Effective inhibition of foot-and-mouth disease virus (FMDV) replication in vitro by vector-delivered microRNAs targeting the 3D gene
© Du et al; licensee BioMed Central Ltd. 2011
Received: 9 April 2011
Accepted: 10 June 2011
Published: 10 June 2011
Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined.
Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV.
Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.
Foot-and-mouth disease (FMD) is an economically important and highly contagious disease of cloven-hoofed animals, most notably of cattle, pigs and sheep, as well as several wild-life species [1, 2]. The ability of FMD virus (FMDV) to spread rapidly in susceptible animals makes FMD a disease that is serious enough to be monitored by the World Organization for Animal Health (OIE). FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. The virus is antigenically highly variable and consists of seven serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3) and multiple subtypes . FMDV contains a positive-sense, single-stranded RNA genome of 8,500 nucleotides (nt) with a 50 nt terminus covalently bound to a small viral polypeptide VPg (3B), and a 30 nt poly(A) tail . The genome contains a long open reading frame (ORF) translated into a single polypeptide that can be cleaved into four structural proteins (VP4, VP2, VP3, and VP1), and 10 non-structural proteins (L, 2A, 2B, 2C, 3A, 3B1, 3B2, 3B3, 3C, and 3D) [3, 5]. Of particular importance to viral replication is the 3D gene encoding the RNA-dependent RNA polymerase (RDRP). In a mechanism catalyzed by two bivalent metal ions, the 3D enzyme elongates a primer to copy the viral RNA template (plus strand). The newly synthesized minus strand folds back on itself to generate a template-primer structure, which is elongated by the 3D gene product to form covalently linked dimeric RNA molecules [6, 7]. Due to its significance in viral replication, the 3D gene was employed as an RNAi target in this study.
RNA interference (RNAi) is an evolutionarily conserved mechanism of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNA (dsRNA). In the process, the cellular complex Dicer cleaves a dsRNA molecule to generate discrete 21-23 nt small interfering RNAs (siRNAs) or microRNAs (miRNAs), which guide the RNAi-induced silencing complex (RISC) to cleave the target mRNAs [8–10]. Because of the high rapidity and specificity of the RNAi effect, this method may complement and improve the traditional tools available to control important animal pathogens. In the past, siRNAs have been widely studied for their effects on FMDV [11–16]. Recently, artificial miRNA has been developed [17, 18]. It has been demonstrated that expression of miRNA vectors is more effective and less toxic than regular siRNA vectors [19–21]. In order to explore a new approach to inhibit FMDV, here we report on vector-delivered miRNA molecules that were studied for their inhibitory effects on FMDV replication. Our results show for the first time that vector-delivered miRNAs are able to efficiently inhibit FMDV replication. This study provides not only an experimental basis for the development of a new anti-FMDV strategy, but also for a new approach to study FMDV infection and replication.
Cell culture and viruses
Baby hamster kidney (BHK-21) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, Invitrogen Corporation, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The cultures were maintained at 37 °C in a 5% CO2 humidified incubator. FMDV isolates of strain O/CHA/99 (GenBank accession number AF506822)  were used for viral challenge. FMDV titers were determined in BHK-21 cells, and 50% tissue culture infective dose (TCID50) was calculated using the Reed-Muench method .
Selection of target sequences
Inserted sequences in miRNA-expressing plasmids
Inserted sequence (5'-3')
Position in 3D gene
Construction of miRNA expression plasmids
Complementary single-stranded DNA oligos (top and bottom strands) encoding four pre-miRNAs were synthesized, annealed, and ligated into pcDNA6.2-GW-miR vectors (Invitrogen, USA), a Pol II miR RNAi expression vector containing specific miR-155 flanking sequences (Figure 1B). The ligation mixture was then transformed into competent E. coli DH5α cells following the manufacturer's protocol. Plasmid DNAs were isolated and purified with Plasmid Miniprep Kit (TaKaRa, Japan). The pcDNA6.2-GW-miR-negative control plasmid contains an insert that can form a hairpin structure, which is processed into mature miRNA, but is predicted not to target any known vertebrate gene. Their corresponding sequences are separately shown in Table 1. The sequences of the inserts were checked by DNA sequencing (TaKaRa, Japan).
Construction of reporter plasmid
To provide a reporting system for detecting miRNA function, the recombinant plasmid p3D-GFP, containing the whole length of 3D gene, was constructed as follows: BHK-21 cells infected with FMDV (O/CHA/99) were lysed by repeated freeze-thaw cycles. Cell debris was removed by centrifugation for 10 min at 4000 rpm. The RNA was extracted from 350 μL of the clarified infected cell culture supernatant using Mini RNeasy Kit (Qiagen, Germany) as per recommendation of the manufacturer. Reverse transcription (RT) was carried out using Avian Myeloblastosis Virus (AMV) reverse transcriptase (TaKaRa, Japan) and an antisense Xba I-adapter primer, 3DR. The reaction mixture was incubated at 42°C for 1 h. Additional incubation at 95°C for 5 min inactivated the enzyme. The PCR amplification of 3D cDNA fragments was carried out using the primer 3DR and a sense Kpn I-adapter primer, 3DF. The PCR products were then cloned into the unique site of Kpn I and Xba I of the pcDNA3.1-CT-GFP vector (Invitrogen, USA). Competent Escherichia coli TOP 10 cells were transformed with the vector by heat shock. The sequences of the inserts were checked by restriction enzyme digestion and DNA sequencing (TaKaRa, Japan). To monitor fusion protein expression as an indicator of interference by miRNAs candidates, the constructed plasmid p3D-GFP was only transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, USA) per the manufacturer's protocol, and the transfected cells were then examined by fluorescence microscopy.
