Báo cáo khoa học: A new and efficient method for inhibition of RNA viruses by DNA interference pptx

A new and efficient method for inhibition of RNA viruses by DNA interference Monika Nowak 1 , Eliza Wyszko 1 , Agnieszka Fedoruk-Wyszomirska 1 , Henryk Pospieszny 2 , Mirosława Z. Barciszewska 1 and Jan Barciszewski 1 1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland 2 Department of Virology and Bacteriology, Institute of Plant Protection, Poznan, Poland Introduction RNA technologies, which began 30 years ago with the antisense oligonucleotides, and progressed through ribozymes and DNAzymes (deoxyribozymes) and their analogues, have not met expectations, as they have failed to deliver a suitable agent that can effec- tively inhibit gene expression at the RNA level. RNA interference (RNAi) technology is the most recent in the long line of nucleic acid-based therapeutic candi- dates. RNAi is induced by long dsRNA processed by the endonuclease Dicer into 21 nucleotide short inter- fering RNAs (siRNAs) or other 19–28 nucleotide small RNAs (sRNAs) [1,2]. These short stretches of RNA with 3¢-overhangs of two nucleotides are incor- porated into RNA-induced silencing complex (RISC), where they unwind, and an antisense strand of siR- NA (guide strand) then binds to the complementary RNA. A target RNA molecule undergoes endonucleo- lytic cleavage or, optionally, translational repression is achieved. After identification of the chemically syn- thesized siRNAs as sufficient effectors for RNAi [3,4], studies focused on the development of siRNAs with improved stability, pharmacokinetic properties and pharmacodynamic properties that were suitable for in vivo applications. Chemical modifications of siRNA include protection of internucleotide phosphodi- ester bonds, ribose residues and nucleobases, and Keywords Dicer; DNA interference; gene silencing; RNA interference; siDNA Correspondence J. Barciszewski, Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12, 61-704 Poznan, Poland Fax. +48 61 8520532 Tel: +48 61 8528503 E-mail: Jan.Barciszewski@ibch.poznan.pl (Received 16 February 2009, revised 2 June 2009, accepted 10 June 2009) doi:10.1111/j.1742-4658.2009.07145.x We report here a new method for inhibition of RNA viruses induced by dsDNA. We demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by com- plementary short interfering DNA. We showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA effected with dsDNA, we coined the term DNA interference technology. Abbreviations Ago, Argonaute; as-DNA, short antisense ssDNA; DNAi, DNA interference; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIV-1, human immunodeficiency virus type 1; ODN, oligodeoxynucleotide; PMMoV, pepper mild mottle virus; RHA, RNA helicase A; RISC, RNA-induced silencing complex; RNAi, RNA interference; s-DNA, short sense ssDNA; siDNA, short interfering DNA; siRNA, short interfering RNA; sRNA, small RNA; TMV, tobacco mosaic virus. 4372 FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS attachment of different tags on either the 5¢-end or the 3¢-end [5]. Despite common features of RNA silencing, there are differences between the animal and plant king- doms. The mechanism of RNA silencing in plants is often known as post-transcriptional gene silencing. Various classes of sRNAs, ranging from 18 to 26 nucleotides, can be found, as well as different forms of Dicer. For example, in Arabidopsis thaliana, dsRNA is processed into sRNA duplexes of specific sizes by one of four Dicer-like proteins [6]. Furthermore, plant RNA silencing can spread from an initially silenced cell to surrounding cells (short-range spread through the plasmodesmata) and over a long distance through the vascular system to different parts of the plant [7,8]. Double-stranded RNAs, which induce RNA silenc- ing, might be derived from virus replication, transcrip- tion, inverted-repeat sequences, or convergent transcripts, or may also be generated endogenously within the cell, e.g. as a transcript with an internal stem–loop structure [6]. Alternatively, dsRNA may be