- Open Access
Multiple microRNAs targeted to internal ribosome entry site against foot-and-mouth disease virus infection in vitro and in vivo
© Chang et al.; licensee BioMed Central Ltd. 2013
Received: 22 September 2013
Accepted: 27 December 2013
Published: 6 January 2014
Foot-and-mouth disease virus (FMDV) causes a severe vesicular disease in domestic and wild cloven-hoofed animals. Because of the limited early protection induced by current vaccines, emergency antiviral strategies to control the rapid spread of FMD outbreaks are needed.
Here we constructed multiple microRNAs (miRNAs) targeting the internal ribosome entry site (IRES) element of FMDV and investigated the effect of IRES-specific miRNAs on FMDV replication in baby hamster kidney (BHK-21) cells and suckling mice.
Four IRES-specific miRNAs significantly reduced enhanced green fluorescent protein (EGFP) expression from IRES-EGFP reporter plasmids, which were used with each miRNA expression plasmid in co-transfection of BHK-21 cells. Furthermore, treatment of BHK-21 cells with Bi-miRNA (a mixture of two miRNA expression plasmids) and Dual-miRNA (a co-cistronic expression plasmid containing two miRNA hairpin structures) induced more efficient and greater inhibition of EGFP expression than did plasmids carrying single miRNA sequences.
Stably transformed BHK-21 cells and goat fibroblasts with an integrating IRES-specific Dual-miRNA were generated, and real-time quantitative RT-PCR showed that the Dual-miRNA was able to effectively inhibit the replication of FMDV (except for the Mya98 strain) in the stably transformed BHK-21 cells.
The Dual-miRNA plasmid significantly delayed the deaths of suckling mice challenged with 50× and 100× the 50% lethal dose (LD50) of FMDV vaccine strains of three serotypes (O, A and Asia 1), and induced partial/complete protection against the prevalent PanAsia-1 and Mya98 strains of FMDV serotype O.
These data demonstrate that IRES-specific miRNAs can significantly inhibit FMDV infection in vitro and in vivo.
Foot-and-mouth disease is an acute, highly contagious and economically important disease that affects domestic and wild cloven-hoofed animals, such as cattle, swine, sheep and goats [1, 2]. The etiological agent, foot-and-mouth disease virus (FMDV), belongs to the genus Aphthovirus in the family Picornaviridae. There are seven serotypes of FMDV and multiple subtypes [4–6]. The viral genome is composed of a positive-sense, single-stranded RNA that functions as an mRNA and contains a unique open reading frame (ORF) encoding a viral polyprotein. This polyprotein is co-translationally processed, largely by virus-encoded proteases, to produce about 15 mature proteins plus many different precursors [7–9]. Initiation of FMDV RNA translation is directed by a large RNA cis-acting element of about 440 nucleotides (nts) termed the internal ribosome entry site (IRES) element [10, 11]. This region is predicted to adopt a secondary structure that mediates RNA–protein interactions essential for ribosome recognition [12, 13]. The RNA genome also has to act as the template for RNA replication . During this process, the genome undergoes rapid mutation at average rates of 10-3 to 10-5 substitution per nucleotide copied, due to the lack of proofreading mechanism of the RNA-dependent RNA polymerase (RdRp) [15–17]. Thus, FMDV populations form as “clouds” of mutants, or mutant distributions, termed viral quasispecies [18–21]. FMDV evolution is strongly influenced by high mutation rates and the dynamics of viral quasispecies, and results in ever-changing targets for antiviral strategies, including vaccination [22, 23].
Although the current FMD vaccines play an essential role in the control of FMD outbreaks, they fail to induce an immediate protective response. There is a “window”, a so-called immune blank period, of susceptibility to FMDV infection in vaccinated animals at 1–7 days post-immunization [24, 25]. Hence, alternative emergency strategies are needed for rapid control of FMDV outbreaks. Small interference RNAs (siRNAs) have been widely studied as a means of inhibiting FMDV replication in vitro and in vivo[26–33]. However, the efficacy and specificity of this inhibition could be completely abolished by a single point mutation in the target sequence [34–37], potentially limiting the usefulness of this approach against rapidly mutating and mutated viruses such as FMDV .
