Journal of Medical Virology 80:1025–1032 (2008)
Development of RNA Interference (RNAi) as Potential Antiviral Strategy Against Enterovirus 70 Eng Lee Tan,1 Kah Fai Ho Marcus,2 and Chit Laa Poh3* 1
School of Chemical and Life Sciences, Singapore Polytechnic, Singapore, Singapore Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 3 Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Victoria, Australia 2
Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1–3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential. J. Med. Virol. 80:1025–1032, 2008. ß 2008 Wiley-Liss, Inc. KEY WORDS: enterovirus 70; acute hemorrhagic conjunctivitis (AHC); RNAi as promising antiviral agent
INTRODUCTION Acute hemorrhagic conjunctivitis (AHC) is a rapidly progressive and highly contagious viral disease that is caused primarily by Enterovirus 70 (EV70) [Kono et al., 1972]. Common to all picornaviruses, EV70 is a positive single stranded RNA virus and possesses a single long open reading frame of 6,582 bases. EV70 has caused epidemics of AHC in tropical coastal regions throughout the world. In 1969 a novel epidemic form of acute hemorrhagic conjunctivitis (AHC) surfaced in Ghana, West Africa. AHC spread rapidly across Africa followed by a second focus causing extensive epidemics across South East Asia [Yin-Murphy, 1984]. The disease ß 2008 WILEY-LISS, INC.
subsequently spread to several other countries in the Middle East, Asia, and Oceania [Higgins, 1982]. More recently, AHC outbreaks have been reported in Okinawa, Japan in 1994, and Rio de Janeiro, Brazil in 2004. Acute hemorrhagic conjunctivitis is characterized by a short incubation period of 24–48 hr preceding a rapid onset of uniocular or binocular symptoms and signs. Patients manifest excessive lacrimation, pain, periorbital swelling, and redness of the conjunctiva, conjunctival congestion, vascular dilatation, and onset of oedema. Viral infections usually elicit a mononuclear cell response. In AHC, a prominent hemorrhagic component soon appears that is characteristic of this infection [Yin-Murphy, 1984]. Keratitis with accompanying pain and possible visual impairment may be seen. The infection is benign and the episode normally resolves without sequelae in 1–2 weeks. It was observed that a proportion of AHC patients who were infected with EV70 eventually develop nonophthalmic symptoms such as neurological dysfunction resembling paralytic poliomyelitis as well as respiratory and gastrointestinal disturbances [Higgins, 1982]. Currently, there is no available treatment for AHC caused by EV70 and patient management consists of symptomatic treatment, and allowing the disease to run its full course of 5–7 days. With the potential risk of EV70 to cause neurological complications, there is a need to develop potential antiviral strategies against this virus. RNA interference (RNAi) has been widely studied over the last few years as a promising strategy against infectious diseases based on specific gene silencing. The phenomenon of RNAi is an ATP-dependent process which occurs in the cytoplasm [Nykanen et al., *Correspondence to: Chit Laa Poh, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street, Hawthorn, Victoria 3122, Australia. E-mail:
[email protected] Accepted 14 March 2008 DOI 10.1002/jmv.21210 Published online in Wiley InterScience (www.interscience.wiley.com)
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2001]. The first step involves the Dicer which has an N-terminal helicase domain, a Piwi/Argonaute/Zwille (PAZ) motif, a dsRNA binding domain and two RNase III motifs at the C-terminus. The dsRNA-specific endonuclease activity of the Dicer cleaves the long dsRNAs to produce short interfering RNAs (siRNAs). These siRNAs are subsequently incorporated into a multi-protein RNA-induced silencing complex or RISC. siRNA molecules require phosphorylation at the 50 end in order to be incorporated into RISC [Nykanen et al., 2001]. The duplex siRNAs is unwound by the ATP-dependent helicase activity of RISC, leaving only the antisense strand to guide RISC to the homologous target mRNA for endonucleolytic