- Open Access
Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells
© Luo et al; licensee BioMed Central Ltd. 2011
Received: 7 June 2011
Accepted: 7 September 2011
Published: 7 September 2011
shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells.
Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV.
Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.
Foot-and-mouth disease virus (FMDV) is a picornavirus, which causes a highly contagious disease in cloven-hoofed animals. Foot-and-mouth disease (FMD) is a threat to the worldwide livestock industry, because FMD can have a devastating effect on a country's economy. FMDV shows a high genetic and antigenic variability, which is reflected in the seven serotypes and numerous variants described to date . The FMDV genome is composed of a positive-sense single-stranded RNA molecule of about 8 500 nucleotides, which contains a unique open reading frame. There are seven distinguishable serological types, i.e., O, A, C, Asia1, SAT1, SAT2, and SAT3, and more than 65 subtypes.
Recently, RNAi has shown promise as a therapeutic in many infections, including, viral diseases of animals and humans. RNAi is a process of sequence specific, post-transcriptional gene silencing (PTGS) in animals and plants, which is conducted using 21 to 33 nucleotides (nt) of small interfering RNA (siRNA), which are homologous in sequence to the silenced gene . In eukaryotic organisms, dsRNA is produced in vivo or introduced by pathogens and processed into 21-23 nucleotide double-stranded short interfering RNA duplexes (siRNA) using an enzyme called Dicer, which is a member of the RNase III family of double-stranded RNA-specific endonucleases [3, 4]. Each siRNA ia then incorporated into an RNA-induced silencing complex (RISC), which is an enzyme complex used to target cellular transcripts complementary to the siRNA for specific cleavage and degradation [5, 6]. In addition to dsRNA, other endogenous RNA molecules have been shown to be capable of triggering gene silencing, including, short temporal RNA (stRNA) and microRNA (miRNA) .
RNAi has been widely studied as an antiviral technology for combating FMDV [8, 9]. FMDV is antigenically variable, undergoing rapid mutation, which allows it to easily escape the host immune system via the high variability of its surface antigens. Existing vaccines and antiviral drugs have limited effectiveness, so the development of new strategies is essential. High genetic variability is a major issue that must be addressed in order to establish RNAi as a viable approach against FMDV . Current measures for the control of FMDV replication include, RNAi plasmid 1D or 3D , VP1 , IRES , all of which are focused on several regions of the FMDV genome . RNAi directed at specific gene sequences of certain FMDV strains might be a risky strategy, especially in the event of an emergent FMD outbreak in the absence of information concerning the serotype or genotype of the isolated pathogen, although early protection is needed. The integrin αv subunit is the FMDV receptor for each serotype or genotype and the integrin αv subunit of the FMDV receptor plays a key role in the attachment of the virus to susceptible cells. Studies have shown a clear correlation between sensitivity of FMDV and expression of integrins αvβ1, αvβ3, αvβ6, and αvβ8. Most integrin subunits have independent functions and are essential for normal development [14, 15]. α1-null mice are viable, fertile and apparently normal, whereas α7-null mice develop muscular dystrophy, and α9-null mice die within 10 day of birth. Mutations in many of the ligands for αv integrins are also viable. Thus, although many of these ligands and αv integrins are widely expressed during development, they do not appear to be essential [16, 17]. In this study, we used "knockdown" rather than "knockout" target gene expression of the porcine integrin αv subunit, the FMDV receptor, to establish a cell line that could inhibit FMDV replication. Therefore, our work on RNAi with FMDV is a step forward on all previous work, particularly with respect to the cell model system.
In this study, we constructed an RNAi lentiviral vector that inhibited target gene expression of the porcine integrin αv subunit and the FMDV receptor in PK-15, and FMDV replication in iαv-PK-15. The vector was capable of inhibiting viral replication in cultured porcine PK-15 cells.
Cells and viruses
293T cells and PK-15 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO™, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (pH 7.4). Cultures were incubated at 37°C with 5% CO2. FMDV O/CHA/99 [GenBank accession number AF506822] was used for viral challenge.