Therefore, the use of microRNA (miRNA), rather than siRNA, may be necessary, to cause mRNA degradation in a sequence-specific manner or gene silencing in an imperfectly base-paired manner [39, 40]. Here we report that multiple vector-delivered, IRES-specific miRNAs effectively and specifically silence EGFP (enhanced green fluorescent protein) expression from IRES-EGFP fusion protein reporter plasmids in BHK-21 cells and inhibit virus replication in FMDV-IRES-specific Dual-miRNA-transformed BHK-21 cells and suckling mice. Additionally, a high-efficiency Dual-miRNA targeted to the IRES element was integrated stably into the chromosomal DNA of goat fibroblasts, for the future creation of transgenic animals resistant to FMDV infection.
IRES-specific miRNAs on plasmids silence reporter gene expression in BHK-21 cells
Specific silencing of EGFP expression by a single miRNA
Oligonucleotides of vector-delivered pre-miRNAs
Single stranded DNA sequences (5′ → 3′)
Efficiencies of miRNAs targeting the FMDV IRES in inhibiting EGFP expression in BHK-21 cells as assayed by flow cytometry
Inhibition efficiency of each miRNA (%)
Enhanced silencing of EGFP expression by Bi-miRNA and Dual-miRNA
pmiR242 and pmiR276 were used for further analysis of effective inhibition of IRES-EGFP reporter expression in BHK-21 cells. Co-transfection of a mixture of these two IRES-specific miRNA expression plasmids (pmiR242 and pmiR276, Bi-miRNA) with any of the three IRES-EGFP reporter plasmids resulted in a 78.4%–88.3% reduction in intensity of EGFP fluorescence, as compared with the individual plasmids of pmiR242 (44.3%–71.4%) and pmiR276 (60.5%–81.4%) (Figure 2, Table 2).
To further improve the specific silencing, we constructed pmiR242 + 276 (Dual-miRNA), a co-cistronic expression plasmid containing two IRES-specific miRNA hairpin structures (Figure 1A). BHK-21 cells were co-transfected with the Dual-miRNA plasmid pmiR242 + 276 and each IRES-EGFP reporter plasmid. Remarkably, the results showed that pmiR242 + 276 was more effective than pmiR153, pmiR220, pmiR242, pmiR276, or Bi-miRNA, and displayed 83.6%–96.6% inhibition of EGFP expression at 48 post-transfection (Figure 2, Table 2).
Stable expression of IRES-specific Dual-miRNA confers effective inhibition of FMDV replication
Selection of stably FMDV-IRES-specific Dual-miRNA-transformed BHK-21 cells and goat fibroblasts
Analysis of inhibition of FMDV replication in Dual-miRNA-transformed BHK-21 cells
Real-time quantitative RT-PCR analysis of the inhibition of FMDV replication in pEGFP-miR242 + 276-transformed BHK-21 cells, compared with normal BHK-21 cells
Ct values (mean)
34.61 ± 0.32/37.73 ± 0.07
36.20 ± 0.09/38.76 ± 0.25
35.98 ± 0.83/36.09 ± 0.12
37.55 ± 0.22/36.75 ± 0.44
37.62 ± 0.51/26.92 ± 0.21
36.08 ± 0.08/28.49 ± 0.39
36.45 ± 0.05/35.65 ± 0.66
36.85 ± 0.57/31.32 ± 0.10
33.41 ± 0.15/28.62 ± 0.40
28.54 ± 0.10/28.82 ± 0.49
26.84 ± 0.16/26.34 ± 0.11
26.34 ± 0.30/26.54 ± 0.22
16.70 ± 0.10/16.27 ± 0.23
15.17 ± 0.02/14.57 ± 0.04
11.81 ± 0.30/12.44 ± 0.14
8.43 ± 0.59/14.16 ± 0.41
7.49 ± 0.31/9.16 ± 0.04
13.68 ± 0.10/15.02 ± 0.59
30.97 ± 0.08/25.23 ± 0.17
36.23 ± 0.78/30.44 ± 0.41
36.36 ± 0.18/35.02 ± 0.79
38.45 ± 1.07/25.21 ± 0.92
36.32 ± 0.94/10.32 ± 0.71
37.13 ± 0.15/11.16 ± 0.31
36.73 ± 0.18/34.16 ± 0.68
37.12 ± 0.11/35.65 ± 0.19
37.28 ± 0.39/35.46 ± 0.04
35.42 ± 0.09/27.39 ± 0.20
36.62 ± 0.57/17.84 ± 0.63
38.59 ± 0.77/16.68 ± 0.44
Antiviral activity of vector-delivered Dual-miRNA in suckling mice
