ j insphys

Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5cells: Stability and interactions with the core RNAi machinery

http://dx.doi.org/10.1016/j.jinsphys.2014.03.0020022-1910/� 2014 Published by Elsevier Ltd.

⇑ Corresponding authors. Tel.: +30 2106503681 (L. Swevers).E-mail addresses: [email protected] (L. Swevers), [email protected]

(J. Sun).

Please cite this article in press as: Swevers, L., et al. Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: Stability and interactiothe core RNAi machinery. Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.03.002

Luc Swevers a,⇑, Anna Kolliopoulou a, Zheng Li b, Maria Daskalaki c, Frederic Verret c,Kriton Kalantidis c, Guy Smagghe d, Jingchen Sun b,⇑a Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research ‘‘Demokritos’’, P. Grigoriou & Neapoleos Str, AghiaParaskevi Attikis, 153 42 Athens, Greeceb Guangdong Engineering Research Center of Subtropical Sericulture and Mulberry Resources Protection and Safety, Guangdong Provincial Key Lab of Agro-animal Genomics andMolecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of Chinac Department of Biology, University of Crete, Voutes University Campus, 700 13 Heraklion, Crete, Greeced Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

a r t i c l e i n f o a b s t r a c t

333435363738394041424344

Article history:Received 31 October 2013Received in revised form 3 March 2014Accepted 7 March 2014Available online xxxx

Keywords:Cytoplasmic polyhedrosis virusBmCPVdsRNARNAiBm5 cells

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While several studies have been conducted to investigate the stability of dsRNA in the extracellular med-ium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has beenintroduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus(BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as adetection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellularenvironment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence hadno effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also foundthat transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, evenwhen co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggestingthat the neutralization by RNAi does not play a role in the establishment of an in vitro culture system.The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the estab-lishment for an in vitro culture system for BmCPV.

� 2014 Published by Elsevier Ltd.

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1. Introduction

Today, RNA interference (RNAi) is used as a tool to achieve po-tent and specific gene knockdown in eukaryotes (Carthew andSontheimer, 2009; Jinek and Doudna, 2009; Moazed, 2009; Siomiand Siomi, 2009; Huvenne and Smagghe, 2010). The trigger of RNAiis dsRNA which, in the canonical pathway, is processed by Dicerenzymes to small interfering RNAs (siRNAs), typically consistingof a 21–22 nucleotide (nt) dsRNA duplex with 2–3 nt 30-overhangs(Myers et al., 2003). The siRNAs are then assembled in RNA-induced silencing complexes (RISCs) that retain one strand of theduplex. RISC complexes, with Argonaute proteins functioning ascatalytic components, subsequently use the retained single-stranded siRNAs as ‘guides’ to search RNA populations for comple-mentary sequences as targets for cleavage (Liu et al., 2004).

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In insects, exogenous dsRNA is preferentially processed by Di-cer-2 in conjunction with a dsRNA-binding protein, R2D2, and gen-erated siRNAs are loaded into RISC complexes with Ago-2 ascatalytic unit (Tomari et al., 2007). Although Dicer-2 by itselfshould be able to directly (non-specifically) degrade RNA targetswith dsRNA structure, it is believed that the generation of smallRNAs and their loading in catalytic Argonaute-containing com-plexes acts as a sequence-specific mechanism to achieve efficientsilencing (van Rij et al., 2006).

Despite its identification as the trigger of RNAi as defense mech-anism, surprisingly little is known regarding the stability of dsRNAin insects. DsRNA stability has been considered as a contributingfactor to explain differences in RNAi efficiency among insects(Terenius et al., 2011; Garbutt et al., 2013; Scott et al., in press).A few studies have investigated dsRNA stability in the hemolymph,the gut content, the saliva and the food source in which it wasexogenously added (Li et al., 2011; Garbutt et al., 2013; Liu et al.,2013; Allen and Walker, 2012; Christiaens et al., in press). It hasalso been demonstrated that dsRNA can be efficiently taken upby cells (Saleh et al., 2006; Swevers and Smagghe, 2012). However,

ns with

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very little is known regarding the stability and fate of dsRNA insidethe cells after uptake, although the fate of siRNA/dsRNA inside thecell is gaining more interest recently [see, for instance, Wang andHuang (2013)].

In this article, the stability inside the cells was investigated fornaturally occurring dsRNA fragments, corresponding to the seg-mented genome of Bombyx mori cytoplasmic polyhedrosis virus(BmCPV; Reoviridae). Besides the study of dsRNA stability, it wasalso of interest to observe whether transfection of the BmCPV gen-ome into B. mori (silkmoth)-derived Bm5 cells would result in thegeneration of a functional virus, as is routinely observed aftertransfection of the DNA genome of B. mori nuclear polyhedrosisvirus (BmNPV) (O’Reilly et al., 1992). BmCPV is one of the majorpathogens of the silkworm and causes enormous damage to thesericulture industry (Wu et al., 2009; Mori and Metcalf, 2010).Infection in larvae is initiated by ingestion of polyhedra which dis-solve in the alkaline environment of the gut to release viral parti-cles (Payne and Mertens, 1983). Usually the infection is limitedto the epithelial lining of the gut although spreading to other tis-sues has also been observed. Unfortunately, no in vitro cell culturesystem for BmCPV has been developed (Hill et al., 1999), and theavailability of such cell-based system which would be of great ben-efit to investigate and understand the pathogenicity of the virusand to develop methods to combat the disease.