cleavage of the target mRNA [Dykxhoorn et al., 2003]. In this study, we evaluated RNAi as a potential antiviral strategy against EV70. Two 19-mer siRNAs were designed to target at specific regions of the 3Dpol gene of EV70, and their efficacies in inhibiting EV70 replication were evaluated and compared in the in vitro system. MATERIALS AND METHODS Cell Culture and Virus Strain Rhabdomyosarcoma (RD) cells were routinely grown in minimum essential medium (Gibco, Boston, MA) supplemented with 5% fetal calf serum, HEPES, 1% sodium pyruvate, and 1.5% sodium bicarbonate. The EV70 strain J670/71 (Accession No. DQ201177) was used in this study. Design and Synthesis of 19-mer siRNAs Two 19-mer siRNAs were designed targeting at two different and specific regions of the 3Dpol region of the EV70 genome and designated as si-3D1 and si-3D2, respectively. The siRNAs were designed according to recommendations made by Elbashir et al. [2001]. Briefly, the duplexes have a G þ C content of about 30–35% and the oligonucleotides were synthesized with 30 -dTT extensions (Table I). The sequences were subjected to BLAST search of the National Center for Biotechnology Information database (http://www.ncbi. nlm.nih.gov/blast) to ensure the specificity of the siRNAs to respective target genes. All the siRNA oligonucleotides were synthesized by Sigma Proligo
(Sigma-Proligo, St. Louis, MO). In addition, a scrambled-sequenced siRNA (3D-scr) with the same base composition as si-3D1 was designed and used as a target specificity control. To determine the transfection efficacies of the siRNAs, another set of si-3D1 and si-3D2 were synthesized and tagged with fluorescein at the 30 end of the sense strand. Transfection and Infection RD cells were first seeded at a density of 5 104 into each well of a 24-well plate and were allowed to grow overnight at 378C in 5% CO2. After 24 hr, the growth medium in each well was replaced with 500 ml of reduced-serum medium, OPTI-MEM I (Gibco, Invitrogen Life Science Technologies, Carlsbad, CA) for another 24 hr before transfection. For viral protein analysis, the RD cells were seeded at the same density in a six-well plates. The transfection of the RD cells was carried out under optimal conditions. The RD cells were transfected with graded concentrations (1, 10, and 100 nM) of either si-3D1 or si-3D2. All the siRNAs were complexed with Lipofectamine 2000CD as the lipid carrier (Invitrogen Life Technologies, Carlsbad, CA). After 48 hr of incubation, each well was infected with EV70 at an MOI of 10. After 1 hr, the cells were replaced with fresh growth medium and incubated at 378C in 5% CO2. At different time points post-infection, cell supernatants and cell lysates were harvested for extraction of total viral RNA and viral proteins, respectively. The harvested viral RNA and viral proteins were stored at 808C for analysis. The therapeutic potential of the 19-mer siRNAs was also evaluated by first infecting the RD cells with EV70 (MOI of 10), followed by transfection with 100 nM of either si-3D1 or si-3D2 at 1, 3, 5, 7, and 9 hr post-infection. Determination of Transfection Efficiency To determine the transfection efficiency of the siRNAs into RD cells, the RD cells were grown in Labtek Permanox1 Chamberslide (Nunc, Germany) and transfection was carried out as described above with 100 nM of fluorescein labeled si-3D1 or si-3D2. After 48 hr, the cells were washed twice with phosphate buffer saline (PBS), and fixed with 4% paraformaldehyde for 30 min.
TABLE I. Nucleotide Sequences of si-3D1, si-3D2, and Scrambled siRNA siRNA si-3D1 Sense Antisense si-3D2 Sense Antisense Scrambled siRNA Sense Antisense
Nucleotide sequence
Target location
50 -GUC-UAG-AUU-GAU-UGA-AGC-UTT-30 50 -AGC-UUC-AAU-CAA-UCU-AGA-CTT-30
6,442–6,460
50 -AUG-CGC-CAG-CUA-AAA-CCA-ATT-30 50 -UUG-GUU-UUA-GCU-GGC-GCA-UTT-30
5,979–5,997
50 -GAU-UGA-AGC-UUU -GUC-UAG-ATT-30 50 -UCU-AGA-CAA-AGC-UUC-AAU-CTT-30
—
The siRNAs were 19 nt long with a 30 -dTT extension. The scrambled siRNA was designed by rearranging the si-3D1 sequence and thus has a similar base composition to si-3D1.