synthesized by one of six RNA-dependent RNA polymerases, which copy antisense RNA from an aber- rant or overexpressed sense transcript. Such RNA- dependent RNA polymerases may also participate in the spread of silencing signals by amplification of dsRNAs. Such a mechanism has been identified in fungi, worms, and plants [1]. Dicer and Dicer-like proteins are approximately 200 kDa multidomain members of the RNase III fam- ily that are responsible for processing long dsRNAs into effector siRNAs. Human Dicer contains a single PAZ domain, a single dsRNA-binding domain, and ATPase ⁄ helicase domains, as well as two RNase III- like domains and a domain of unknown function (DUF238) [9]. Dicer appears to be a complex and dynamic enzyme that interacts with other cellular pro- teins. In mammalian cells, Dicer is associated with TAR-RNA-binding protein [10] and Argonaute (Ago) proteins [11]. It is thought that Dicer–Ago protein complex formation is crucial for siRNA channelling into RISC and mediating the transition between the initiation and execution phases of RNAi. Ago proteins are key components of RISC responsible for the cleav- age of the target RNA. The cleavage is catalysed by a Piwi domain of Ago proteins, the structural homolog of RNase H [12,13]. Both Dicer and Ago proteins con- tain PAZ domains, which recognize and bind to the ends of dsRNA molecules [14]. In a previous article, we described the viral RNA degradation induced by a short (16 nucleotide) ssRNA that binds to tobacco mosaic virus (TMV) RNA, forms the leadzyme structure and, in the presence of lead ions, causes the cleavage of virus genomic RNA [15]. Here, we extend our studies by analysing the antiviral effects of short and long DNA and RNA molecules specific for TMV genomic RNA. We have found that both long dsRNA and long dsDNA are potent inhibitors of viral RNA. A long dsDNA frag- ment (470 bp) that is homologous with TMV RNA suppressed TMV infectivity in Nicotiana tabacum.We also demonstrated that siDNAs are capable of sequence-specific inhibition of viral RNA both in tobacco plants and in HeLa cells. Furthermore, Dicer has been shown to be capable of DNA cleavage, which suggests that the silencing of viral RNA was due to a mechanism analogous to RNAi, which we have called DNA interference (DNAi). Results and discussion The dsDNA (470 bp) and ssDNAs, with either sense or antisense orientation, were tested for their ability to inhibit the development of local lesions elicited by TMV in N. tabacum cv. Xanti-nc. Half-leaves were inoculated with the virus supplemented with one of the following: sense strand DNA, antisense strand DNA, dsDNA, or TMV-specific siRNA. Five experiments were performed, comprising several assays: (a) plants inoculated with only TMV; (b) plants inoculated with TMV and long dsDNAs; (c) plants inoculated with TMV and siRNA; (d) plants inoculated with short antisense ssDNA (as-DNA); (e) plants inoculated with TMV and short sense ssDNA (s-DNA); and (f) unin- oculated control tobacco (Table 1). For each single assay, three to five plants were inoculated with the same inoculum. All tested DNAs as well as siRNA were homologous to the region including nucleotides 203–674 of TMV RNA within the first ORF encoding replicase [15]. The necrosis lesions caused by TMV spread were monitored 5 days postinfection. Reduced infectivity was observed in tobacco leaves inoculated with long dsDNA (at 330 nm) or siRNA. It was mani- fested as a decreased number of local lesions (necrotic symptoms) when compared with control plants treated only with TMV (Fig. 1A,B). Neither sense nor anti- sense DNA strands alone affected local lesion forma- tion by TMV. These macroscopic observations were confirmed with RT-PCR analysis (Fig. 1C), which showed a lack of TMV RNA accumulation in tobacco plants treated either with dsDNA or with siRNA homologous to a part of the TMV RNA (Fig. 1C, lanes 5 and 6), in contrast to infected leaves treated with sense and antisense DNAs (Fig. 1C, lanes 3 and 4) as well as TMV alone (lane 2). The level of glyceral- dehyde-3-phosphate