In this study, the stability of the segmented dsRNA of BmCPVgenome was investigated after transfection in Bm5 cells in theabsence or presence of dsRNA targeting RNAi core machinerygenes, such as Dicer-2 (Dcr2) and Argonaute-2 (Ago2). Transfectionof BmCPV dsRNA genome in silkworm-derived Bm5 cells was alsocarried out to see whether an in vitro culture system for this path-ogen could be established. Simultaneously, we investigatedwhether transfection of BmCPV genomic dsRNA or expression oftwo genes of the BmCPV genome, RNA-dependent RNA polymerase(RdRp), and non-structural 5 (NS5) could affect RNAi efficiency ina luciferase-based silencing assay. Our results provide the basisfor a discussion of the mechanism of dsRNA stability in Bm5 cells.

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2. Materials and methods

2.1. Isolation and purification of BmCPV polyhedra

BmCPV(C) was cultivated and purified by established protocolsat South China Agricultural University (Tan et al., 2001). SilkmothB. mori 4th instar larvae were orally infected with BmCPV usingsmall pieces (2 cm � 2 cm) of mulberry leaves that were soakedin BmCPV solution (106 polyhedra/ml) for a few minutes. Larvaewere left to feed for 6 h on BmCPV-soaked mulberry leaves andwere provided normal leaves after that period. At 6–10 days afterinfections, the midgut of diseased silkworm was dissected afterremoval of head, gut content and silk glands, homogenized indistilled water and centrifuged at 10000g for 10 min. The freevirions remained in the supernatant; the polyhedra were containedin the pellet. Polyhedra were purified by sucrose density ultracen-trifugation as described (Hayashi and Bird, 1969).

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2.2. Preparation of BmCPV dsRNA genomic fragments

A purified polyhedra solution (108 polyhedra/ml) was mixed at1:1 (v:v) with urea/SDS lysis buffer (Drevet et al., 1995) and incu-bated at 37 �C for 30 min. Then nucleic acid was extracted by phe-nol/chloroform and precipitated with 1/10 volume of 2.5 Mammonium acetate and 2.5 volumes of ethanol. To remove con-taminating DNA, BmCPV dsRNA was treated with RNase-freeDNase I (Thermo Scientific) for 30 min at 37 �C (2 U per lg of

Please cite this article in press as: Swevers, L., et al. Transfection of BmCPV genthe core RNAi machinery. Journal of Insect Physiology (2014), http://dx.doi.org

nucleic acid) in the presence of RiboLock RNase inhibitor (ThermoScientific) (4 U per lg of nucleic acid).

2.3. Expression and reporter plasmids

BmCPV ORFs were amplified by RT-PCR using BmCPV genomeinformation available at the Reoviridae website (http://www.reoviridae.org/dsRNA_virus_proteins/Cypovirus.htm). For the iso-lation of BmCPV genes, BmCPV genomic dsRNA was first denaturedat 60 �C for 30 min in 50% DMSO. To isolate the ORF of the NS5 gene,reverse transcription reactions were carried out using the primer50-AGTAAATCCCAGGCGTAAACCG-30 (42 �C for 1 h) and PCR reac-tions were performed using the primers 50-ACGGATCATCATG-GAAGCCTTC-30 and 50-ATCAGCGACACTATTCTGATAC-30 (35 cyclesof 94 �C for 45 s, 55 �C for 45 s and 72 �C for 1 min). To isolate theORF of the RdRp gene, reverse transcription reactions were carriedout using the primer 50-CGTCTGAAATGTTACCGAACAC-30 and PCRreactions were performed using the primers 50-CGTCTGAAATGTTACCGAACAC-30 and 50-ACTCATCTACACGTTATCTGCC-30 (40 cyclesof 94 �C for 1 min, 55 �C for 1 min and 72 �C for 3 min). PCR reac-tions were performed using a mixture of Taq polymerase (HyTest;1.5 U per reaction) and Pfu polymerase (Thermo Scientific; 1 Uper reaction). PCR fragments were cloned in pBluescript vectorand their identities were confirmed by sequencing.

To construct the expression vectors, the NS5 and RdRp ORFswere amplified with the primer pairs NS5F: 50-AATTGGATCCCAACATGGAAGCCTTCTTACTC-30/NS5R: 50-ATCAGCGACACTATTCTGATAC-30 and RdRpF: 50-AATTGGATCCCAACATGTTACCGAACACAAAACTAC-30/RdRpR: 50-ACTCATCTACACGTTATCTGCC-30, respectively(stop codons in reverse primers in bold). In both cases, the forwardprimer contains a BamHI-site (underlined), a Kozak translation ini-tiation sequence (italic) upstream of the start codon (bold), and theconfiguration allows in-frame cloning with the N-terminal Myc se-quence of the pEA-Myc expression vector (Douris et al., 2006), thusgenerating the expression plasmids pEA-Myc-NS5 and pEA-Myc-RdRp.

For the isolation of the ORF encoding the B2 RNAi inhibitor pro-tein from nodavirus, RNA isolated from Hi5 cells was used for re-verse transcription reaction using primer Noda-U7 (Li et al.,2007). The ORF of B2 was subsequently amplified by primers 50-AAACGATGCCAAGCAAACTCGCG-30 and 50-CACCTACAGTTTTGCGGGTG-30 and cloned in pBluescript. For in-frame cloning with theN-terminal Flag tag of the pEA-Flag vector the primers 50-AATTG-GATCCCAACATGCCAAGCAAACTCGCG-30 and 50-AATTGGATCCCTACAGTTTTGCGGGTGGGGGGTC-30 were used.

2.4. Bm5 tissue culture cells and transfections

Bm5 cells, derived from ovarian tissue (Kolliopoulou and Swe-vers, 2013), were maintained in IPL-41 medium (Gibco) supple-mented with 10% fetal bovine serum (FBS) and cultured at 27 �C.Transfection was carried out according to established protocols(Johnson et al., 1992) using Escort IV (Sigma) as transfection agent.

