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Short Communication |
CIRAD, Département Systèmes Biologiques, UR-15, Campus International de Baillarguet, 34398 Montpellier, France
Correspondence
Renata Servan de Almeida
renata.almeida{at}cirad.fr
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ABSTRACT |
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), PPRV has now become endemic in west, central and eastern Africa, in the Middle East and in south-west Asia (Lefèvre & Diallo, 1990; Shaila et al., 1996). RP has also caused devastating losses of ruminant populations in Africa, Europe and Asia for several centuries (Rweyemamu & Cheneau, 1995). Despite large eradication campaigns, this disease remains endemic in some areas of the Somalian ecosystem. Given their animal health and economic relevance, it is of major interest to control PPR and RP efficiently. Extensive studies on vaccination of ruminants against both diseases have been carried out and have shown the economic advantages of this practice. However, the current vaccines are only preventive tools that induce a protective immune response before the animals are infected. A curative tool should therefore be developed that could help in the control of virus replication when animals are newly infected or are at high risk of becoming infected in the next few days. A promising approach is the possibility to knock down the expression of virus genes by the mechanism of RNA interference (RNAi).
RNAi is a process whereby introduction of double-stranded RNA (dsRNA) into cells results in the degradation of homologous mRNA and, consequently, post-transcriptional gene silencing (Fire et al., 1998; Hammond et al., 2001; Hannon, 2002). The dsRNA is processed into 21–25 nt short interfering RNA (siRNAs) with 2 nt 3' overhangs by the RNase III-like protein DICER in an initiation phase. Afterwards, the cleavage products are separated into single-stranded RNA and incorporated into the RNA-induced silencing complex (RISC). RISC, which consists of several proteins and the antisense strand of siRNA, mediates the recognition and cleavage of its mRNA target.
In this study, we tested chemically synthesized siRNAs targeting the nucleocapsid (N) genes of PPRV and RPV for specific inhibition of virus replication. As indicators of siRNA functionality, we measured reduction in viral RNA and protein expression, reduction in virus particle production and the cytopathic effects induced in infected cell culture. Our strategy for siRNA design was to target conserved regions of the N gene among different morbilliviruses identified on multiple alignments of the N gene sequences. Within these regions, 19 nt long siRNAs were designed, taking into account different criteria, including a 35–60 mol% G+C content of the duplex and the presence of specific bases in strategic positions (Amarzguioui & Prydz, 2004; Reynolds et al., 2004; Ui-Tei et al., 2004; Huesken et al., 2005). All designed siRNAs were purchased from Ambion. First, five siRNAs specific for the N gene of PPRV were tested (NPPRV5 sense, 5'-GAGAACUCAAUUCAGAACAtt-3', position 1001–1019; NPPRV6 sense, 5'-GGCGGUUCAUGGUAUCUCUtt-3', position 741–759; NPPRV7 sense, 5'-GCAUUAGGCCUUCACGAGUtt-3', position 899–917; NPPRV8 sense, 5'-GUAUCAACAGCUAGGAGAGtt-3', position 958–976; and NPPRV9 sense, 5'-GAACUUUGGCAGGUCAUAUtt-3', position 1102–1120). Afterwards, the regions silenced efficiently on PPRV (functional regions) were evaluated on RPV, using siRNAs homologous to the N gene of RPV (NRPV6 sense, 5'-GCAGAUUUAUGGUGGCAUUtt-3', position 741–759; and NRPV7 sense, 5'-GCACUGGGCCUGCAUGAAUtt-3', position 899–917).