J. Med. Virol. DOI 10.1002/jmv
RNA Interference (RNAi) Against Enterovirus 70
The cells were then washed and mounted with Vectashield mounting medium which contain propidium iodide (Vector Laboratories, Boston, MA). The cells were observed under the Olympus BX60 fluorescent microscope.
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instructions (Invitrogen Life Technologies, Carlsbad, CA), with the use of EV70 VP1 monoclonal antibody (1:200 dilution; Chemicon International, Madison, WI), the anti-PKR antibody (1:2,000 dilution; Sigma– Aldrich, St. Louis, MO), and the b-actin antibody at 1:4,000 dilution (Sigma–Aldrich, St. Louis).
Real-Time Reverse Transcription (RT) PCR At 48 hr post-infection, the cells were lysed with 150 ml of CelLyticTM Cell Lysis Reagent (Sigma–Aldrich, St. Louis, MO). The mixture of CelLytic reagent and lysed cells were then centrifuged at 12,000g for 15 min to remove cell debris. This was followed by viral RNA extraction using the QIAamp1 Viral RNA Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). The efficacies of si-3D1 and si-3D2 in inhibiting EV70 replication were then analyzed using real-time TaqMan RT-PCR. Specific primers and a TaqMan probe were designed to amplify a specific region within the 3Dpol gene of EV70 genome (Table II). The real-time TaqMan RT-PCR was carried out using the LightCycler and a one-step LightCycler RNA Amplification Hybridization Probe kit (Roche Molecular Biochemicals, Mannheim, Germany). The enzyme mix contains a mixture of reverse transcriptase and ‘‘Faststart’’ Taq Polymerase that allows reverse transcription of RNA template and subsequent cDNA amplification. Each 10 ml reaction contained 1.0 ml of RNA, 4 mM MgCl2, 0.4 mM of the forward primer, 0.5 mM of the reverse primer, 0.4 mM of TaqMan probe, 2.0 ml hybridization probe reaction mix, 0.2 ml enzyme mix and PCR grade water. The cDNA was first synthesized from the RNA template by reverse transcription for 10 min at 558C and subsequently amplified for 45 cycles at 958C for 30 sec, 608C for 15 sec and 728C for 9 sec. SDS–PAGE and Western Blot The transfection of siRNAs, followed by infection with EV70 was performed according to the experimental set up as described. At 24 hr post-infection, the cells were harvested for determination of total viral proteins. The cells were first lysed using 150 ml of CelLytic M Cell Lysis Reagent (Sigma, TX). The mixture of CelLytic reagent and lysed cells were then centrifuged at 12,000g for 15 min to remove cell debris and the lysate was collected. An aliquot of 20 ml of each lysate was then electrophoresed in a denaturing 10% polyacrylamide gel. Western Blot was then carried out using the Western Breeze detection kit according to the manufacturer’s
Cell Viability Assay MTS assay was carried out to assay for the toxicity of the 19-mer siRNAs. RD cells were seeded in a 24-well plates and transfection was carried out as described. At 48 hr post-transfection, cells were trypsinized, and 100 ml of the resuspended cells were transferred to the wells of a 96-well plates. A total of 20 ml of the MTS/PMS reagent (Promega, Madison, WI) were then added into each well. After 3 hr of incubation at 378C, the absorbance at 490 nm was measured. All assays were performed in triplicates and carried out as two independent experiments. RESULTS Design of 19-mer siRNAs and Transfection Efficiency Into RD Cells Both si-3D1 and si-3D2 were designed to target specifically at the 3Dpol gene of EV70 and the BLAST search showed that the 19-mer siRNAs designed were specific and there was no homology with human genes. The transfection efficiencies of the siRNAs were determined by transfecting the RD cells with 100 nM of fluorescein-labeled si-3D1 or si-3D2. Our results showed that there was high delivery of si-3D1 or si-3D2 into the RD cells and the