To investigate the stability of dsRNA, Bm5 cells were transfec-ted with 0.5 lg/ml dsRNA of BmCPV in the presence of 0.5 lg/mldsRNA specific to malE (dsmalE), to Dicer-2 (dsDcr2) or to Argona-ute-2 (dsAgo2). Preparation of dsRNA targeting the RNAi machin-ery gene Dicer-2 (dsDcr2) and Argonaute-2 (dsAgo2) was carriedout as described before (Kolliopoulou and Swevers, 2013). In an-other series of experiments, 0.2 lg/ml of BmCPV genomic dsRNAwas transfected in the presence of 1.0 lg/ml of pEA-Myc-RdRp orpEA-PAC (negative control) expression vector and 0.2 lg/ml ofpBmIE1. Cells were collected at different times after transfection(from 2 h up until 8 days; see Section 3) for RNA extraction andpreparation for RT-PCR.

omic dsRNA in silkmoth-derived Bm5 cells: Stability and interactions with/10.1016/j.jinsphys.2014.03.002

http://www.reoviridae.org/dsRNA_virus_proteins/Cypovirus.htm



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For expression studies, 1.0 lg/ml of pEA-Myc-NS5, pEA-Myc-RdRp or pEA-Flag-B2 expression vector was used together with0.5 lg/ml of pBmIE1 helper plasmid encoding the i.e. -1 gene fromBmNPV (Lu et al., 1996). Cells were harvested at 2–3 days aftertransfection for expression analysis by Western blot.

To investigate the capacity of BmCPV genomic dsRNA to triggergene silencing, 1.0 lg/ml of pEA-Myc-NS5 expression vector and0.2 lg/ml of pBmIE1 were co-transfected with 0.05 lg/ml or0.01 lg/ml of BmCPV genomic dsRNA (the latter in the presenceof 0.04 lg/ml of dsmalE).

2.5. Nucleic acid extraction and RT-PCR

Cell pellets were dissolved in urea/SDS lysis buffer and total nu-cleic acid was extracted as described (Drevet et al., 1995). Thequantity of extracted nucleic acid was assessed with a NanoDrop1000 Spectrophotometer (Thermo Scientific) and/or by electropho-resis on 1% (w/v) agarose gels. First-strand complementary DNA(cDNA) synthesis was performed using RevertAid reversetranscriptase (Thermo Scientific) and oligo-dT or BmCPV segment10-specific primer 50-AGTAAAAGTCAGTATCTTACCGGC-30 (S10F).Oligo-dT primed cDNA was used to detect cellular actin mRNAby PCR using actin primers (Machado et al., 2007). BmCPVpolyhedrin gene was detected with the primers 50-AGGATCATGGCAGACGTAGC-30 and 50-GCAATCACTGACGGTTACTCAG-30 usingS10-primed cDNA as template. Cycling conditions were 94 �C for30 s, 55 �C for 30 s and 72 �C for 30 s for actin mRNA and were94 �C for 1 min, 55 �C for 1 min and 72 �C for 1 min for BmCPVpolyhedrin (25–40 cycles).

Quantification of PCR fragments in agarose gel was carried outby BioCapt image quantification software (Vilber Lourmat, Eber-hardzell, Germany). Quantities of PCR fragments correspondingto the housekeeping gene actin were used for normalization. Rela-tive amounts of the dsRNA fragments were compared with theirlevels at 2–2.5 h after transfection (set as 100%). Statistically signif-icant differences in normalized amounts of dsRNA in differentexperimental conditions or at different time points after transfec-tion were determined by ANOVA analysis.

2.6. Protein extracts preparation and Western blot

Transfected cells cultures were collected and separated as solu-ble cellular extracts and insoluble cellular pellets by centrifugationand freeze–thawing as described (Liu et al., 2012). Protein fractionswere electrophoresed on 10% polyacrylamide gels and subjected toWestern blot analysis as described (Georgomanolis et al., 2009).Proteins were transferred to Hybond ECL membranes (Amersham)and the membrane was blocked with 10% non-fat milk. To detect,Myc- and Flag-tagged proteins, Western blot analysis was carriedout using mouse anti-Myc (Cell Signalling; at 1:1000) or rabbitanti-Flag (Sigma; at 1:1000) as primary antibody and HRP-conju-gated anti-mouse or anti-rabbit (Chemicon; at 1:2000) as second-ary antibody. To detect the GW body marker Gawky, total cellextracts were prepared in cracking buffer (Georgomanolis et al.,2009) and subjected to SDS–PAGE and Western blot. Gawky pro-tein was detected using anti-Gawky (Schneider et al., 2006) andanti-guinea pig HRP (Chemicon) antibodies (both at 1:1000).

2.7. RNAi response assay in Bm5 cells

RNAi experiments were carried out as described before (Swe-vers et al., 2011; Kolliopoulou and Swevers, 2013).

To investigate possible interference of BmCPV dsRNA on RNAiefficiency, Bm5 cells were transfected with 3.3 lg/ml of ecdy-sone-responsive luminescence reporter pERE.bA.luc, 0.17 lg/mlof ecdysone-responsive fluorescence reporter pEcRE.bA.gfp, as well


as with 0.8 lg/ml of BmCPV genomic dsRNA. DsMalE, which is spe-cific for maltose binding protein mRNA of Escherichia coli (Zhuet al., 2011), was used as non-specific control dsRNA. Moreover,all samples were transfected with 0.01–0.1 lg/ml of luciferase spe-cific dsRNA (dsLuc).