miRNAs play an important role in post-transcriptional gene silencing (PTGS), inhibiting translation at and/or following initiation, as RNA interference (RNAi) [42–44]. It is believed that miRNAs are essential regulators of cell fate determination, such as in early development, and of cellular proliferation and differentiation, apoptosis, and pathogen-host interactions [45, 46]. There are few reports of the use of miRNAs as anti-FMDV agents, although miRNA has more potential than siRNA to silence FMDV replication . In this study, the FMDV IRES was selected as the target of vector-delivered miRNAs and the inhibitory effects of these FMDV-specific miRNAs on IRES-EGFP expression, replication of the genomic RNA, and the pathogenicity of FMDV were examined in BHK-21 cells and/or suckling mice.
To analyze the antiviral effect, all serotypes of FMDV (including vaccine strains and the prevalent strains) isolated from China, except for the SEA topotype of FMDV serotype A , were used to inoculate the IRES-specific Dual-miRNA-transformed BHK-21 cells and for virus challenge in suckling mice. In the suckling mice, the Dual-miRNA plasmid was delivered with the virus challenge, differing from previous studies [30, 33, 38, 61]. Unexpectedly, the IRES-specific Dual-miRNA had no inhibitory effect on the RNA replication of FMDV O/CHN/Mya98/33-P in vitro despite its efficacy in vivo (Table 3, Figure 4E). The potential for the rapid, selective replication of the virus in vitro would increase the possibility of genetic changes and diversity in the populations of progeny virus (Table 3) [62, 63]. Consequently, the antiviral effect was inversely proportional to the number of mismatches between the miRNA and the targeted IRES sequence, although the predicted secondary structure was tolerated (Figure 5A) [64, 65]. In addition, the different gene silencing efficiencies and expression levels of the mature IRES-specific miRNAs could not guarantee complete inhibition of FMDV replication in the Dual-miRNA-transformed BHK-21 cells, and suggested that the tandem arrangement of pre-miRNAs and the reporter gene might influence the antiviral efficacy of FMDV-specific miRNA-expressing plasmids (Figure 1A) .
Our data demonstrate that FMDV replication can be significantly inhibited by FMDV-specific miRNAs targeted to the IRES element in vitro and in vivo. BlasticidinR clones of goat fibroblasts with chromosomally integrated FMDV-IRES-specific Dual-miRNA genes have also been obtained, in order to produce transgenic animals resistant to FMDV. We propose that multiple miRNAs could be effective new tools for the control of rapidly spreading FMD outbreaks in the future.
Cells, animals, and viruses
BHK-21 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone). Goat fibroblasts were kindly provided by Prof. Baohua Ma (Northwest Agriculture & Forestry University) and cultured in DMEM/F12 nutrient mixture (Gibco) (containing 1.5 g/L sodium bicarbonate) supplemented with 10% FBS. All cell lines were incubated at 37°C with 5% CO2. Kunming White suckling mice, 2–3 days old and weighing 3–4 g, were purchased from Lanzhou Institute of Biological Products. Five FMDV isolates, O/Tibet/China/1/99 [PanAsia-1 strain of ME-SA (Middle East-South Asia) topotype, AF506822], O/HN/CHA/93 (vaccine strain of Cathay topotype) , O/CHN/Mya98/33-P [Mya98 strain of SEA (South-East Asia) topotype, JQ973889], and AF72 (vaccine strain of Asia topotype) , Asia 1/Jiangsu/China/2005 (vaccine strain of SEA topotype, EF149009), were preserved and provided by OIE/National Foot-and-Mouth Disease Reference Laboratory of China.