fluorescein-labeled siRNAs were localized in the cell cytoplasm (data not shown). Protection of RD Cells From EV70-Induced CPE by si-3D1 and si-3D2 In order to evaluate the efficacies of the 19-mer siRNAs in inhibiting EV70 replication, the RD cells were first transfected with si-3D1 or si-3D2 at final concentrations of 1 and 100 nM. After 48 hr, the RD cells were infected with EV70 at a MOI of 10. Microscopic observations showed that there was significant protection of the RD cells from EV70-induced CPE when they were treated with 1 nM of si-3D1 or si-3D2 (Fig. 1C). The protection against CPE was more significant when the RD cells were treated with 100 nM of si-3D1 or si-3D2, indicating a dosage dependent inhibition of EV70
TABLE II. Nucleotide Sequences of the Specific Primers and TaqMan Probe Designed for the Detection of EV70 Primers/probe Primers EV70_3DFP EV70_3DRP TaqMan Probe EV70_3DTaq
Nucleotide sequence (50 –30 )
Position
AGC-AAG-GAC-AAG-AGA-CTG-A AGG-TTT-CCA-AAT-GCC-ACT-C
6,058–6,076 6,506–6,488
AGC-TTC-AAT-CAA-TCT-AGA-CTT-GCC-C
6,460–6,436 J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Protection of RD cells against EV70-induced CPE by 19-mer siRNAs. The RD cells were first transfected with 19-mer siRNAs (si3D1), followed by infection with EV70. The morphological changes of the RD cells were observed at 48 hpi under the light microscope at 40 magnification. A: Uninfected RD cells. B: Infected RD cells without any
siRNA treatment. C: EV70-infected RD cells treated with 1 nM of si-3D1. D: EV70-infected RD cells treated with 100 nM of si-3D1. E: EV70-infected RD cells treated with 100 nM scrambled 19-mer siRNAs (3D-scr). Similar results were observed in RD cells treated with si-3D2. The tests were carried out in two independent experiments.
replication by the 19-mer siRNAs (Fig. 1D). The inhibition of CPE was observed to last up to 48 hr post infection. In contrast, the scrambled siRNA (3D-scr) failed to protect the RD cells from CPE, even when it was used at 100 nM (Fig. 1E). To determine whether there is any cytotoxic effects on the RD cells as a result of treatment with 19-mer siRNAs, MTS assay was performed to measure the viability of the RD cells after they were treated with 100 nM of si-3D1 or si-3D2 for 48 hr. Our results showed that there was no deleterious effect on the growth and viability of the transfected RD cells (Fig. 2).
Inhibition of EV70 Replication by 19-mer siRNAs
J. Med. Virol. DOI 10.1002/jmv
We next determined the effects of the 19-mer siRNAs in inhibiting EV70 replication by transfecting the RD cells with graded concentrations (1, 10, and 100 nM) of either si-3D1 or si-3D2, followed by infections with EV70 at a MOI of 10. The inhibitory effects of si-3D1 and si3D2 on EV70 replication were analyzed by real-time TaqMan RT-PCR and Western blots. Real-time RT-PCR showed that transfection of RD cells with 1, 10, and 100 nM of si-3D1 resulted in a
RNA Interference (RNAi) Against Enterovirus 70
Fig. 2. Viability of RD cells was determined by the MTS assay after transfection with 1, 10, and 100 nM of synthetic 19-mer siRNAs (si-3D1 and si-3D2) targeted at the 3Dpol gene of the EV70 genome for 48 hr. The untransfected RD cells, RD cells treated with transfection agent (TA) and the RD cells treated with 100 nM of scrambled siRNAs (3D-scr) were included as controls. The absorbance was measured at 490 nm. The data shown represent the mean SD from two independent experiments. The ANOVA analysis for comparison between different concentrations of each 19-mer siRNAs was found to be P < 0.05. The ANOVA analysis for comparison between the untransfected RD cells and RD cells treated with 3D-scr was P < 0.05. The ANOVA analysis for comparison between the RD cells treated with transfection agent (TA) and the RD cells treated with 3D-scr was P < 0.05.