To investigate the expression of viral genes on RNAi efficiency,Bm5 cells were transfected with 0.9 lg/ml of pERE-Luc, 0.2 lg/mlof pERE-gfp, 0.9 lg/ml of expression plasmid pEA-PAC (negativecontrol), pEA-Myc-NS5, pEA-Myc-RdRp or pEA-Flag-B2, and0.05 lg/ml of dsLuc or non-specific dsMalE as a negative control.Two days after transfection, the ecdysone agonist tebufenozidewas added to a final concentration of 500 nM to induce the ecdy-sone reporter genes pERE-luc and pERE-gfp. Soluble cellularextracts of transfected cell populations were prepared as describedand directly used for fluorescence measurements or processed forluminescence measurements using the Steady-Glo� LuciferaseAssay System kit (Promega) according to the manufacturer’sinstructions. Specific luciferase activities were calculated as lumi-nescence/fluorescence ratios. Both fluorescence and luminescencemeasurements were carried out with an Infinite M200 lumino-meter (Tecan, Switzerland). Statistical analysis of differences innormalized luciferase activity was carried out by unpaired t-testanalysis.

2.8. Agro-infiltration

ORFs of BmCPV NS5 and nodavirus B2 were cloned as BamHI-fragments in the primary cloning vector pART7, downstream ofthe cauliflower mosaic virus 35S promoter. The 35S expression cas-sette was subsequently cloned as a NotI-fragment into the T-DNAvector pART27 (binary vector system; Gleave, 1992). Agro-infiltra-tion of GFP only or in combination with either BmCPV NS5, noda-virus B2 or P19 ORFs were carried out in leaves of Nicotianabenthamiana transgenic line 16C stably expressing a GFP trans-gene. Phenotype of co-suppression of the endogenous GFP trig-gered by the additional infiltrated GFP has been reported inHamilton et al. (2002). Co-infiltration of GFP with the well-knownsuppressor of RNA silencing P19 sustains GFP fluorescence and isused as a positive control.

2.9. Subcellular localization of fluorescent dsRNA andimmunofluorescence

DsRNA specific for GFP (dsGFP) was synthesized as described(Liu et al., 2013) and labelled with fluorescein using the SilencerSiRNA Labeling Kit (Ambion) according to the instructions of themanufacturer. Based on absorbance measurements at 260 nmand 492 nm, the base:dye ratio was calculated as 132. This indi-cates the presence of approximately 5–6 fluorescein groups oneach dsGFP molecule. Fluorescein-labelled dsGFP (2.5 lg/ml) wastransfected in Bm5 cells and cells were collected after 16 h for fluo-rescence and immunofluorescence staining (Labropoulou et al.,2008). Cells were fixed with 4% formaldehyde in PBS for 20 minand subsequently permeabilized with PBS supplemented with0.1% (v/v) Triton X-100 (PBS-T) for 10 min. Cells were stained over-night at 4 �C with anti-Gawky antibody (Schneider et al., 2006) at1:500. Following five washes in PBS, AlexaFluor-conjugated goatanti-guinea pig secondary antibody (Invitrogen; at 1:2000) wasadded for 1 h at room temperature. Cells were mounted in Mowiol4–88 (Sigma) and examined under a fluorescence microscope(Zeiss Axiovert 25 inverted microscope). For confocal microscopy,the cells were treated as described above and observed in a Bio-Rad confocal microscope (MRC 1024 ES) equipped with Lasersharpsoftware (Bio-Rad) and a kryptonargon laser.



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3. Results

3.1. Stability of BmCPV dsRNA in transfected Bm5 cells

Analysis of nucleic acid extracts of purified BmCPV polyhedraby agarose gel electrophoresis revealed the 10 dsRNA segmentsthat constitute the BmCPV genome and range in size from 4.2 kbuntil 0.95 kb (sequences available at http://www.reoviridae.org/dsRNA_virus_proteins/Cypovirus.htm) (Fig. 1A). BmCPV dsRNAgenome was transfected at 0.5 lg/ml into Bm5 cells in the pres-ence of dsRNA that targets the RNAi core machinery genes Dcr2and Ago2 (also at 0.5 lg/ml). Our previous studies demonstratedthat these dsRNAs could specifically knock-down the expressionof the targeted genes and influence the RNAi efficiency in reporterassays (Kolliopoulou and Swevers, 2013). DsRNA targeting malE(the maltose-binding protein of E. coli) was used as a negative con-trol. At different times after transfection (2.5 h, 2.5 days, 5 days,8 days), aliquots were collected from the transfected cells and pro-cessed for RNA extraction (N = 3). As shown in Fig. 1B, RT-PCRexperiments showed that the polyhedrin gene of BmCPV could bedetected by RT-PCR efficiently for a period of up to 8 days aftertransfection. While the amount of BmCPV dsRNA has dropped sig-nificantly between 2.5 h and 2.5 days post infection (to 43 ± 16%,69 ± 6% and 39 ± 8% for the dsmalE, dsDcr2 and dsAgo2 conditions,respectively), the intracellular levels remained stable between2.5 days and 8 days (Fig. 1B). Statistically significant differencesin the amount of BmCPV dsRNA were found between samplesco-transfected with dsDcr2 and dsAgo2 at 2.5 days after

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Fig. 1. Stability of BmCPV dsRNA genome in transfected Bm5 cells. Panel A: Ethidium broand a preparation of BmCPV dsRNA genome. Panel B: BmCPV dsRNA genome at 0.5 lg/dsAgo2, as indicated. BmCPV polyhedrin dsRNA and cellular actin mRNA was detected bethidium bromide-stained agarose gel showing the amplified PCR fragments. Amplificagraph with the quantification of relative amounts of dsRNA normalized to actin mRNA (three different conditions (co-transfection with dsmalE, dsDcr2 and dsAgo2) is set at 100%amount.


transfection (P = 0.006) and between samples co-transfected withdsmalE and dsDcr2 at 5 days after transfection (P = 0.037). No sta-tistically significant differences in the amount of BmCPV dsRNAwere observed in the different experimental conditions at 8 daysafter transfection (Fig. 1B).