Construction of Lentivirus vector and establishment of the iαv-PK-15 cell line
Double-stranded oligo  (ds oligo)
Assay for knockdown of the iαv-PK-15 cells
RNA isolation and real-time qRT-PCR 
Primer sequences used in this study
Indirect immunofluorescence (IIF) assay was carried out to detect expression of the targeted αv protein on the cell surface of an established iαv-PK-15 cell line at passage number 20, which was cultured in DMEM supplemented with 10% heat inactivated FBS. Cells were grown on acid-washed cover slips to ensure adherence of cells. The culture media was carefully aspirated from the culture dish using a pipette, because vacuum aspiration might also suck up cells from the cover slip surface. Cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature and were permeabilized for 5 min with 0.1% triton X-100 in 1× PBS at room temperature. Cells were then incubated in freshly prepared 50 mM NH4Cl for 15 min to quench free aldehyde groups from PFA, before fixation after washing three times with 1× PBS. Cultures were blocked with 1% BSA in 1× PBS for 30 minutes at room temperature, before incubation with primary antibody diluted in blocking buffer (1% BSA in PBS) for 1 h at room temperature. After primary incubation, cover slips were returned to the dishes and washed in PBS five to six times, for 5 min each wash. Secondary incubation was for 1 h at room temperature. The diluted antibody was centrifuged before use to pellet aggregates and covered in foil to protect from light. A control was included, which was comprised of the secondary antibody alone. The cover slips were returned to the dish and washed five times, for 5 min each wash using PBS, ensuring that samples were protected from the light. Cover slips were mounted by dipping the cover slips in distilled water to remove salt from PBS, before blotting the edge using filter paper to dry off excess water, before placing the cover slip cell-side down on a drop of mounting medium on a glass slide. The mounting medium used was DAPI (USA). The cover slip was stabilized on the slide using nail lacquer and left to dry overnight before viewing.
Indirect cell ELISA for adherent Cells
We added 100 μL of the cell suspension (1.0 × 105 cells in each well of a 96-well culture plate) and incubated in 5% CO2 at 37°C, for at least 24 h allowing cells to attach. We removed the residual culture medium by inversion and by gently tapping the inverted plate on a paper towel. We dispensed the primary antibody (50 μL), which was diluted in ice-cold ELISA buffer at the optimal concentration, whilst holding the pipette tip against the walls of the wells, then incubated for 1 h at 4°C. The unbound primary antibody was removed by vacuum aspiration, whilst taking care to avoid drying. The plate was washed by gently adding 200 μL of ice-cold washing buffer, whilst holding the pipette tip against the wall of the well to prevent detachment and loss of cells. The washing step was repeated four more times. After the last washing, the washing buffer was removed by inversion, whilst gently tapping the inverted plate on a paper towel. We then added the HRP-conjugated to the specific antibody for the primary antibody (50 μL) diluted in ice-cold ELISA buffer at the optimal concentration before incubating the plate for 1 h at 4°C. After five washings with PBS containing 1% BSA, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well and incubation was continued for an additional 10 min at room temperature. We stopped the enzyme reaction by adding 50 μL of 1.25 M H2SO4, and the optical density (OD) was measured with an ELISA reader (iEMS-Reader; Labsystems, Helsinki, Finland) at A450 nm.
Viral challenge assay for the iαv-PK-15 cells
Virus infectivity was determined by serial dilution of cell samples grown in 96-well plates. The virus titer was calculated as the 50% tissue culture infective dose (TCID50) using the Reed-Muench formula . A viral suspension titrated at 104.41 TCID50 was used in the experiment. Monolayers (about 50% confluent) were grown in 96-well plates to assess the capacity of viruses to grow in PK-15 cells expressing siRNAs. Cells in one well of the 96-well plates were infected with 100 TCID50 of virus per 0.1 ml. After 1 h of adsorption, the inoculum was removed and cells were washed twice with DMEM. The infection then proceeded in DMEM supplemented with no-fetal bovine serum and the virus titers were determined after two days. Statistical analysis was performed with the Microsoft Excel program.
Target gene expression of the porcine integrin αv subunit (the FMDV receptor) constructed with an RNAi lentivirus vector and established as an iαv-PK-15 cell line
We constructed the iαv-pENTR™/U6 entry clone and iαv-pLenti6/BLOCK-iT™ expression clone by producing a lentiviral stock (containing the packaged iαv-pLenti6/BLOCK-iT™-DEST expression construct, or the packaged pLenti6/BLOCK-iT™-DEST expression construct) and co-transfecting the optimized ViraPower™ Packaging Mix (3.0 μg/μl) and iαv-pLenti6/BLOCK-iT™-DEST expression construct or pLenti6/BLOCK-iT™-DEST expression construct (3 μg) into the 293FT producer cell line, then harvested the viral supernatant, and determined the lentiviral stock titer with blasticidin. We picked blasticidin-resistant clones and cultured then in DMEM supplemented with 10% heat-inactivated FBS at passage number 20. The sequencing results demonstrated successful cloning of the iαv-pENTR™/U6 entry clone and the iαv-pLenti6/BLOCK -iT™ expression clone construct. The functional titer of the virus obtained was 1.0 × 106 TU/mL. We established stabled iαv-PK-15 cells line at passage number 20, which inhibited target gene expression in the porcine integrin αv subunit, the FMDV receptor.