dehydrogenase (GAPDH) mRNA M. Nowak et al. Virus inhibition by DNA interference FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS 4373 used as a control was similar in all samples. In a previ- ous study of Tenllado and Diaz-Ruiz [16], it was shown that long dsRNA (977 bp) but not cDNA (1409 bp) with the sequence corresponding to part of the replicase gene reduced the infectivity of pepper mild mottle virus (PMMoV) in a Nicotiana benthami- ana host. Although the authors did not observe reduc- tions of PMMoV expression in tobacco plants in response to cDNA treatment, northern blot analysis showed lower intensities of the virus RNA band in extracts prepared from leaves inoculated with cDNA in comparison with control leaves inoculated only with the virus and sense or antisense long RNAs. The effect of PMMoV inhibition by treatment with cDNA was visible as a weaker northern blot signal in leaves inoc- ulated with specific nucleic acids, but was not observable in the uppermost systemic leaves of N. benthamiana. The weaker response of infected tobacco plants to dsDNA treatment in these experi- ments could be explained by the length of the cDNA (1409 bp), which was longer than that of the dsRNA and dsDNA used in our experiments (470 bp). Such long molecules probably had a limited ability to enter the cell. To further characterize the antiviral effect of specific nucleic acids, we prepared anti-TMV long dsRNA (280 bp) and short interfering DNA (siDNA, 21 bp), as well as long, scrambled, nonspecific dsDNA (384 bp) (Table 1). All anti-TMV nucleic acids used were located within 203–674 nucleotides of TMV RNA, as in the previous experiments. Whereas the scrambled dsDNA did not influence viral expression, both dsRNA and siDNA reduced TMV accumulation in tobacco plants (Fig. 1D,E). This was also observed in RT-PCR analysis (Fig. 1F). The level of viral RNA inhibition induced by long dsRNA was greater than in the case of siDNA, but comparable with the long dsDNA effect. Silencing of viral RNA was observed to be most efficient in cases of long dsRNA (89%), long dsDNA (84%), and siRNA (89%), and less efficient for siDNA (70%), whereas long scrambled dsDNA and short antisense and sense ssDNAs had no effect on viral expression (Table 1). It was not surprising that siRNA and long dsRNA were potent silencers of specific RNA, but the fact that both long and short dsDNAs showed similar effects is intriguing. To determine whether such a dsDNA- induced silencing effect on RNA could be observed in cultured human cells, we analysed the influence of siD- NAs on HIV-1 gp41 RNA expression in HeLa cells. The cells were cotransfected with the pEGFP-N3- gp41-323 vector encoding a gp41 protein and siDNA at 10, 25, 50, 100 and 250 nm, respectively. Cells with green fluorescence expressed the gp41–EGFP fusion protein. After treating the cells with increasing concen- trations of siDNA, we observed a decrease in the expression of gp41 (Fig. 2A). In order to prove that the silencing effect was due to degradation of the tar- geted gp41 RNA, we carried out RT-PCR analysis and found degradation of gp41–EGFP RNA in the presence of siDNA (Fig. 2B). The semiquantitative analysis showed decreases of approximately 50% and 78% in the expression level of gp41 when 25 nm and 100 nm siDNA was applied, respectively (Fig. 2C). In a analogous experiment on HeLa cells transfected with siRNA with a sequence identical to that of siDNA, we observed a strong decline in viral expression when cells were treated with 5 nm siRNA (84%) (Fig. 2D– F). Scrambled siRNA did not affect gp41–EGFP expression (data not shown). Therefore, we can con- Table 1. Comparison of RNA and DNA silencing triggers. The effectiveness of specific DNA and RNA molecules in silencing TMV or gp41 expression was evaluated as an average percentage of TMV or gp41 reduction on the basis of a number of local lesions in infected tobacco plants (Fig. 1) and semiquantitative IMAGEQUANT analysis of RT-PCR results (Fig. 2), respectively. Silencing effects were evaluated for specific DNA