3.2. Interactions of BmCPV genomic dsRNA with the core RNAimachinery

We also tested whether co-transfection of BmCPV dsRNA gen-ome could interfere with dsLuc-mediated silencing of the lucifer-ase reporter. In the presence of 0.8 lg/ml of BmCPV dsRNA,dsLuc silenced luciferase activity to 18.7 ± 3.5% (0.01 lg/ml) and2.8 ± 0.5% (0.1 lg/ml) (Fig. 2A). However, similar values were ob-tained when 0.8 lg/ml of control dsRNA (dsmalE) was co-transfec-ted (reduction to 21.6 ± 0.6% at 0.01 lg/ml of dsLuc and to2.1 ± 0.3% at 0.1 lg/ml of dsLuc). Statistical analysis showed no dif-ference between the two types of dsRNA that were co-transfected(P values of 0.25 and 0.47).

However, transfected BmCPV dsRNA was recognized by theRNAi machinery and could induce a silencing response. Whenlow amounts of BmCPV dsRNA genome were co-transfected withthe expression plasmid for Myc-tagged NS5 (encoded by segment9 of the BmCPV dsRNA genome), clear interference with theexpression of the protein was observed (Fig. 2B). The silencing ofotherwise efficiently expressed Myc-NS5 (Fig. 4A) was dose-dependent and occurred at a similar sensitivity as that was ob-served for dsLuc-mediated silencing of the luciferase reporter.

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dsDcr2 dsAgo2

mide-stained agarose gel showing Lambda DNA double-digested with HincII/HindIIIml was transfected to Bm5 cells in the presence of 0.5 lg/ml of dsmalE, dsDcr2 ory RT-PCR at different times after transfection. At the top is shown a representativetion cycles were 35� for BmCPV polyhedrin and 28� for actin. Below is shown theN = 3). In the graph, the amount of dsRNA detected at 2.5 h after transfection in the

. The quantities of dsRNA detected at later time points are expressed relative to this





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Fig. 2. Interaction of BmCPV dsRNA with the RNAi machinery in Bm5 cells. Panel A: BmCPV dsRNA does not interfere with silencing of a luciferase reporter by dsLuc. Doses ofdsLuc were 0.01 or 0.1 lg/ml while the concentration of dsmalE or BmCPV dsRNA was 0.8 lg/ml. Panel B: Co-transfection of BmCPV genomic dsRNA at the indicated doses(10–50 ng/ml) interferes with expression of Myc-NS5 protein from the pEA-Myc-NS5 expression vector. Western blot shows expression of Myc-NS5 and tubulin.

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Fig. 3. Expression of viral RdRp in Bm5 cells and functional assays. Panel A: Western blot detection of Myc-RdRp in soluble and insoluble extracts of the Bm5 cells. Tubulindetection is used as control in the Western blot analysis. Panel B: Stimulation of BmCPV dsRNA amplification by co-expressed Myc-RdRp. BmCPV polyhedrin is amplified byRT-PCR (40 cycles) and actin amplification (25 cycles) is used as control. At the top is shown a representative ethidium bromide-stained agarose gel showing the amplifiedPCR fragments. Below is shown the graph with the quantification of relative amounts of dsRNA normalized to actin mRNA (N = 3). In the graph, the amount of dsRNA detectedat 2 h after transfection in the different conditions (co-transfection with pEA-PAC and pEA-Myc-RdRp) is set at 100%. The quantities of dsRNA detected at later time points areexpressed relative to this amount. Panel C: Expression of Myc-RdRp does not interfere with silencing of a luciferase reporter by dsLuc (at 50 ng/ml). In all cases, Bm5 cellstransfected with EA-PAC were used as negative control for Myc-RdRp expression and functional analysis.



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Silencing of Myc-NS5 was indeed observed at concentrations of10–50 ng/ml of BmCPV dsRNA which corresponds to 0.5–2.5 ng/ml of segment 9 dsRNA (total length of BmCPV dsRNA geno-me = 24758 bp; length of segment 9 dsRNA = 1186 bp, of which963 bp correspond to the NS5 ORF; http://www.reoviridae.org/dsRNA_virus_proteins/Cypovirus.htm).

3.3. Expression of viral RdRp and investigating its capacity to amplifyBmCPV dsRNA and to modulate dsRNA-mediated gene silencing

The viral RdRp was cloned in the pEA-N-Myc expression vectorwhich was subsequently used to transfect Bm5 cells in the pres-ence of the pBmIE1 helper plasmid (Douris et al., 2006). Westernblot analysis revealed a cross-reacting protein to the Myc antibodythat is somewhat smaller (approximately125 kDa) than the pre-dicted size (138.6 kDa) in the soluble extracts of the transfectedbut not the untransfected cells (Fig. 3A).


To test whether expression of viral RdRp could stimulate ampli-fication of BmCPV dsRNA genome, co-transfections were carriedout in the presence of 0.2 lg/ml of BmCPV genomic dsRNA. At3 days after transfection, BmCPV dsRNA was detected at 3.1(±0.3) -fold higher amounts in cells co-transfected with the expres-sion vector for viral RdRp than in the cells transfected with a con-trol construct (Fig. 3B; P = 0.0002). Also at 7 days after transfection,higher amounts of BmCPV dsRNA were detected in cells co-trans-fected with RdRp expression vector (P = 0.005).