Design and generation of vector-delivered miRNA plasmids
Four potential miRNAs were developed from the complete IRES nucleotide sequence of FMDV O/HN/CHA/93 strain by using the miRNA design tool on Invitrogen’s web site tool (http://rnaidesigner.invitrogen.com/rnaiexpress/, Table 1). Oligonucleotides of the pre-miRNAs forward and reverse strands were synthesized, annealed, and cloned into pcDNA™6.2-GW/miR vector (Invitrogen) under the control of PCMV and a transcriptional termination signal (TK pA), following the manufacturer’s protocol. These plasmids were designated pmiR153, pmiR220, pmiR242, and pmiR276 (Figure 1A). For subcloning, Bam H I/Xho I digested products from pmiR276 were inserted into pmiR242 at its Bgl II/Xho I sites, resulting in pmiR242 + 276, a Dual-miRNA plasmid containing two IRES-specific miRNA hairpin structures (Figure 1A). Then, Bam H I/Xho I fragments were digested from pmiR242 + 276 and cloned into pcDNA™6.2-GW/EmGFP-miR using a BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen), to generate the recombinant plasmid pEGFP-miR242 + 276 expressing EGFP (Figure 1A). The pcDNA6.2-GW/miR-negative control plasmid (pmiR-NC) was provided by Invitrogen (Table 1) and has no sequence homology with FMDV. All of these plasmids were confirmed by DNA sequencing.
Construction of reporter plasmids
To provide a reporting system for monitoring miRNA function, three recombinant reporter plasmids pHN/IRES-EGFP, pFC/IRES-EGFP, and pJS/IRES-EGFP were constructed as follows. Briefly, IRES fragments of each FMDV of vaccine strains of serotypes A, O, and Asia 1 were obtained using RT-PCR amplification with a sense Bam H I-adapter primer and an antisense primer, from genomic RNAs extracted from BHK-21 cell-adapted FMDV strains (O/HN/CHA/93, AF72, and Asia 1/Jiangsu/China/2005). The EGFP sequence was amplified from the pEGFP-N1 vector (Clontech) using specific primers, and the amplification products of the FMDV-IRES fusion with EGFP were constructed by use of overlapping PCR (PrimeSTAR; TaKaRa). The PCR products were then cloned into Bam H I/Xho I-degested pcDNA™6.2-GW/miR vector (Figure 1B). The sequences of the inserts were confirmed by restriction enzyme analysis and DNA sequencing. The reporter plasmid p3D-GFP used as a control for nonspecific effects was kindly provided by Dr. Junzhen Du .
Cell transfection and miRNA silencing of EGFP expression
BHK-21 cells were seeded in 6-well plates (Nunc) within 24 h before transfection. Monolayers (about 90–95% confluent) of BHK-21 cells were transiently co-transfected with 5, 10, or 20 μg of each reporter plasmid and 5, 10, or 20 μg of each miRNA expression plasmid (including a mixture of pmiR242 and pmiR276, Bi-miRNA) or pmiR-NC construct at an optimal ratio with 10 μL Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The cells were examined by fluorescence microscopy (Leica) for EGFP expression at 12, 24, 36, and 48 h post-transfection.