and RNA concentrations as shown. Type of nucleic acid Length Target Localization of target sites (nucleotides) Silencing effect %nM dsDNA 470 bp TMV RNA 203–674 of TMV RNA 84 330 as-DNA (ssDNA) 22 nucleotides TMV RNA 483–504 0 5 · 10 3 s-DNA (ssDNA) 20 nucleotides TMV RNA 390–409 0 5 · 10 3 siRNA 21 bp TMV RNA 420–440 89 4.5 · 10 3 dsRNA 284 bp TMV RNA 390–674 89 330 siDNA 21 bp TMV RNA 549–569 70 4.5 · 10 3 Scrambled dsDNA 384 bp TMV RNA Non-TMV 0 330 siRNA 21 bp HIV1gp41 257–277 of gp41 RNA 96 100 siDNA 21 bp RNA 257–277 78 100 Virus inhibition by DNA interference M. Nowak et al. 4374 FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS clude that siDNAs are less potent inhibitors of specific RNAs in cultured human cells than are siRNAs. The same effect was observed in a tobacco plant model (Fig. 1). The ability of siDNA molecules to specifically inhibit gene expression has been demonstrated before in mammalian cell culture [17]. Our findings clearly demonstrate that both exoge- nous long dsDNA and siDNA molecules can be applied for silencing of homologous RNA. To deter- mine the mechanism of this process, we tested dsDNA processing in plant extract. Products of 20–26 bp were observed (Fig. 3A), suggesting the involvement of Dicer in dsDNA processing. Interestingly, dsDNA (160 bp) incubated with Dicer also gave short dsDNA fragments of 21–30 bp, with the most abundant being 17 bp (Fig. 3B, lane 3). Incubation with smaller amounts of the enzyme provided intermediate products of dsDNA hydrolysis (Fig. 3B, lane 2). We also A B C E F D Fig. 1. Inhibition of TMV infection in N. tabacum cv. Xanti-nc leaves with various nucleic acid-based effectors complementary to the repli- case gene of TMV RNA. (A) Half-leaves were inoculated with TMV and either 22 nucleotide sense strand DNA, 20 nucleotide antisense strand DNA, 470 bp dsDNA, or siRNA. Tobacco leaves with symptoms of TMV infection are shown. Uninfected plants and plants inoculated only with the virus were used as controls. Leaves were photographed at 5 days postinoculation. (B) Diagram showing changes in local lesions observed on tobacco leaves after inoculation of virus with TMV-specific as-DNA, s-DNA, dsDNA, and siRNA. Data (±standard devia- tion) from three independent experiments are shown. (C) Analysis of RT-PCR products for TMV and GAPDH on 1.5% agarose gel. The reac- tion products were amplified from total RNA extracted from the infected tobaco leaves and uninfected control. TMV cDNA (470 bp) was amplified with primers corresponding to the 203–674 fragment of TMV RNA. GAPDH cDNA (300 bp) amplified with primers specific for tobacco GAPDH was used as a reference. (D) Half-leaves of tobacco leaves were inoculated with TMV alone, TMV and scrambled dsDNA (384 bp), TMV and siDNA, and TMV and dsRNA (280 bp). Symptoms of infection are shown. (E) Graph showing the amounts of necrotic symptoms observed on tobacco leaves after inoculation of virus with scrambled dsDNA and TMV-specific siDNA or dsRNA. Data (±standard deviation) from three independent experiments are shown. (F) Analysis of RT-PCR products for TMV and GAPDH on 1.5% agarose gel. The reaction products were amplified from total RNA extracted from tobaco leaves inoculated with TMV and scrambled dsDNA, TMV-specific siDNA, and dsRNA. M. Nowak et al. Virus inhibition by DNA interference FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS 4375 observed some nonspecific degradation of a substrate DNA of 9–10 nucleotides. From these results, it is clear that Dicer shows a broad nuclease specificity with the potential to cleave dsDNA substrates, like other enzymes, e.g. S1 and Neurospora crassa nucleases, which efficiently process both DNA and RNA [18,19]. The ability of proteins with PAZ domains to bind DNA has been confirmed in crystallographic studies A B C D E F Fig. 2. RT-PCR analysis of HIV-1 gp41 mRNA expression in HeLa cells transfected with gp41-specific siDNA or siRNA. (A) Fluorescence microscopy of HeLa cells expressing gp41–EGFP protein. Cells were cotransfected with pEGFP-N3-323 and