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Short Communication |
6 RNA-dependent RNA polymerase
1 Department of Virology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland
2 Institute of Biotechnology and Department of Biological and Environmental Sciences, Biocenter 2, Viikinkaari 5, PO Box 56, FIN-00014 University of Helsinki, Finland
3 Department of Microbiology, Aapistie 5A, 90014 University of Oulu, Finland
Correspondence
Michaela Nygårdas
mnygarda{at}abo.fi
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ABSTRACT |
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6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837–7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the 6 RdRP (6–siRNAs) was considerably more effective than single-site siRNAs. The 6–siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9. A supplementary table is available with the online version of this paper.
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; Klingel et al., 1995; Leslie et al., 1989; McManus et al., 1991; Woodruff, 1980). The disease is often self-limiting and subclinical, but some acute infections are severe and lethal. Enteroviruses, and CBVs in particular, may have a role in the pathogenesis of type 1 diabetes (Hyöty & Taylor, 2002). There are no virus-specific therapies for enterovirus infections.
Bacteriophage 6 is a complex double-stranded RNA (dsRNA) virus of the family Cystoviridae, infecting the plant-pathogenic bacterium Pseudomonas syringae pv. phaseolicola HBY10 (Vidaver et al., 1973). The polymerase complex of the virus is composed of four phage-specific protein species, P1, P2, P4 and P7, which are all encoded by the genomic L-segment (Mindich et al., 1988). The RNA-dependent RNA polymerase (RdRP) P2 has several features which can be exploited as biotechnical tools. Firstly, it acts as both replicase and transcriptase in vitro using single-stranded RNA (ssRNA) and dsRNA templates, respectively (Makeyev & Bamford, 2000a, b). Secondly, it initiates RNA synthesis without a primer, starting the polymerization at the very 3'-terminus of the template (using both phage-specific and heterologous ssRNA templates) and may thus be used as a sequencing tool, especially for dsRNA viruses (Makeyev & Bamford, 2001). Thirdly, 6 RdRP can be used for production of large quantities of high-quality dsRNA (Aalto et al., 2007). This can be accomplished by two separate mechanisms, either by combining T7 RNA polymerase in vitro transcription and 6 RdRP for full-length dsRNA synthesis, or by an in vivo RNA replication system in which carrier state bacterial cells contain the 6–polymerase complex (Aalto et al., 2007). The first method produces dsRNA molecules of almost unlimited length, whereas the second method can produce high amounts of dsRNA up to 4.0 kb. These dsRNA molecules could serve as a source of short interfering RNAs (siRNAs) for RNA interference (RNAi) studies.
dsRNAs and various RNAi applications have been used to investigate gene functions, to target disease-related genes and as antiviral agents in cell culture and in vivo (Carthew & Sontheimer, 2009; Castanotto & Rossi, 2009). The RNAi approach also has potential for antiviral therapy of virus infections (Haasnoot et al., 2007; López-Fraga et al., 2008).
Enterovirus infections, such as poliovirus (Gitlin et al., 2002), enterovirus 70 and 71 (Lu et al., 2004; Sim et al., 2005; Tan et al., 2007a, b, 2008), coxsackievirus A16 (CAV16) (Wu et al., 2008) and CBV3 (Merl et al., 2005; Merl & Wessely, 2007; Schubert et al., 2005; Werk et al., 2005; Yuan et al., 2005) infections have been successfully inhibited using RNAi in cell culture and in animal models (Fechner et al., 2008; Kim et al., 2008; Merl et al., 2005; Tan et al., 2007b). These studies have employed sequence-specific siRNAs or hairpin RNAs, synthesized chemically or transcribed by DNA-dependent RNA polymerases. Since identifying target sequence candidates for RNAi can be difficult and expensive, it would be beneficial to find an alternative way of producing siRNAs. One mechanism would be to digest long dsRNA into siRNA pools representing multiple sequences of the viral gene or genome. EndoRNase-produced siRNAs have been used previously against hepatitis C virus (Krönke et al., 2004) and other targets (Buchholz et al., 2006). In the present study, dsRNA covering a wide area of the CBV3 genome was synthesized with the 6 RdRP and then Dicer-digested into siRNAs (Fig. 1). Their efficiency in silencing the virus propagation was then compared with that of previously published siRNAs targeting single sites in the essential CBV3 RNA polymerase gene. In addition, we studied the possible effects of the 6–CBV3 siRNA pool on other enteroviruses. As controls, we used unrelated siRNAs against green fluorescent protein (GFP), both as a single-site siRNA molecule as well as a 6 siRNA pool.