Specific silencing of target genes to restrain EGFP expression was also examined by flow cytometry at 48 h post-transfection as follows. The co-transfected cell monolayers were dissociated with 200 μL of 0.25% trypsin after washing with 1 × PBS two times, and resuspended in a total volume of 1 mL 1 × PBS/well. After three washes with 1 × PBS, they were diluted to 1 × 105–1 × 107 cells/mL in 1 × PBS for analysis by FACSCalibur (Becton-Dickinson), according the manufacturer’s protocol. The EGFP fluorescence was detected by optimal excitation at 488 nm and emission at 508 nm, and the fluorescence intensity values were calculated as the percentage of the cell populations.
Analysis of FMDV replication in Dual-miRNA-transformed BHK-21 cells
To establish BHK-21 cells stably transformed with Dual-miRNA, 10 μg pEGFP-miR242 + 276 plasmid was used to transfect 95% confluent BHK-21 cells in 35-mm plates using Lipofectamine 2000 as described above. At 4–6 h post-transfection, the OptiMEM-I (Gibco) suspended transfection complex was removed and the cells were trypsinized, diluted 10-fold, and seeded on microtitre plates (Greiner Bio-one). The cells were maintained under DMEM containing 10% FBS and 3 μg/mL Blasticidin (Invitrogen), by means of selection of resistant forms. The selection medium was changed every 2–3 days until the resultant BHK-21 cell cultures reached 100% confluency.
The stable cell monolayers were grown at a cell density of 1–2 × 105/well in 6-well plates, and washed twice with 1 × PBS. Viral suspensions titrated at 30–100 plaque forming units (PFU) per 1 mL were used for virus challenge. A multiplicity of infection (MOI) of 5–50 PFU of each virus per 200 μL in DMEM was added to each well. After 1 h of adsorption, the inoculum was removed and the cells were washed twice with DMEM. Then, 2 mL of DMEM supplemented with 2% FBS and 1% antibiotic (50 μg/mL Spectinomycin, Sigma) was added to each well and the plates was incubated at 37°C with 5% CO2. Subsequently, supernatants were collected at designated time points, and frozen at −80°C for later real-time quantitative RT-PCR analysis as described previously .
Virus challenge assay in suckling mice
To investigate the anti-FMDV activity of vector-delivered IRES-specific Dual-miRNA plasmid in vivo, suckling mice were used for virus challenge assay as previously described . Four suckling mice in each group were treated by subcutaneous injection in the neck of mixtures of a total volume of 200 μL comprising 50 or 100 LD50 of each virus in 50 μL 1 × PBS mixed with 200 μg of pmiR242 + 276 plasmid in 150 μL 1 × PBS. Control mice were inoculated subcutaneously in the neck with the same titer of FMDV (positive control), or 1 × PBS (negative control). All mice were monitored every 6 hours up to 7 days.
Establishment of FMDV-specific Dual-miRNA-transformed clones of goat fibroblasts
Goat fibroblasts were plated in 60-mm diameter dishes with 5 × 105 cells in 10% FBS-containing DMEM/F12 24 h before transfection. The cells were transfected with 10 μg pmiR242 + 276 plasmid. After 4–6 h, the transfection complex was removed, and 10% FBS in DMEM/F12 with Blasticidin (3 μg/mL) was added to the cells. Cells resistant to Blasticidin were selected for one week, with medium changes about every 2–3 days. Independent BlasticidinR clones were picked and expanded in the presence of Blasticidin (2 μg/mL) to avoid loss of the integrated DNAs. Cell stocks of IRES-specific Dual-miRNA-transformed clones were identified by PCR assay, and kept frozen in liquid nitrogen for further study.
We thank Prof. Baohua Ma (Northwest Agriculture & Forestry University) and our colleague Dr. Junzhen Du for providing goat fibroblasts and the p3D-GFP plasmid, respectively. We are also thankful to OIE/National Foot-and-Mouth Disease Reference Laboratory of China for providing the virus isolates, and Division of Quality Management of China Agricultural Veterinary Biological Science and Technology Co., LTD., for the necessary facilities for our experiments. This work was supported by grants from China’s Ministry of Agriculture, Genetically Modified Organisms Breeding Technology Major Program (no. 2009ZX08008-010B) (2009ZX08008-010B).
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