anti-gp41 siDNA at the concen- trations indicated, and harvested after 24 h for total RNA isolation. (B) RT-PCR amplification products of total RNA from HeLa cells transfect- ed as indicated. Products were fractioned by electrophoresis on 1.5% agarose gel with ethidium bromide. (C) Diagram showing an evaluation of gp41 mRNA inhibition with siDNA in HeLa cells. Numbers below the diagram represent concentrations of anti-gp41 siDNA (10, 25, 50, 100 and 250 n M, respectively) cotransfected with 1 lg of pEGFP-N3-323. The analysis was performed using IMAGEQUANT (Molecular Dynamics). Data are represented as mean ± standard deviation. (D) HeLa cells expressing gp41–EGFP protein after transfection with anti- gp41 siRNA. (E) RT-PCR analysis of gp41 and GAPDH from HeLa cells transfected with siRNA as indicated. (F) Graph showing semiquantita- tive analysis of gp41 mRNA expression in HeLa cells transfected with siRNA. Virus inhibition by DNA interference M. Nowak et al. 4376 FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS on PAZ domains of Ago proteins from Drosophila [14,20,21]. Thus, we can assume that the PAZ domain is able to recognize RNA as well as DNA and there- fore directs the cell protein machinery for RNA interference or, alternatively, DNAi pathway. Post- transcriptional gene silencing induced by dsDNA has been observed previously in Nicotiana species [22,23] and ferns [24,25]. Our hypothesis that DNAi, like RNAi, acts at the RNA level is consistent with the previous observation that long dsDNAs with sequences homologous to those of the targeted RNAs caused knockout phenotypes in Adiantum gametophytes, and the lack of a silencing effect when dsDNA corre- sponded to the intron sequence of the target gene [24]. Hydrolysis of dsDNA with Dicer makes it possible to channel the siDNA molecules into RISC by the action of Ago protein. Recently, RNA helicase A (RHA) was identified as a human RISC-associated fac- tor contributing to loading and unwinding of siRNA [26]. It has been shown that RHA unwinds RNAÆRNA duplexes as well as RNAÆDNA heteroduplexes. It also shows an affinity for ssDNA [27]. Thus, we cannot exclude the involvement of RHA in siDNA processing or the existence of an equivalent DNA helicase. The unwound antisense strand of siDNA binds a homolo- gous target RNA, and such a complex is cleaved by the Piwi domain of Ago protein. Because Piwi is the structural homologue of RNase H [12,13], the siDNA antisense strand–target RNA complex can be effi- ciently hydrolysed. Recently, the crystal structure of eubacterial Thermus thermophilus Ago protein com- plexed with 21 nucleotide guide DNA and its 20 nucle- otide target RNA was reported [28,29]. In the cleavage activity assays, it was shown that Ago protein com- plexed with the DNA guide strand efficiently bound and cleaved the target RNA, even when single muta- tion in the target RNA was introduced [28]. The cleav- age rate was reduced after insertion of a dual bulge at the same position on the target RNA. It seems that siDNA works in a similar way as siRNA, simply because DNA resembles a modified siRNA molecule deprived of the 2¢-OH group of ribose. We showed the silencing effect of targeted RNA in HeLa cells to be at the level of approximately 78% when 100 nm siDNA was used (Fig. 2). Applica- tion of higher concentrations of siDNAs than of siRNAs is necessitated by the lower stability of RNAÆDNA than of RNAÆRNA duplexes formed within RISC. On the other hand, DNA oligomers are more resistant to intracellular nucleases than oligoribo- nucleotides, and they do not need to be modified. A different mechanism has been proposed for oligode- oxynucleotides (ODNs), which efficiently reduced retroviral replication both in human cell culture and a mouse model [30,31]. The ODNs (54 nucleotides) were designed to form hairpin–loop-structured DNA, which binds viral RNA and forms a triple helix. The authors suggested that the antiviral effect of the examined ODNs was due to the action of viral RNase H. In summary, our data suggest that the DNAi path- way may have