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

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

by DNA interference

Monika Nowak1, Eliza Wyszko1, Agnieszka Fedoruk-Wyszomirska1, Henryk Pospieszny2,

Mirosława Z Barciszewska1and Jan Barciszewski1

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.

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 cleavcleav-age 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 observaforma-tions 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

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-anahost 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.

Localization of target sites (nucleotides)

Silencing effect

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

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.

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 indicattransfect-ed Products were fractiontransfect-ed 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 anti-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.

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 concentraApplica-tions 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

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 c32P-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.

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 20C temperature (first day),

and then at 25C 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 hybridizaTranscrip-tion 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

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 [32P]dATP[aP] was obtained in a PCR reaction on TMV cDNA template by the addition of [32P]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· 104

c.p.m.⁄ reaction) was determined in 90 lL of 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 25C, 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

[32P]dATP[cP], using recombinant human Dicer (Ambion) was performed in a 10 lL reaction mixture It contained

8· 104c.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

Hepes, 5 mm MgCl2, pH 9) DNA incubated only in buffer,

in the absence of the enzyme, served as a control Reaction

mixtures were incubated at 37C 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· 105

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

37C under a 5% CO2atmosphere After 1 day of culture,

cells (70% confluence) were washed with NaCl⁄ Pi(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 lL of

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 37C under a 5% CO2

atmosphere Total RNA was isolated using Trizol reagent

(Invitrogen), according to the manufacturer’s instructions

Anti-gp41 siDNA was obtained by hybridization of sense

strand (5¢-GTTGCTCTGGAAAACTCATTT-3¢) and

anti-sense strand (5¢-ATGAGTTTTCCAGAGCAACTT-3¢), and

siRNA was synthesized using DNA templates (sense strand,

5¢-AAATGAGTTTTCCAGAGCAACCCTGTCTC-3¢;

anti-sense strand, 5¢-AAGTTGCTCTGGAAAACTCATCCTG

TCTC-3¢) and a Silencer siRNA Construction Kit (Ambion)

Acknowledgements

This work was supported by grants from the Polish

Ministry of Science and Higher Education 501-0724 and

501-07-26 M Nowak’s doctoral scholarship is funded

by the President of the Polish Academy of Sciences The

critical comments of M Szymanski are acknowledged

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