Real-time qRT- PCR assay of inhibition of RNA replication
Level of inhibition of integrin αv subunit expression by IIF and indirect cell ELISA for adherent cells
Inhibition of FMDV O/CHA/99 replication in iαv-PK-15 cells
FMDV is the prototypic member of the Aphthovirus genus of the Picornaviridae family. The viral capsid is made up from 60 copies each of four viral encoded proteins, VP1 to VP4: VP1, VP2 and VP3 form the outer clasped shell; and VP4 makes up the interior surface . FMDV enters the cell by receptor-mediated endocytosis, in a process that begins with the initial attachment of the virus to cell-surface receptors. The G-H loop forms a major antigenic site on the virion, which includes at its apex an Arg-Gly-Asp (RGD) motif [23, 24], a sequence that has been implicated in receptor binding on the basis of synthetic peptide inhibition studies . FMDV can enter cells using different pathways; the main pathway is by binding integrins via a conserved RGD located within the GH loop of VP1 [26, 27]. One subgroup within the integrin family is the αv integrin, which recognizes RGD as a binding motif on natural ligands. The αv subunit is subdivided into two groups based on certain structural differences . The first group is formed by α-1, α-2, α-L, α-M, and α-X. The second group is formed by α-3, α-5, α-6, α-7, α-8, α-IIb, α-V and αIEL. 24 functional integrins have been identified in mammalian species, which result from different pairings among 18 α subunits and 8 β subunits . Different integrins have roles as FMDV receptors (αvβ1, αvβ3, αvβ6, αvβ8) . Members of this group all undergo post-translational cleavage of their precursor into a heavy and a light chain. The light chain is composed of the cytoplasmic domain, the transmembrane region, and a part of the extracellular domain (about 25 kDa), whilst the heavy chain contained the rest of the extracellular domain (about 120 kDa) .
RNAi is a powerful gene silencing mechanism operating in most eukaryotic cells . The effector molecules comprise short duplex RNA sequences of 21-23 bp that direct inhibition of homologous genes. This natural pathway is important for processing of regulatory micro RNAs (miRs) . FMDV is antigenically highly variable and consists of seven serotypes and multiple subtypes  that underlie the rapid spreading nature of the clinical disease as early as 2-3 days post-exposure. This means that there is a need to develop new tools that provide early protection, thereby containing the disease spread. RNAi is used as an antiviral technology and has been widely studied for its affect on FMDV [9, 35, 36]. A multiple-siRNA expression system was developed that focused on several regions of the viral genome . Focusing on the conserved regions of the viral genome [38, 39], or simultaneously targeting several viral sequences are different approaches to resolving this issue. The multiple-siRNA-expressing plasmid, the pCWN11 system, is a feasible strategy to overcome the issue of high genetic variability in FMDV and viral escape [40, 41]. This approach may be used to deal with several viruses, which would be especially useful in the treatment of coinfections by multiple pathogens . Current measures for the control of FMD outbreak include routine vaccination, control of animal movement, and slaughter. FMD vaccines based on inactivated virus and adjuvants are effective in eliminating the disease, but risk the escape of live viruses from animal facilities or as a consequence of improper vaccine preparations [33, 42]. The development of a recombinant peptide vaccine  and a synthetic peptide vaccine ) both of which are safe and effective, has been reported. The limited effectiveness of existing vaccines and antiviral drugs means the development of new strategies is essential. The RNAi approach has been reported as an ideal tool to inhibit infectious virus replication in host cells, because siRNA can target and silence important viral genes. However, RNA viruses are likely to evolve mechanisms to suppress or escape an RNAi response. Long-term silencing of viral protein expression by siRNAs has been reported to result in the emergence of viruses resistant to RNA interference [40, 45]. In particular, integrin αv-heterodimers have been previously shown to mediate cellular entry of field strains of the virus. In contrast, cell surface expression of αβ3 or mRNA for the αβ1, αβ3 or αβ5 subunits did not appear to contribute to sensitivity of cells to FMDV . We constructed an RNAi lentiviral vector to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor.