converging steps with RNAi, or exploit the RNAi protein machinery. There are some features of DNAi that resemble RNAi, e.g. sequence-specific inhibition of targeted RNA, processing of long dsDNA molecules to shorter triggers, and the potential of siDNA to induce silencing. Although dsDNA cleav- age induced by Dicer is slightly less efficient than in the case of dsRNA, and a higher dose of siDNA is required for efficient silencing, dsDNA shows higher cellular stability than dsRNA. The other advantages of AB Fig. 3. Hydrolysis of dsDNA in tobacco leaf extract and in vitro with Dicer. (A) Autoradiogram of 10% polyacrylamide gel with 7 M urea of a 32 P-labelled dsDNA (470 bp) cleavage products. Incubation of DNA in plant extract was carried out at 25 °C, and 10 lL por- tions of the incubated mixture were removed at the following times: 0 min (lane 1), 2 min (lane 2), 5 min (lane 3), 15 min (lane 4), 30 min (lane 5), 1 h (lane 6), 2 h (lane 7), 5 h (lane 8), 8 h (lane 9), and 24 h (lane 10). For cleavage product evaluation, a DNA size marker of 29 nucleotides was used. (B) In vitro hydrolysis of dsDNA with Dicer. Autoradiogram of 10% polyacrylamide gel with 7 M urea of c 32 P-labelled dsDNA (160 bp) incubated with human recombinant Dicer (Ambion). Lane 1: control DNA incubated in 1· Dicer reaction buffer (Ambion). Lane 2: DNA with 1 U of Dicer. Lane 3: DNA with 3 U of Dicer. A 29 nucleotide DNA size marker (M) was used for product determination. M. Nowak et al. Virus inhibition by DNA interference FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS 4377 DNAi are the lower cost of dsDNA synthesis and the lack of necessity for further DNA modification. These observations may open a new path towards the use of dsDNA or siDNAs as reverse genetics and therapeutic tools in mammalian cells and plant models. Experimental procedures Inoculation of tobacco leaves Before inoculation, carborundum was sprayed onto leaves of N. tabacum cv. Xanti-nc plants. Inoculations were car- ried out on one-half of a fully expanded leaf of at least three tobacco plants for one assay by gently rubbing the leaf surface with the inoculum. The opposite half of the leaf served as an uninoculated control. All leaves chosen for inoculation were at the same developmental stage. The inoculum used throughout experiments contained TMV suspension (strain U1 at 5 lgÆmL )1 ) mixed immediately before inoculation with either dsDNA corresponding to TMV RNA (470 bp, at concentrations ranging from 65 to 330 nm), ssDNA (as-DNA of 22 nucleotides and s-DNA of 20 nucleotides at 5 lm), siRNA (4.5 lm), siDNA (4.5 lm), dsRNA (284 bp, 330 nm), or scrambled dsDNA (384 bp, 330 nm). After infection, the inoculated plants were kept in a growth chamber, initially at 20 °C temperature (first day), and then at 25 °C with 12 h light and 12 h dark cycle. Silencing of TMV RNA was monitored by the observation of local lesion formation. Inoculated leaves were harvested and photographed 5 days postinoculation. Double-stranded DNA (470 bp) corresponding to TMV genomic RNA nucleotides 203–674 (RNA fragment-encod- ing replicase gene) was synthesized on the TMV RNA tem- plates isolated from the infected tobacco leaves by RT-PCR, using primers TMV1 (5¢-GCCCAAGGTGAACT TTTCAA-3¢) and TMV2 (5¢-TAGCGCAATGGCATACA CTC-3¢). The sequence of as-DNA is 5¢-CAATACTGTCT TTCTGGCCTTC-3¢, and that of s-DNA is 5¢-ATAGGCG GGAATTTTGCATC-3¢. Anti-TMV siRNA was synthe- sized using the DNA templates 5¢-AAGGGACGAGCA TATGTACACCCTGTCTC-3¢ and 5¢-AAGTGTACATAT GCTCGTCCCCCTGTCTC-3¢ and a Silencer siRNA Con- struction Kit (Ambion, Austin, TX, USA). The sequence of the anti-TMV siDNA sequence sense strand is 5¢- AACTTCCAAAAGGAAGCATTT-3¢, and that of the antisense strand is 5¢-ATGCTTCCTTTTGGAAGTTTT-3¢. Specific anti-TMV dsRNA (284 bp) was obtained by T3 and T7 in vitro transcription (MEGAscript T3 High Yield Transcription Kit, MEGAscript T7 High Yield Transcrip- tion Kit; Ambion) and hybridization of the obtained RNA strands. The templates for the T3 and T7 transcriptions were prepared by RT-PCR with primers