Finally, we investigated whether expression of viral RdRp couldinterfere with the RNAi process, using assays in which a luciferasereporter was silenced by co-transfected dsRNA targeting the lucif-erase ORF (dsLuc). Low amounts of dsLuc (0.05 lg/ml; achievingapproximately a knockdown of 50%) were used in the experimentto reveal more clearly possible modulations of the silencing re-sponse. In the presence of control expression plasmid (pEA-PAC),dsLuc silenced the luciferase reporter to 50.7 ± 2.7%, while in the





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Fig. 4. Expression of viral NS5 in Bm5 cells and functional assay. Panel A: Western blot detection of Myc-NS5 and nodaviral Flag-B2 in soluble and insoluble extracts of theBm5 cells. Tubulin detection is used as control in the Western blot analysis. Panel B: Expression of Myc-NS5 or Flag-B2 does not interfere with silencing of a luciferasereporter by dsLuc (at 50 ng/ml). In all cases, Bm5 cells transfected with EA-PAC were used as negative control for Myc-NS5 expression and functional analysis.



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presence of RdRP expression vector luciferase was reduced to49.0 ± 8.4%, indicating no modulation of the RNAi response byRdRp (P = 0.86, Fig. 3C).

3.4. Expression of viral NS5 and investigating its capacity to modulatedsRNA-mediated gene silencing in Bm5 cells

Transfection of their respective expression vectors in Bm5 cellsresulted in efficient expression of BmCPV-derived Myc-tagged NS5and nodavirus-derived Flag-tagged B2 proteins (Fig. 4A). The B2protein is derived from the nodavirus that persistently infectsHi5 cells (Li et al., 2007). It was demonstrated that B2 functionsas an inhibitor of RNAi because of its propensity to bind dsRNAand siRNA (Lingel et al., 2005). On the other hand, NS5 is a non-structural protein that is encoded by BmCPV genomic dsRNA seg-ment 9 and for which dsRNA-binding activity has been demon-strated (Hagiwara et al., 1998).

When expressed in Bm5 cells, we found that neither nodaviralB2 nor BmCPV-derived NS5 could interfere with the silencing ofa luciferase reporter by co-transfected dsLuc (Fig. 4B). DsLuc at0.05 lg/ml suppressed luciferase activity to 32.9 ± 3.4% in controlcells, to 24.7 ± 3.0% in B2-expressing cells, and to 29.3 ± 0.8% inNS5-expressing cells. Neither for B2 (P = 0.14) nor for NS5(P = 0.36), the repression level differed significantly from thecontrol.

3.5. Investigating the capacity of viral NS5 to modulate dsRNA-mediated gene silencing in agro-infiltrated tobacco leaves

Agro-infiltration in N. benthamiana leaves is a powerful ap-proach to characterize putative suppressors of RNA silencing(Helm et al., 2011). Co-expression of NS5 in agro-infiltrated leavesdid not delay silencing of the GFP reporter, in contrast to expres-sion of P19 of tombusvirus (Fig. 5A). On the other hand, nodavirusB2 could function as a strong RNAi suppressor in plants (Fig. 5B).Similar results were found for the tagged versions of B2 (N-termi-nally Flag-tagged and C-terminally MycHis-tagged; data notshown).

3.6. Distribution of transfected dsRNA in the intracellular environment

Although amplification of dsRNA can be considered as a factor(see Section 4), the most important factor for the stability of dsRNAin transfected cells likely is the inefficiency of the intracellular RNAdegradation machinery to recognize and degrade transfecteddsRNA. In Drosophila and mammalian cells, cytoplasmic bodieshave been identified that are implicated in the regulation of mRNA


stability, translation and the RNAi pathway (Schneider et al., 2006).These cytoplasmic bodies (called P-bodies in mammalian cells;GW-bodies in Drosophila cells) can be visualized by antibodies di-rected against GW182 protein, which is an effector in the RNAipathway (Jakymiw et al., 2005).

To investigate whether transfected dsRNA could localize to GWbodies, we transfected Bm5 cells with FITC-labeled dsRNA and per-formed co-localization studies using an antibody directed againstGawky, the Drosophila homolog of GW182 (Schneider et al.,2006). When used in Western blot, the anti-Gawky antibody recog-nized a protein of approximately 160 kDa in Drosophila-derived S2cells, and a protein of 120 kDa in Bm5 cells (Fig. 6A). The size dif-ferences are consistent with predicted sequence information: Dro-sophila Gawky isoforms consist of around 1380–1385 AA while thehomolog in the monarch butterfly, Danaus plexippus, is predicted tobe considerably smaller (1088 AA; accession EHJ71059). While thehomolog in Bombyx has not been completely annotated yet, it issafe to assume that it will correspond in size to the protein pre-dicted in Danaus.

In co-staining experiments, transfected FITC-dsRNA and Gawkyshowed a different intracellular localization: Gawky-reactivebodies showed mostly a peripheral localization in the cell whiledsRNA was detected more centrally (Fig. 6B). While some dsRNAstaining overlapped with that of Gawky at the periphery, it is clearthat the majority of transfected dsRNA had a different localizationin the cell.

4. Discussion

In this work, evidence is provided for long-term stability ofdsRNA in the intracellular environment after its initial transfectioninto silkmoth-derived Bm5 cells. At 8 days after transfection,considerable amounts of dsRNA could still be detected by RT-PCR,and the amount of intracellular dsRNA remained remarkablystable between 2.5 days and 8 days after transfection (Figs. 1 and2). Although in these experiments, a special type of dsRNA samplewas used, corresponding to the dsRNA genome of BmCPV, anotherstudy has also demonstrated rather long-term persistence (up toat least 4 days) of dsGFP in another lepidopteran cell line, Hi5 (Liuet al., 2012).