To assess the antiviral activity of the siRNA sequences, Vero cells [European Collection of Cell Cultures (ECACC) 84113001] with 48 h growth were trypsinized and plated at 1x105 cells per well in 24-well plates. Twenty-four hours later, different final concentrations of siRNAs (100, 50, 25 and 12.5 nM) were complexed with 2 µg Lipofectamine 2000 (Invitrogen) per well in 100 µl OptiMEM I serum-free medium (Invitrogen). After 20 min incubation at room temperature, the siRNA–Lipofectamine 2000 complexes were added to cell-culture wells containing 200 µl MEM serum-free medium. Plates were incubated for 6 h at 37 °C with 5 % CO2 and then the well supernatant was replaced with 1 ml MEM supplemented with 5 % fetal bovine serum. Twenty-four hours after transfection, cells were infected by using an m.o.i. of 0.1 of either PPRV or RPV. Four days later, the siRNA silencing effect was evaluated by observation of cytopathogenic effect (CPE) and flow cytometry. CPE scores were defined as (+) for a CPE 
10 %, (++) for a CPE between 10 and 50 % and (+++) for a CPE >50 %. Virus titration and real-time PCR were also carried out for functional siRNAs identified by the two previous methods. Inhibition levels of 70 % for flow cytometry and 50 % for CPE were established as cut-off values for siRNA efficacy. Controls were done by using irrelevant siRNAs and non-transfected cells. Viral proteins in infected cells were labelled by using specific monoclonal antibodies (mAbs) against the N proteins of PPRV and RPV, the matrix (M) protein of PPRV and the fusion (F) protein of RPV, and a secondary fluorescein isothiocyanate (FITC)-conjugated antibody. In brief, cells were trypsinized and plated in 96-well plates. mAbs specific for each of the viral proteins were diluted appropriately and added to each well. After 30 min incubation at 4 °C and two washing steps, cells were incubated for 30 min at 4 °C with a second FITC-conjugated antibody (Bio-Rad). Finally, cells were washed and fixed with 1 % paraformaldehyde. All dilutions and washings were carried out using a PBS solution containing 0.015–0.025 % saponin for cell permeabilization and intracellular staining. Silencing of viral protein production by siRNAs was measured by microscopic observation of intracellular fluorescence and by flow cytometry using a FACSort (Becton Dickinson). To evaluate the reduction in infectious viral progeny, supernatants from siRNA-transfected and virus-infected cultures were recovered and subsequently titrated by TCID50 assay in Vero cells as described by Reed & Muench (1938). Total cellular and viral RNA was isolated from siRNA-transfected and virus-infected Vero cells by using an RNeasy Mini kit (Qiagen). RT-PCR and quantitative PCR were carried out in one step by using a Brilliant SYBR green QRT-PCR Master Mix kit, One-Step (Stratagene), according to the manufacturer's protocols. The primers for the N gene of PPRV were NP3bis, 5'-GTCTCGGAAATCGCCTCACAG-3' (forward, position 1232–1252), and NP4bis, 5'-CCTCCTCCTGGTCCTCCAGAA-3' (reverse, position 1563–1583). The primers for the N gene of RPV were NP 223, 5'-AGCATCTTATCACTGTTTGTC-3' (forward, position 275–295), and NP 388r bis, 5'-ATCTATCAGCCTCGTCATC-3' (reverse, position 420–438). Each reaction mixture contained 12.5 µl 2x SYBR QRT-PCR master mix, 100 nM each primer, 0.0625 µl StrataScript reverse transcriptase, 1 µl sample and water up to 25 µl. For relative RNA quantification, an external standard curve was created by using spectrophotometrically determined copy-number standards of plasmid pBluescript or pcDNA4/HisMax B containing the N gene of PPRV or RPV, respectively. Real-time PCR was carried out in an Mx 3000 cycler (Stratagene) using the following thermal-cycling profile: 50 °C for 30 min, 95 °C for 10 min, followed by 35 cycles of amplification (95 °C for 30 s, 55 °C for 1 min, 72 °C for 30 s) and a final cycle of 95 °C for 1 min, 55 °C for 30 s, 95 °C for 30 s. All samples were run in duplicate.
The results of the siRNA activity assay demonstrate that siRNAs NPPRV6 and NPPRV7, targeting two conserved regions of PPRV, clearly inhibited the replication of PPRV, as assessed by the marked decrease in CPE and immunofluorescence staining (Fig. 1), viral protein, viral titres and viral RNA. Flow cytometry showed a dramatic decrease of >80 % in fluorescence staining in cells treated with these siRNAs (Fig. 2). Production of PPRV progeny was shut down efficiently by approximately 100-fold in cells treated with the siRNAs NPPRV6 and NPPRV7, as demonstrated by virus titration. These siRNAs also caused an approximately 20- to 50-fold decrease in copy number of virus target genes, as demonstrated by real-time PCR (Fig. 3). Additionally, we have demonstrated that siRNAs NRPV6 and NRPV7, targeting the same conserved regions of the N gene of RPV, clearly inhibited replication of RPV, as assessed by the marked decrease in CPE (Fig. 1) and viral protein (Fig. 2), a reduction of >1000-fold in RPV titre and of 10- to 70-fold in RPV RNA copy number (Fig. 3).