reduction of EV70 RNA by 47.56 7.07%, 92.56 2.78%, and 97.48 1.41%, respectively. On the other hand, treatment with similar concentrations of si-3D2 led to a corresponding 88.94 2.60%, 97.25 1.37%, and 99.98 0.00% reduction of EV70 RNA (Fig. 3A). To correlate with the dosage dependent decrease in the EV70 viral RNA shown by real-time TaqMan RTPCR, Western blotting using specific antibodies against EV70 VP1 structural protein was carried out. It was observed that as the concentrations of si-3D1 or si-3D2 increased, there was a significant decrease in the EV70 VP1 viral capsid protein (Fig. 3B). The reduction in VP1 detected correlated with increased concentrations (1, 10, 100 nM) of si-3D1 or si-3D2. In addition, there was a greater reduction in VP1 protein detected when the cells were treated with si-3D2 compared to treatment with si-3D1, augmenting the results established by the real time RT-PCR in that si-3D2 was more efficient at inhibiting EV70 replication when compared to si-3D1. siRNA Secondary Structure Prediction To investigate the possible role of secondary structures on the efficacies of the siRNAs, the secondary structures of the sequences for both si-3D1 and si-3D2 were predicted using Oligotech software version 1.0. The results showed that a relatively strong hairpin structure (G ¼ 2.8 kcal/mol) was formed in the nucleotide sequence of si-3D1. This structure was found to be stable up to 388C (Fig. 4). On the other hand, no stable secondary structure (G ¼ 0.5 kcal/mol) was formed in the nucleotide sequence of si-3D2 (Fig. 4). Therapeutic Potential of 19-mer siRNAs in Inhibiting EV70 Replication The therapeutic potential of si-3D1 and si-3D2 in inhibiting on-going EV70 replication was evaluated by first infecting the RD cells over a period of 9 hr. At 1, 3, 5,
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Fig. 3. Dose dependent inhibition of EV70 replication using 19-mer siRNAs targeted at the 3Dpol gene of the EV70 genome. The RD cells were transfected with 1, 10, and 100 nM of either si-3D1 or si-3D2, followed by infections with EV70 at a MOI of 10. The untreated EV70infected RD cells (þ), infected RD cells which were treated with 100 nM of scrambled siRNAs (3D-scr), infected RD cells treated with transfection agent only (TA) and untransfected () RD cells were used as controls. The cell lysates and cell supernatants were harvested to determine the viral RNA titers and VP1 protein levels. A: Real-time RTPCR was performed to analyze the viral RNA levels in the RD cells after they were treated with the various concentrations of either si-3D1 or si3D2. The results represent the mean percentage reduction in RNA levels (SD) from two independent experiments. The ANOVA analysis for comparison between different concentrations of each 19-mer siRNAs was found to be P < 0.05. The ANOVA analysis for comparison between the untransfected RD cells and the treated RD cells was P < 0.01. The ANOVA analysis for comparison between the RD cells treated with 3D-scr and the treated RD cells was P < 0.01. The ANOVA analysis for comparison between the untransfected RD cells and RD cells treated with 3D-scr was P < 0.05. The ANOVA analysis for comparison between the RD cells treated with transfection agent (TA) and the RD cells treated with 3D-scr was P < 0.05. B: Western blot analysis was performed using monoclonal antibodies against the VP1 structural proteins of EV70. b-actin was used as an internal control, using anti b-actin monoclonal antibodies. The tests were carried out in two independent experiments.
7, and 9 hr post-infection, the infected-RD cells were treated with 100 nM of si-3D1 or si-3D2. The RD cells were observed for CPE over a period of 48 hr. The EV70 viral RNA were extracted at 48 hpi and analyzed by realtime TaqMan RT-PCR. Survival of the RD cells treated with 100 nM of si-3D1 or si-3D2 was still observed at 7 hr post-infection. In contrast, significant CPE was observed in the untreated EV70-infected cells (data not shown). Real-time RT-PCR showed a significant decrease of viral RNA in the infected RD cells treated with either si-3D1 or si-3D2. Treatment with 100 nM of si-3D1 at 1 hpi showed a reduction of 66.10% 7.25. A reduction of 61.90% 1.13, 33.21% 5.82, and 20.52% 7.23 of EV70 viral RNA was observed when the RD cells were treated with 100 nM of si-3D1 at 3, 5, and 7 hpi, respectively. No significant reduction in the EV70 viral RNA was observed when the RD cells were treated with 100 nM of si3D1 at 9 hpi. Similar reductions of viral RNA were obtained when the RD cells were treated with si-3D2 at J. Med. Virol. DOI 10.1002/jmv
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Fig. 4. Predicted secondary structure of si-3D1 and si-3D2. The structures of the siRNAs were determined from the OligoTech software version 1.0. The local free energy of motives of the involved nucleotides is indicated.