Although the cause for the persistence remains unknown, it canbe speculated that the cells do not express many nucleases that candegrade long perfect duplexes of dsRNA efficiently, in contrast tossRNA. However, it is well established that dsRNA is recognizedby Dicer enzymes and becomes incorporated in RISC complexessince it will trigger homologous gene silencing (Figs. 2–4; Swevers



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Fig. 5. Assay for RNAi suppressor activity in agro-infiltrated leaves. Panel A: Suppression of RNAi is observed in agro-infiltrated leaf areas with tombus-virus P19 expression,but not in agro-infiltrated leaf areas with BmCPV NS5 expression. Panel B: Strong suppression of RNAi in agro-infiltrated leaf areas with nodaviral B2 expression. In bothpanels, ‘‘GFP’’ shows co-suppression by additional infiltrated GFP in the absence of additionally expressed protein (P19, NS5 or B2).

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Fig. 6. Detection of Gawky protein in Bm5 cells and co-localization with transfected FITC-labeled dsRNA. Panel A: Western blot analysis of Gawky expression in Drosophila S2cells and Bombyx Bm5 cells. Tubulin expression is shown as control. Panel B: Immunofluorescence detection of Gawky protein (AlexaFluor) and fluorescence detection ofFITC-dsRNA in Bm5 cells. Shown are three representative stained cells. Magnification = 200�.



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et al., 2011; Liu et al., 2012; Kolliopoulou and Swevers, 2013). It isnoted that efficient silencing can be triggered by very smallamounts of dsRNA (5–10 ng/ml) and only small fractions of trans-fected dsRNA in our experiments therefore need to be processed byDicer-2 to cause efficient silencing by RISC complexes. Processingby Dicer-2 may therefore be a minor factor that affects dsRNA sta-bility. This result is corroborated by the observation that co-trans-fection with dsRNA targeting Dicer-2 has only a minor impact onthe abundance of dsRNA (Fig. 1B). The suggestion that dsRNA-degrading enzymes are limiting in lepidopteran cell lines is alsostrengthened by the observation that expression of dsRNA-degrad-ing enzymes can significantly accelerate the degradation of dsRNA(Liu et al., 2012).

Another possible factor may be amplification by intracellularunknown RNA-dependent RNA polymerases which could counter-balance degradation activity. This is expected to occur since trans-fected cells are dividing which would lead to loss of dsRNA unless acompensatory mechanism occurs. An extra factor playing in ourexperiments relates to the use of BmCPV dsRNA which could trig-ger expression of BmCPV proteins that can affect amplification ofdsRNA, such as the viral RdRp. Although we observed that viralRdRp could stimulate BmCPV replication up to 3-fold (Fig. 4), itshould be kept in mind that the polymerase was expressed from


an expression vector plasmid and that expression from transfecteddsRNA likely has much lower efficiency.

It is known that dsRNA readily can undergo editing andunwinding by RNA-editing enzymes such as adenosine deaminasesthat act on RNA (ADARs; Keegan et al., 2011). Since edited dsRNA,but not non-edited dsRNA, is a target for degradation by Tudor-SNenzymes, editing can be considered as the trigger for an alternativedegradation pathway for dsRNA (Scadden, 2005). However, thecytoplasm of Bm5 cells does not contain unwinding (and thereforeediting) activity although it exists in the nuclei (Skeiky and Iatrou,1991). Thus, the majority of cell’s volume may in this case be de-void of this degradation pathway and this could be a contributingfactor to explain the stability of dsRNA.

It is possible that cellular or viral dsRNA-binding factors protectdsRNA from degradation. For instance, Hi5 cells are persistently in-fected with a nodavirus which produces the B2 antiviral suppres-sor (Li et al., 2007; Lingel et al., 2005). B2 protein can sequestratedsRNA and siRNA from viral origin to prevent their further process-ing by the RNAi machinery (Lingel et al., 2005). However, Bm5 cellsdo not seem to be infected by nodavirus, in contrast to Hi5 cells(unpublished results), and the accumulation of B2 protein cantherefore not explain dsRNA persistence in this cell line (see alsobelow for further discussion regarding B2).



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In the experiments presented in this work, dsRNA is introducedinto the cells by transfection. Transfection can be considered as anunnatural way to introduce dsRNA into the cells. By contrast,dsRNA from the extracellular medium enters the cells naturallyby endocytosis and evidence has accumulated that late endo-somes/multivesicular bodies constitute a critical point in the endo-cytosis pathway where RNAi is activated (Lee et al., 2009;Rocheleau, 2012). Evidence exists that only a very small amountof dsRNA (1–2%) can escape from the endosomes while the remain-der will undergo exocytosis or degradation in lysosomes (Wangand Huang, 2013). Since the RNAi machinery is associated withthe endosome pathway, dsRNA that has escaped from the endo-some may be processed immediately and not allowed to spread in-side the cells. A similar argumentation can be applied to othernatural sources of dsRNA such as viral infection and productionof RNA hairpins at genomic loci: locally produced dsRNA structuresmay not have the opportunity to spread throughout the cell be-cause of strict surveillance. By contrast, transfected dsRNA occursthroughout the cell (Fig. 6); as an example, very little overlap is ob-served with GW-bodies, where the RNA-degradation machinery(including RISC components) is concentrated (Schneider et al.,2006).