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). This 3562 bp fragment includes the P2b, P2c, P3A, 3B, 3C and 3D genes (Fig. 1
). This sequence was cloned into pBluescript II KS+ (Stratagene), the plasmid was linearized with ClaI and ssRNA was synthesized using T7 RNA polymerase. dsRNA was synthesized from this template with the 6 RdRP and the resulting dsRNA was digested using recombinant Dicer enzyme (Stratagene). The siRNAs were purified from undigested long dsRNA using Waters Gen-Pak FAX HPLC column. The 6–GFP siRNA pool was synthesized in a similar manner from a plasmid containing the entire enhanced GFP (eGFP) gene (Aalto et al., 2007).
Studies using single 21 nt siRNAs against CBV3 have previously targeted the RNA polymerase 3D (Schubert et al., 2005; Werk et al., 2005; Yuan et al., 2005), the viral protease 2A (Merl et al., 2005; Yuan et al., 2005) and VP1 (Ahn et al., 2005; Yuan et al., 2005). For comparison, we used two previously studied siRNAs specific to the 3D region: Sirev3, 5'-AATTAGGACACTAATGCTA-3' and siRdRP2, 5'-CTAAGGACCTAACAAAGTT-3' (Schubert et al., 2007), targeting the CBV3 sequence at nt 6825–6843 and 6315–6333, respectively (Qiagen). GFP-22 (Qiagen library item no. 1022064) siRNA and 6–GFP siRNA pool, both reactive against GFP, were used as non-specific controls. We also used a transfection control which contained water instead of siRNAs.
To test the inhibition of CBV3-, CBV4- and CAV9-infections with the 6–CBV3 siRNAs, LLC-MK2 cells (ATCC) were grown to a confluence of 
90 % and transfected with 100 ng siRNA targeting CBV3 (6–CBV3 siRNAs, Sirev3 or siRdRP2) or with control siRNAs (6–GFP siRNAs or GFP-22) per well, or with plain water, using Lipofectin reagent (Invitrogen). After a 24 h incubation at 37 °C, the cell culture medium was replaced with fresh growth medium (2 % fetal calf serum in Dulbecco's modified Eagle's medium+gentamicin). At 20–24 h after transfection, the cells were infected with 30 p.f.u. per well (m.o.i. of 0.0003) of CBV3 (Nancy strain), CBV4 (JVB strain) or CAV9 (Griggs strain) in a total volume of 100 µl and incubated for 1 h at 37 °C. Cells were washed four times with medium and incubated for 24, 48 and 72 h. The appropriate m.o.i. for the infections were determined in preliminary experiments (data not shown). Supernatants were collected and the cells were fixed with methanol for immunoperoxidase (IPS) detection and stained with a polyclonal antibody known to recognize the virus strains used in this study (Vuorinen et al., 1999).
IPS detection of the fixed cells (Fig. 2) treated with 6–CBV3 siRNAs showed no infected cells for CBV3, CBV4 or CAV9 as late as 72 h post-infection (p.i.). 6–GFP siRNAs also had a effect on the infection efficiency, but foci of infected cells were seen for all three viruses at 72 h p.i. No difference in numbers of infected cells was found between 6–GFP siRNA-treated and untreated cells in CAV9 infection. CBV3- and CBV4-infected cells transfected with the 6–GFP siRNAs showed a non-significant reduction in numbers of infected cells in comparison with untreated cells. No clear reduction was seen after treatment with Sirev3 in either CBV3-, CBV4- or CAV9-infected cells at 72 h p.i. (Fig. 2b, d, f). Treatment with siRdRP2 reduced the number of CBV3-infected cells but not CBV4- or CAV9-infected cells. CBV3 grows well in LLC cells and therefore cells were already detaching by 72 h p.i. This was seen also for Sirev3- and GFP-22-treated, infected cells. Sirev3 appeared to be somewhat toxic to the cells, as the cells detached sooner from the wells than did the untreated cells. All treated and untreated cells infected with CBV4 and CAV9 showed high numbers of infected cells by 72 h p.i. and no detectable difference could be seen between the different single-site siRNA treatments.