1 hpi (63.37% 6.34), 3 hpi (61.06% 1.38), 5 hpi (53.34% 4.32), and 7 hpi (33.95% 6.98) (Fig. 5). Only 10.20% (5.88) reduction of the EV70 viral RNA was observed when the RD cells were treated with 100 nM of si-3D2 at 9 hpi. Thus, siRNA can still effectively inhibit viral replications when introduced to EV70 infected RD cells, but the protective effects of the siRNAs decreased gradually with prolonged infection of the RD cells. Specificity of siRNA-Mediated Viral Inhibition To test that the reduction in viral replication is due to RNAi and not off-target interferon response, detection of
PKR by western blots using anti-PKR antibodies were performed. As a positive control for the induction of PKR, the RD cells were treated with human alpha interferon (Sigma, TX) as described previously [Kanda et al., 2004]. No increase in PKR expression was observed in the RD cells treated with either si-3D1 or si-3D2 (Fig. 6). This ruled out the possibility that inhibition of viral replication was due to activation of PKR and induction of the interferon response. We concluded that viral inhibition is due to the specific action of both siRNAs. DISCUSSION
Fig. 5. Therapeutic potential of 19-mer siRNAs in protecting RD cells against EV70 infection shown by real-time RT-PCR. The RD cells were first infected with EV70 (MOI of 10), followed by transfection with 100 nM of either si-3D1 or si-3D2 at 1, 3, and 5 hr post-infection. The untreated EV70-infected RD cells (þ), infected RD cells which were treated with 100 nM of scrambled siRNAs (3D-scr), infected RD cells treated with transfection agent only (TA) were included as controls. The ANOVA analysis for comparison between the untransfected RD cells and the treated RD cells was P < 0.01. The ANOVA analysis for comparison between the RD cells treated with 3D-scr and the treated RD cells was P < 0.01. The ANOVA analysis for comparison between the untransfected RD cells and RD cells treated with 3D-scr was P < 0.05. The ANOVA analysis for comparison between the RD cells treated with transfection agent (TA) and the RD cells treated with 3Dscr was P < 0.05. The results represent the mean percentage reduction in RNA levels (SD) from two independent experiments.
J. Med. Virol. DOI 10.1002/jmv
It is widely accepted that RNAi serves as a natural antiviral mechanism in plants [Gitlin and Andino, 2003]. In 2001, Elbashir et al. [2001] established that the RNAi silencing machinery was conserved in mammals. Indeed, introduction of siRNAs into cells in tissue culture and in animal models have been shown to inhibit replication of several human pathogenic viruses such as human immunodeficiency virus [Coburn and Cullen, 2002], influenza virus [Ge et al., 2003], human papillomavirus [Jiang and Milner, 2002], hepatitis B virus [McCaffrey et al., 2003], hepatitis C virus [Kapadia et al., 2003], enterovirus 71 [Lu et al., 2004; Sim et al., 2005; Tan et al., 2007] and the poliovirus [Gitlin et al., 2003]. A study by Kierzek et al. [1999] showed that the RNAi machinery is extremely sensitive to the nature of the mismatches. This finding was further supported by Gitlin et al. [2005] who showed that certain positions, particularly the 9–12 bp from the 50 end of the siRNA, are critical for recognition and RNAi function. This highlighted the importance of specificity of the siRNAs. Therefore, it is apparent that the RNAi machinery does not rely only on the thermodynamic characteristics of the duplex to select target RNAs. Instead, RISC seems to discriminate based on the architectural and structural properties of the siRNA-target RNA duplex. Analysis of a large dataset of target mRNA structures showed that
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Fig. 6. PKR levels in the RD cells as shown by Western blotting. The RD cells were transfected with various concentrations of either si-3D1 or si-3D2 for 48 hr, followed by EV70 infections at a MOI of 10. The RD cells treated with alpha interferon, scrambled siRNAs (3D-scr) and untransfected () RD cells were used as controls. Western blot analysis was performed using monoclonal antibodies against the PKR. b-actin was used as an internal control, using anti-b-actin monoclonal antibodies. The tests were carried out in two independent experiments.