The presence of BmCPV genomic dsRNA clearly does not affectRNAi efficiency, despite its recognition by the RNAi machinery(Fig. 2). This suggests that the RNAi machinery in Bm5 cells has arather large capacity to respond to dsRNA which cannot be satu-rated easily. Transfection of BmCPV dsRNA also represents a spe-cial case because of its encoding of ORFs which, if expressed,could interfere with RNAi at the protein level. To test this possibil-ity, two ORFs of the BmCPV genome, RdRp and NS5, were cloned inexpression vectors to check for possible interference with RNAi.Although expression was observed, no effect on silencing of a lucif-erase reporter could be observed (Figs. 3 and 4). However, no effectin this Bm5 cell-based assay was also observed for B2, a knownRNAi inhibitor from nodavirus (Lingel et al., 2005; Singh et al.,2009). When tested in another assay based on agro-infiltration ofN. benthamiana leaves, high suppressor activity of B2 (but not ofNS5) was observed (Fig. 5). The different outcomes between thetwo assays (Bm5 cell transfection and N. benthamiana leaf agro-infiltration) can be explained by two mechanisms: (1) the relativeamount of the targeting transgene in agro-infiltrated tissues ishigher than in transfected Bm5 cells, due to multiple insertionsof T-DNA, and strong and constitutive transgene expression con-trolled by the p35S promoter; and (2) amplification of and expres-sion from agro-infiltration vectors occur locally and geneticelements as RNA hairpin and RNAi inhibitor are more likely tointeract. To achieve better results in RNAi inhibition experimentsemploying Bm5 cells, we have tried to stimulate expression ofRNAi inhibitors by co-transfection with pIE1 plasmid (Douriset al., 2006), but were unsuccessful. Regarding the second possibil-ity, there is evidence that the B2 protein acts locally rather thanthroughout the cell during nodavirus infection; it may preferen-tially interact with viral RdRp and through this interaction seques-trate emerging dsRNA structures during viral replication andtranscription (Aliyari et al., 2008). Thus, protein inhibitors of RNAimay affect the RNAi efficiency only at high concentrations, whichcan be achieved only locally during persistent viral replicationand only globally in special circumstances.

A second observation in this work relates to the generation ofan in vitro culture system for BmCPV virus. Such in vitro culturesystem would be of great value for the genetic analysis of BmCPVfunction and the generation of recombinant virus. However, nofunctional virus was obtained after transfection with BmCPV geno-mic dsRNA. The failure is not likely caused by the degradation ofgenomic dsRNA or viral transcripts by the RNAi machinery(Fig. 1B), since co-transfection with dsRNA targeting Dicers and


Argonaute proteins did not result in an increase in dsRNA abun-dance. In these experiments, it is assumed that Dicer enzymes willdegrade dsRNA directly while Argonaute proteins will silence theviral mRNAs after the generation of viral siRNAs (Siomi and Siomi,2009). In our previous study, efficient silencing of RNAi machinerygenes by similar doses of dsRNA was achieved that resulted in clearinhibition of RNAi in a luciferase reporter assay (Kolliopoulou andSwevers, 2013).

Two additional hypotheses are therefore considered to explainthe failure to generate an in vitro culture system for BmCPV: first,it is possible that BmCPV only replicates efficiently in specializedcells of the midgut, its main infection site (Wu et al., 2011). How-ever, BmCPV infection is known to spread to other tissues in somecases (unpublished results; Payne and Mertens, 1983) and is there-fore not restricted to one cell type. A second possibility is that geneexpression from transfected dsRNA is very inefficient and thatdsRNA amplification and virion formation only can be achievedby co-expression of high amounts of viral proteins. While a smallincrease in dsRNA abundance was observed after co-expressionwith viral RdRp (Fig. 3), it is probably necessary to express mostof the 10 proteins of the BmCPV genome at sufficiently high levelstogether with the efficient introduction of the 10 dsRNA genomicsegments in order to establish a culture system. This strategy hasalready been used to establish an in vitro production system forinfluenza virus, the genome of which consists of 8 segments of(-)-ssRNA. In this approach, 8 plasmids are transfected in a mam-malian cell line which generate 8 viral mRNAs by RNA polymeraseII concomitant with the production of the 8 genomic (-)-ssRNAs byRNA polymerase I (Hoffmann et al., 2002).

In summary, our study records the remarkable stability ofdsRNA in the intracellular environment after transfection, whichcontrasts to the reported instability of dsRNA in some extracellularmedia, such as gut content, hemolymph and saliva (Li et al., 2011;Garbutt et al., 2013; Liu et al., 2013; Christiaens et al., in press).This stability is possibly explained by the transfection procedurein which dsRNA is spread throughout the cell volume, while themachinery that directs RNA degradation, including RNAi, has alocal distribution. It is also established that a BmCPV culture sys-tem cannot be established by simple transfection of genomicdsRNA (in contrast to transfection of DNA of BmNPV), even in con-ditions of knock-down of RNAi genes.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (grant number 31172263), Guangdong Natural Sci-ence Foundation of China grant number (S2013010016750), theFund for Scientific Research (FWO-Vlaanderen, Belgium), and bythe General Secretariat for Research and Technology, HellenicRepublic Ministry of National Education and Religious Affairs, inGreece. Thanks are due to D. Stefanou for technical support inmolecular cloning and Western blot techniques and to Dr. T. The-odossiou and Dr. M. Sagnou for expert technical support at the La-ser Confocal Subunit, Institute of Biosciences & Applications, NCSR‘‘Demokritos’’. We also thank Dr. A. Simmonds (University of Alber-ta, Canada) for a sample of the polyclonal antibody against Dro-sophila GW (CG31992).

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Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: Stability and interactions with the core RNAi machinery