containing pro- moters for T3 and T7 RNA polymerases, respectively. The characteristics of all DNA and RNA molecules (both with target sites for specific RNA DNA binding) used in the experiments are shown in Table 1. All ODNs were pur- chased from IBB (Warsaw, Poland). RNA analysis Total RNA was extracted from inoculated leaves at 5 days postinoculation, as described previously [15]. TMV RNA was detected by RT-PCR using the TMV-specific primers TMV1 and TMV2. Reverse transcription and PCRs were performed as described previously [15]. The specific primers used for detection of tobacco GAPDH were GAPDH_t1 (5¢-TGGAAGAATTGGGCGATTAG-3¢) and GAPDH_t2 (5¢-GCAGCCTTGTCCTTGTCAGT-3¢). The gp41-specific primers were gp41-A (5¢-CCAAGGCAAAGAGAAGAGT GG-3¢) and gp41-B (5¢-CTCCATCCAGGTCGTGTG A-3¢); the human GAPDH-specific primers were G1 (5¢-GGGTGGAGCCAAACGGGTC-3¢) and G2 (5¢-GGA GTTGCTGTTGAAGTCGCA-3¢). All of the primers were designed using primer3 (http://frodo.wi.mit.edu/). Equal volumes of amplified products were separated on 1.5% aga- rose gels, and stained with ethidium bromide; the products were detected and quantified by phosphoimager analysis (imagequant, version 5.1; Molecular Dynamics, Sunny- vale, CA, USA). In vitro DNA cleavage in plant extract Double-stranded DNA radiolabelled with [ 32 P]dATP[aP] was obtained in a PCR reaction on TMV cDNA template by the addition of [ 32 P]dATP[aP] to the reaction mixture. The radiolabelled PCR products (470 bp) were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The pattern of digestion of radiolabelled dsDNA (6 · 10 4 c.p.m. ⁄ reaction) was determined in 90 lLof tobacco leaf tissue extract. The extract was obtained by homogenization of leaf tissue in 10 mm Tris buffer (pH 7.5), sonication (3 · 15 s), and centrifugation at 16 000 g for 3 min. The reaction mixture containing DNA and plant extract was incubated at 25 °C, and 10 lL portions were removed at specific times (untreated, 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 5 h, 8 h, 24 h). Reactions were stopped by adding loading solution with 0.1 m EDTA and freezing in liquid nitrogen, and analysed by 10% PAGE with 7 m urea in the presence of a 29 nucleotide mass marker. Double-stranded DNA cleavage analysis with Dicer Digestion of 160 bp DNA, radiolabelled with [ 32 P]dATP[cP], using recombinant human Dicer (Ambion) was performed in a 10 lL reaction mixture. It contained 8 · 10 4 c.p.m. DNA and 1 U or 3 U of Dicer in 1· Dicer reaction buffer (300 mm NaCl, 50 mm Tris ⁄ HCl, 20 mm Virus inhibition by DNA interference M. Nowak et al. 4378 FEBS Journal 276 (2009) 4372–4380 ª 2009 The Authors Journal compilation ª 2009 FEBS Hepes, 5 mm MgCl 2 , pH 9). DNA incubated only in buffer, in the absence of the enzyme, served as a control. Reaction mixtures were incubated at 37 °C for 65 h and analysed by 10% PAGE with 7 m urea in the presence of 29 nucleotide DNA oligonucleotide as a size marker. Cell culture and transfection HeLa cells were seeded at a density 2 · 10 5 cells per well in 24-well tissue culture plates. The cells were grown in RPMI- 1640 medium (Sigma, Munich, Germany) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK), 1% antibiotics (Sigma) and 1% RPMI vitamin mix (Sigma) at 37 °C under a 5% CO 2 atmosphere. After 1 day of culture, cells (70% confluence) were washed with NaCl ⁄ P i (Sigma), placed in fresh growth RPMI-1640 medium without supple- ments, and cotransfected with 1 lg of pEGFP-N3-gp41-323 vector (encoding a fragment of gp41 protein) and siDNA or siRNA at an appropriate concentration (Fig. 2). Transfec- tion was carried out in the presence of 1.5 lL of Lipofecta- mine 2000 (Invitrogen, Paisley, UK) with 500 lLof Opti-MEM (Invitrogen), according to the manufacturer’s protocol. After 4 h, medium was replaced with fresh RPMI-1640 growth medium with supplements, and the cells were incubated for 24 h at 37 °C under a 5% CO 2 atmosphere. Total RNA was isolated using Trizol reagent (Invitrogen), according to the manufacturer’s instructions. 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