siRNAs targeting mRNA with unpaired 50 and 30 ends exhibited greater silencing than when targeting unpaired regions in the center or paired regions throughout the gene [Gredell et al., 2008]. These factors were considered during the designing of the siRNAs. In this study, both si-3D1 and si-3D2 were designed to target the 3Dpol region of EV70 region. This region was chosen since it is the most conserved among the Picornaviruses [Gromeier et al., 1999], and has shown to be highly effective in inhibiting viral replication in other enteroviruses such as EV71 [Lu et al., 2004; Sim et al., 2005; Tan et al., 2007] as compared to other regions of the viral genome. The 3Dpol gene encodes the viral RNAdependent RNA polymerase which oligomerizes into a form most suited to the elongation and RNA-binding activity of polymerase. Since it is an indispensable factor in RNA synthesis, we expect that a knockdown of this region would effectively inhibit viral replications. The results from real-time RT-PCR and the Western blot analysis showed a dosage dependency of the siRNAs in inhibiting EV70 replication. In addition, both si-3D1 and si-3D2 conferred protection of the cells for up to 48 hr, suggesting the high prophylactic potentials of both si-3D1 and si-3D2 against EV70 infections. Results from the post-infection study also showed a significant reduction in the detection of EV70 RNA from cells treated with either si-3D1 or si-3D2 at 1, 3, and 5 hr after EV70 infection, highlighting the therapeutic potentials of si-3D1 and si-3D2. However, it appeared that both si-3D1 and si-3D2 were not as efficient when administered 5 hr after infection, as shown by only a 33% and 53% reduction in viral titers, respectively. Thus, early treatment of AHC is important to ensure effective inhibiting EV70 replication. We have not evaluated the efficacies of the 19-mer siRNAs in inhibiting EV70 replication in an animal model. If the data obtained from the in vitro system could be correlated with an in vivo system, early administration of the siRNAs during EV70 infection could prevent more serious manifestations of AHC due to a reduction of viral titer. As such, it is possible to prevent or manage an AHC outbreak by administering siRNAs to high risk clusters or individuals. It is apparent from both the pre-infection and postinfection studies that si-3D2 is more effective than si3D1 in inhibiting viral replication. A likely reason could
be due to the formation of a strong hairpin structure in the nucleotide sequence of si-3D1, which could have resulted in steric hindrance and impeded the Dicer from binding to si-3D1 in order to guide it to RISC. As a result, the efficiency of si-3D1 might have been compromised. It is possible that the inhibition of the viral replication could be due to an interferon-mediated response which could be activated by dsRNAs [Sledz et al., 2004], subsequently leading to inactivation of the cellular transcriptional machinery [Persengiev et al., 2004; Sledz et al., 2004]. The main mechanism in the interferon pathway is the induction of dsRNA dependent protein kinase R (PKR). PKR is a serine-threonine kinase which is normally inactive. Upon activation by interferon or dsRNA, PKR are phosphorylated and the levels are upregulated. The activated PKR will then phosphorylate the cellular proteins, notably eIF2a, which are downstream of the interferon pathway, and subsequently leading to translational arrest [Khabar et al., 2003; Persengiev et al., 2004; Sledz et al., 2004]. Thus, Western blotting was carried out using a monoclonal antibody directed against PKR to assess the possibility of interferon activation as a result of siRNAs introduction. Since no increase in the endogenous PKR was detected, our data indicated no engagement of the interferon pathway in the RD cells when they were transfected with either si-3D1 or si-3D2, and that inhibition of EV70 replication was due specifically to RNA interference. In conclusion, we have developed a potential therapeutic strategy based on RNAi to target at specific regions within the non-structural 3Dpol gene of the EV70 genome. Significant reduction of EV70 replication in the RD cell line treated with chemically synthesized siRNAs prior to and after EV70 infection was observed in this study, indicating the potential of siRNA serving both as a prophylactic and therapeutic intervention approach against EV70-causing AHC. REFERENCES Coburn GA, Cullen BR. 2002. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 76:9225–9231. Dykxhoorn DM, Novina CD, Sharp PA. 2003. Killing the messenger: Short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457–467.
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