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; Reynolds et al., 2004; Yoshinari et al., 2004; Huesken et al., 2005; Jagla et al., 2005; Pekarik, 2005; Shabalina et al., 2006), despite the loss of two others (A3 and A6; Amarzguioui & Prydz, 2004; Reynolds et al., 2004; Schubert et al., 2005; Pekarik, 2005). However, this study does not suggest that motifs U10, S11 and U18 guarantee siRNA activity; rather, we propose that special attention should be paid to these motifs, which may contribute substantially to siRNA functionality. Alternatively, the better activity of NRPV7+1 could also be the consequence of the addition of an A/U base pair at the 5' end of the antisense strand. This could result in decreased thermodynamic stability at this terminus, thus facilitating the selective incorporation of this strand into the RISC (Khvorova et al., 2003; Schwarz et al., 2003; Heale et al., 2005; Hutvagner, 2005; Shabalina et al., 2006).
The morbillivirus genome is a single, negative-stranded RNA molecule wrapped intimately in the N protein. The resultant nucleoprotein complex associates with the RNA-dependent RNA polymerase subunits to initiate intracellular virus replication. However, the nucleocapsid, rather than the free genome RNA, is the template for all RNA synthesis (Lamb & Parks, 2007). As the N protein is essential for paramyxovirus replication, it would be a good target for the RNAi strategy to inhibit replication of morbilliviruses. Indeed, we demonstrated in the present study that siRNAs targeting selected regions of N mRNA of PPRV and RPV inhibited replication of the viruses effectively in infected cell cultures, as evidenced by the results described above. Moreover, the fact that the N protein plays an essential role in paramyxovirus replication was supported by the reduced expression of the M protein of PPRV and the F protein of RPV. The siRNAs NPPRV6 and NPPRV7 inhibited expression of the M protein by 85 and 83 %, respectively, whereas siRNAs RPV6, RPV7 and RPV7+1 inhibited expression of the fusion protein of RPV by 83, 81 and 86 %, respectively (data not shown).
All inhibitory sequences showed specific activity, as the siRNAs directed against the PPRV genome did not show any inhibitory effect on RPV and vice versa (data not shown). The effect of all functional siRNAs was sustained for 5 days after siRNA administration, given that their antiviral activity was always verified after this interval. Four of five functional siRNAs presented a dose-dependent activity, but siRNA NRPV6 was more effective when weaker concentrations were used. This means that, above certain concentrations of siRNA, no further increase in effect can be achieved. The same behaviour was described by Far & Sczakiel (2003) when they used siRNA directed against ICAM-1 mRNA in a human endothelial cell line. Quantitative analyses have suggested that this phenomenon occurs because the siRNA effect may reach a plateau with increasing siRNA concentration, indicating that RISC in cells is saturable (Barik, 2004).
Our group has selectively inhibited the N genes of PPRV and RPV by using synthetic siRNAs directed against one gene region (position 480–498; D. Keita, R. Servan de Almeida, G. Libeau & E. Albina, unpublished data). Here, we have identified two novel regions in the N genes of PPRV and RPV. The most effective inhibition of virus expression was observed with siRNA NPPRV6 for PPRV and siRNA NRPV7+1 for RPV. All of these defined siRNAs may be used alone or in association for targeting multiple viral regions in order to prevent the emergence of escape mutants. The application of RNAi as an in vivo therapy requires an efficient delivery of siRNA to the appropriate tissues. For this purpose, plasmids or viral vectors, both expressing short hairpin RNA specific for virus genes, may be administered. Currently, we are studying delivery methods of siRNAs to be applied in infected animals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 March 2007;
accepted 25 April 2007.
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, PPRV; 
, RPV) was shut down efficiently, as demonstrated by real-time PCR and virus titration. The RNAs from three independent experiments were analysed. The siRNAs were transfected by using their optimal dilution, as described in the text.