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. 6–CBV3 siRNAs inhibited the CBV3 infection almost completely, with only very low virus titres detected in the supernatants even at 72 h p.i. The CBV3 siRNA pool made using the 6 RdRP also had an inhibitory effect on two related coxsackieviruses, CBV4 and CAV9, both belonging to the enterovirus B species. The reduction in virus titre was significant at all three time points (P<0.05 compared with untreated cells) for all three viruses (CBV3, CBV4 and CAV9) treated with the 6–CBV3 siRNAs. Cells transfected with 6–GFP siRNAs showed a significant reduction in virus titre for CAV9-infected cells at the earlier time points, but not at 72 h p.i. These effects were not significant in the CBV3- and CBV4-infected cells. Reduction was also seen for GFP-22-transfected cells infected with CBV4. However, there is no detectable sequence similarity between the GFP-22–siRNA sequence and the CBV3, CBV4 or CAV9 genome. siRdRP2-treated cells infected with CBV3 showed a significant decrease in virus titre at all three time points (Fig. 3d–f). At 72 h p.i., there was a 10-fold reduction compared with non-transfected cells. For the other viruses, the reduction was less pronounced but was significant for CBV4. No significant decrease in virus titre was seen following CAV9 infection. Sirev3 reduced the amount of virus significantly only at 24 h p.i. in CBV3- and CBV4-infections. The 6–siRNA pools had a stronger effect than the single-site siRNAs, because at 72 h p.i., the reduction caused by siRdRP2 in CBV3-infected cells was 1 : 10 compared with the reduction of 1 : 104 observed for 6–CBV3 siRNA-treated cells.
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6–CBV3 siRNA treatment on the different human enterovirus B species could be explained by the fact that the siRNAs span a large CBV3 genome segment, and this part of the genome is 80 % conserved between these related viruses. The IPS data support the findings obtained by titrating the supernatants. The results of a quantitative (q)PCR for the presence of enteroviral genome (modified from the method described by Lönnrot et al., 1999) in the cells were in accordance with the IPS and plaque titration (data not shown). The statistical analyses were performed with StatView Mann–Whitney U-test and ANOVA. siRNA treated samples were analysed in comparison with untreated infected cells. P values <0.05 were considered statistically significant.
It has been shown that some siRNAs activate the interferon (IFN) system (Kim et al., 2004; Rossi, 2009; Sledz et al., 2003) and it was long believed that an induction of the innate immune system would be an obstacle for possible therapeutic applications of RNAi. However, it has also been shown that siRNAs do not always trigger an IFN response (Judge & MacLachlan, 2008). We performed qRT-PCR for IFNs in our cell cultures and no induction of type 1 IFNs was observed, which could be attributed to the 6–siRNA pools (data not shown). In addition, we studied the IFN-inducible MxA response in the cells with qRT-PCR. The 6–CBV3 siRNA pool did upregulate the MxA response in CBV3 infected or uninfected cells. We observed, however, that some single-site siRNAs and the 6–GFP siRNAs may upregulate MxA expression. The IFN response may explain why the GFP–single-site siRNA or 6–GFP siRNA pool occasionally inhibited CBV4 infection. RNA viruses use dsRNA intermediates during transcription and therefore also trigger IFN responses. Using siRNAs spanning a wider genome area may have negative consequences for normal cell machinery. Such siRNAs may also target transcription of the cellular genes. However, we did not detect an effect of the 6–siRNA pools on transcripts of housekeeping genes such as GAPDH, β-actin or 18S rRNA (Supplementary Table S1, available in JGV Online).
We observed that 6–siRNA pools against CBV3 were three orders of magnitude more efficient at reducing viral replication compared with single-site siRNAs. In addition, they also had a profound effect on CBV4 and CAV9 replication. Such an effect was not observed using the single-site siRNAs. The target sites of these single-site siRNAs are not found in the CBV4 and CAV9 genomes, but are present in the CBV3 genome. The 6–siRNA pools are also more likely to inhibit viruses, which may escape single-site siRNA silencing by point mutations. It might also be possible to prevent human enterovirus B infections by targeting siRNA to highly conserved ‘Cre elements’(Lee et al., 2007). No off-target effects were detected that could be attributed to the 6–siRNA pools, probably due to the low concentration of each individual siRNA within the pool. It seems that the use of pooled siRNAs is a favourable means to target viral infections and may offer a viable alternative for single-site siRNAs.
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ACKNOWLEDGEMENTS |
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6–siRNAs. |
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Received 26 February 2009;
accepted 19 June 2009.
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