Silencing effect of miRNAs on reporter gene expression
Vector-delivered miRNAs were initially tested for sequence-specificity for the target 3D gene by employing a transient transfection of a reporter plasmid p3D-GFP expressing 3D. BHK-21 cells were seeded into 24-well cell culture plates without antibiotics for about 24 h before transfection at a cell confluence of approximately 80-90% and co-transfected in triplicate with Lipofectamine 2000 and Opti-MEM I Reduced Serum Medium (Invitrogen, USA) in the presence of 0.2 μg of reporter plasmid p3D-GFP and 0.5 μg of each miRNA expression plasmid. At 24 and 48 h after transfection, cells were examined under a fluorescence microscope and photographed using a video camera.
Inhibitory effect of miRNAs on FMDV replication
To detect the inhibitory effect of vector-delivered miRNAs on FMDV replication, BHK-21 cells were cultured in 24-well cell culture plates and transfected with miRNA-expressing plasmids in triplicate. After incubation for an additional 24 h, the transfection complex was removed and cells were washed twice with DMEM. A viral suspension titrated at 10-6.0 TCID50 per 0.1 ml was used for viral challenge. The transfected cells in one well of the 24-well plates were then infected with 500 μl of 100 TCID50 of FMDV. After 1 h of absorption, the inoculum was removed and the cells were washed twice with DMEM. The infection then proceeded in DMEM without FBS. At 24 h and 48 h after infection, cell cultures were harvested by three freeze-thaw cycles and stored at -80°C, until virus titer values were measured according to the TCID50 method.
Identification of miRNA-expression plasmids and reporter plasmid
Silencing effects of reporter gene expression by miRNAs
Effective inhibition of FMDV replication by miRNAs
RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections [26–28]. siRNAs, derived by processing long double-stranded RNAs, are often of exogenous origin, degrade mRNAs bearing full complementary sequences, and are currently being extensively evaluated as potential antiviral tools. In contrast, miRNAs, which are endogenously encoded and derived by processing of long hairpin RNA precursors, can either cleave mRNAs bearing full complementary sequences or inhibit translation of mRNAs bearing partial complementary sequences [29, 30]. It is believed that miRNAs are essential regulators of various processes, such as cellular differentiation, proliferation, development, apoptosis and pathogen-host interactions [30–32]. The antiviral potential of siRNAs has been comprehensively discussed in numerous reviews [26, 28, 33, 34]. Thus far, there is no report available for FMDV inhibition by vector-delivered miRNA, though miRNA is believed to have more potential than siRNA/shRNA [35, 36]. In the present study, we systematically evaluated the effects of miRNA-based RNAi on FMDV expression and replication in BHK-21 cells. Our results showed that miRNA-based RNAi could inhibit FMDV 3D protein expression and FMDV replication in vitro. This study is the first report to apply vector-delivered miRNA to inhibit FMDV replication.
Several researchers have shown that siRNA/shRNA targeting the 3D gene could efficiently inhibit FMDV replication. Moreover, according to their reports, viral inhibition triggered by siRNA/shRNA and targeting the 3D gene seems more efficient compared to other genes within the same genome [11, 14, 37]. Here we demonstrated that plasmid-based miRNAs designed against the FMDV 3D gene could strongly inhibit virus replication in the infected BHK-21 cells. Together with the results from previous studies, we are convinced that the 3D gene could be a good target for intervention in FMDV replication. It remains to be tested whether genes other than 3D could be miRNA targets. It has been shown that siRNAs against VP1, 2B, 3C and 5'UTR were highly effective inhibiting viral replication [11, 13, 15, 37]. In our experiment, only four sequences of 3D were tested; therefore, we cannot exclude the significance of other genes of FMDV as effective targets for inhibition.
By incorporating sequences encoding miRNAs specific to the 3D gene of FMDV into a murine miR-155 pre-miRNA backbone under control of Pol II promoter (CMV), we were able to intracellularly express miRNAs in cells transfected with miRNA plasmids coding pre-miRNAs. Theoretically, this type of vector provides unique benefits in designing antiviral therapies [17, 18, 36]. This strategy allows multiple miRNAs to be expressed coordinately from a single precursor RNA and processed into individual miRNAs [17, 38]. It remains to be investigated whether combining different miRNA-3D targets can improve the inhibitory effect beyond what we observed with miRNA-3D alone. To facilitate effective miRNA selection, the reporter vector p3D-EGFP was used to estimate gene silencing effects in transfected BHK-21 cells. Fluorescence microscopy showed dissimilar, but significant, decreases in GFP-positive cell numbers by co-transfection with all four 3D-specific miRNA expression vectors, but not by the control miRNA expression vector, indicating the high confidence of the web-based tool for miRNA prediction and the specificity of the gene silencing effects of the vector-delivered miRNAs. The efficiency of gene silencing varied between miRNAs targeted to different regions of the 3D gene. At present, there is no information available about the mechanisms that determine the gene-silencing efficiency of a given miRNA. Further work needs to be completed to test the relationship between miRNA silencing efficiency and targeted genes.
Our results indicate that vector-delivered miRNAs targeting the 3D gene effectively inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection. Further studies are required to determine whether the technology offers protection against FMDV infection in vivo. However, this work represents a significant advancement, describing another approach to trigger anti-FMDV pathways through actions of miRNAs.
This work was supported by National Major Special Project on New Varieties Cultivation for Transgenic Organisms (No. 2009ZX08007-008B, No. 2009ZX08006-002B, and No. 2009ZX08008-010B); National Natural Science Foundation of China (No. 30800833); and Nature Science Foundation of Gansu Province, China (No.1010RJZA003).
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