It was previously shown that siRNAs inhibited virus yield by 10-fold to 1000-fold. The level of viral replication (IND 63/72) inhibition varied depending on the siRNAs produced by the Cocktail Kit. Here, the maximum inhibition (120-fold reduction in virus titer) was observed with the 2A-2C product compared with both VP3-VP1 (100-fold reduction in virus titer) and 3D-3' UTR (40-fold reduction in virus titer) at 24 h. In this case, there was a > 99% reduction in virus titer with all the serotypes up to 24 h . pCWN11 was a plasmid constructed to express siRNAs with multiple-targeting of the VP1 genes in FMDV. To investigate the effect of pCWN11 on FMDV replication, approximately 103 TCID50 of HKN/2002 virus progeny was detected in the supernatant and collected from cells transfected with pCWN11 at 19 h p.i. Over 105 TCID50 of virus was determined in the supernatant collected from control cells . However, a 1000-fold reduction was found in virus titer 24 h post-infection of BHK cells expressing small hairpin RNA (shRNA) under the control of a mouse U6 promoter from a plasmid construct . We observed a significant cross-inhibition of FMDV replication in BHK-21 cells by siRNAs targeted at various conserved regions of the viral genome (5VNCR, VP4, VPg, POL, and 3VNCR). The results showed that siRNAs generated in vitro by human recombinant dicer enzyme resulted in a viral yield inhibition of 10-fold to 1000-fold in both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection . A sequence corresponding to the critical 30 bp region that forms a stem-loop structure in IRES was also selected for investigation. The regions selected for shRNA were designated as D1, D2, D3, and D4. The virus in the supernatant was titrated in BHK-21 cells to determine infectivity. At 12, 18, and 24 h, the D4-transfected cells yielded a viral titer (10-5 TCID50) that was almost equal to the control. The virus titer in the D2- and D3-transfected cells was less than 10-2 TCID50 at 18 and 24 h. D1-transfected cells yielded 101.4, 101.7, and 102.9 TCID50 at 12, 18, and 24 h, respectively . Kahana et al. demonstrated 100% inhibition of virus growth in BHK-21 cells transfected with a mixture of several anti-FMDV siRNAs . In this study, there was reduction in virus titer of greater than 1000-fold in iαv-PK-15 cell for O/CHA/99. The results indicate that the reduction in virus titer was either better than the previous study or showed the same effect.
In this study, we constructed an RNAi lentiviral vector containing the packaged iαv-pLenti6/BLOCK-iT™-DEST expression construct to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor. We co-transfected the ViraPower™ Packaging Mix and the iαv pLenti6/BLOCK-iT™-DEST expression construct containing the U6 RNAi cassette into 293FT cells to produce a replication-incompetent lentivirus, which was transduced into the mammalian cell line of interest. Once the lentivirus enters the target cell the viral RNA is reverse-transcribed, actively imported into the nucleus, and stably integrated into the host genome. The lentiviral construct contains a deletion in the 3' LTR that leads to self-inactivation of the lentivirus after transduction into mammalian cells. Once the lentiviral construct has integrated into the genome, the lentivirus can no longer produce packageable virus and the shRNA of interest is constitutively expressed allowing the performance of transient RNAi analysis, or blasticidin selection to generate a stable cell line for long-term knockdown studies. To determine whether this system can be used to inhibit gene expression of the integrin αv subunit gene, the expression construct and packing plasmid were transduced into PK-15 cells, and we then established an iαv-PK-15 cell line. We found that co-transduction resulted in a significant reduction in the corresponding integrin αv subunit transcription by real-time qRT-PCR, and integrand αv subunit expression by cell ELISA and IIF. Cell ELISA is a useful technique for the quantitative analysis of cell surface antigen expression, which was developed on the basis of enzyme immunohistochemistry (EIH) and ELISA. Since its development, which was facilitated by the establishment of monoclonal antibody technology, a wide range of cell types and surface molecules have been analyzed by cell ELISA, and other methods for the quantitative detection of cell surface molecules. Cell ELISA is not appropriate for analyzing mixed-cell populations. In cell ELISA, labeling of immunoreactant molecules is achieved using an enzyme, such as horseradish peroxidase (HRP). IIF is a rapid sensitive method, which uses cell culture for the detection of integrin αv subunit expression in cell surface. At present, there is no information available; further work is needed to test the relationship between anti-FMDV efficiency and integrand β1, β3, β6 and β8.
Our results suggest that the interference integrin αv subunit expressing system is a feasible strategy for overcoming the issue of high genetic variability of FMDV, and viral escape. This approach may be developed to combat several viruses, which would be especially useful in the treatment of co-infections by FMDV. In addition, the new strategy would be beneficial in combating viruses with a high viral mutation rate. Our study provides a foundation for the cultivation of new varieties of transgenic organisms. However, this work represents a significant advancement, describing a new approach to trigger anti-FMDV pathways through inhibiting the FMDV receptor gene expression of the porcine integrin αv subunit.
This work was supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms (2009ZX08006-002B, 2009ZX08007-008B and 2008ZX08011-004).
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