A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.


Introduction
The members of Picornaviridae including, foot-and-mouth disease virus (FMDV), human enterovirus 71, and hand, foot-and-mouth disease virus, are positive-sense single-stranded RNA viruses. The genomic RNA of approximately 8500 nucleotides is enclosed within a non-enveloped capsid with an icosahedral structure [1,2]. In infected cells, the viral RNA genome encodes only a single polyprotein, which is post-translationally processed region, when transfected with the two helper plasmids, could produce infectious FMDVs. We believe that not only is this system suitable for FMDV but it can also be applied for other picornavirus minigenomes. Moreover, it can be used for foreign protein expression such as a DNA vector but working solely in the cytoplasm.

RNA Isolation and cDNA Synthesis
Viral RNA was extracted using a viral nucleic acid extraction kit (Geneaid ® , Taipei, Taiwan) following the manufacturer's protocol. cDNA was generated from the RNA template using Superscript III RT (Invitrogen™, Waltham, MA, USA) according to the manufacturer's instruction. Briefly, the reverse transcription (RT) reaction contained 200 ng of random hexamer primers, 1 µL of 10 mM dNTP, 1 µg of RNA, and dH 2 O up to 13 µL. The RT master mix consisting of 4 µL of 5× FS buffer, 1 µL of 0.1 mM DTT, 40 Units of RNase OUT, and 200 units of Superscript III RT was prepared before adding into the reaction tube. The reaction was incubated at 55 • C for 1 h prior to being stopped by incubation at 70 • C for 15 min.

FMDV Growth Kinetics Analysis
Growth kinetics of the FMDVs, including type A; A/Lopburi/2012, type O; O/TAI/ 189/1987, and type Asia1; Asia1/2013, were studied. Briefly, overnight seeded BHK-21 cells in 6-well plates were inoculated with each FMDV strain at a multiplicity of infection (MOI) of 0.01. Three replicates were included for each virus. After an hour of virus adsorption, the inoculum was then removed, and the cells were washed once with MEM and maintained in 2 mL of MEM containing 2% FBS. The infected cells were incubated at 37 • C with 5%CO 2 for 24 h. The supernatants were collected at 6 different time points with 4-h intervals. The virus titers were calculated using the Reed and Muench method and reported as TCID 50 / mL . The log10 of virus titers were analyzed for the difference between mean titers of all 6-time points of each isolate with the repeated measure ANOVA using the generalized linear model (GLM) method available in the SAS ® University Edition (SAS Institute Inc., 2015, Cary, NC, USA). Once the analysis showed at least one mean with a statistically significant difference, the post-hoc comparison with the Tukey method was applied to demonstrate the different one. In addition, the difference of mean titers at each time point was also evaluated with the GLM and Tukey post-hoc comparison. The p-value less than 0.05 was considered as a statistical significance. The viral growth kinetics were plotted between the virus titers and hours post-inoculation (hpi). The procedures containing live viruses were performed at the Bureau of Veterinary Biologics, DLD, Thailand.

Construction of FMDV Minigenome Vectors: pKLS3 and pKLS3_GFP
To generate the FMDV minigenome, the entire FMDV O189 UTR was PCR amplified, cloned, and sequenced. The 5 UTR fragment was divided into three overlapping DNA fragments-S-fragment, poly C tract, and the large fragment (l-fragment)-for amplification by fusion PCR. In the first reaction, the S-fragment, poly C, and l-fragment were amplified with each specific primer pairs (Table 1) using the corresponding plasmids as the templates. In the second reaction, the purified S-fragment and poly C tract were used as the templates for amplification to generate the fused S-poly C fragment. In the third reaction, the fused S-poly C DNA and l-fragment were used as the templates for amplification to produce the -full-length 5 UTR flanked by XhoI and StuI recognition sequences. The pKLS3 vector (Patent Application Number: 1901006625) was modified from pKLS1. Briefly, the sequence of human polymerase I (Pol I) promoter was amplified using pDZ_NP2 [29] as the template and cloned into pGEM-T easy (Promega, Madison, WI, USA) downstream T7 RNA polymerase promoter resulting in pPol I. Subsequently, the full length 5 UTR of FMDV O189 was cloned into pPol I downstream of the Pol I promoter. In addition, the sequences of FMDV O189 3 UTR, a long poly A tail, and the hepatitis delta virus (hdv) ribozyme were synthesized (GenScript ® , Piscataway, NJ, USA), which was subsequently cloned into the plasmid pUC57 vector (GenScript ® , Piscataway, NJ, USA), resulting in pUC57_3 UTR. The DNA cassette containing the sequence T7 promoter-pol I promoter-FMDV O189 5 UTR in 5 to 3 direction was excised with NruI/StuI restriction enzymes (New England Biolab, Ipswich, MA, USA) from the pKLS1 vector and then ligated into pUC57_3 UTR upstream the 3 UTR, to create pKLS3 vector ( Figure 1A). The enhanced green fluorescence protein gene (GFP) was amplified from pEGFP-N1 (Takara Bio, Mountain View, CA, USA) by using primers EGFP_F and EGFP_R ( Table 2). The GFP PCR product was purified and ligated into the pKLS3 vector at the StuI restriction enzyme site, which is the junction between FMDV 5 and 3 UTRs, resulting in pKLS3_GFP ( Figure 1B).

Evaluation of Hepatitis Delta Virus Ribozyme (hdv) Activity by In Vitro Transcription
The activity of the hdv ribozyme was examined by in vitro transcription. The closedcircular plasmid DNA, pKLS, was used as the template for in vitro transcription with the application of the Riboprobe ® -T7 in vitro Transcription Systems (Promega, Madison, WI, USA) to produce an RNA molecule containing the Pol I-FMDV_5 UTR-GFP-FMDV_3 UTRpoly A sequence with the length of approximately 2.2 kb. Briefly, 500 ng of the plasmid was mixed with 4 µL of the 5× Transcription Optimized Buffer, 0.1 M DTT, 20 U of Recombinant RNasin ® Ribonuclease Inhibitor, 2.5 mM of each rNTP, 15 U of T7 RNA Polymerase, and nuclease-free water to the final volume of 20 µL. The reaction was incubated at 37 • C for 1 h, and the RNA product was analyzed by agarose gel electrophoresis. The DNA cassette containing T7 promoter and FMDV O189 5′UTR in pKLS1 vector was removed by digesting with the restriction enzymes, NruI and StuI, and placed upstream of FMDV O189 3′UTR, in plasmid pUC57_3′UTR, resulting in the pKLS3 vector. The complete pKLS3 construct is an FMDV minigenome containing the essential FMDV cis-acting elements for viral replication/transcription and translation and a StuI site for foreign gene insertion. (B) The enhanced green fluorescence protein (GFP) gene was inserted into pKLS3 at the StuI site between FMDV 5′and 3′UTRs to generate a minigenome with a transfection reporter pKLS3_GFP.

Evaluation of Hepatitis Delta Virus Ribozyme (hdv) Activity by In Vitro Transcription
The activity of the hdv ribozyme was examined by in vitro transcription. The closedcircular plasmid DNA, pKLS, was used as the template for in vitro transcription with the application of the Riboprobe ® -T7 in vitro Transcription Systems (Promega, Madison, WI, USA) to produce an RNA molecule containing the Pol I-FMDV_5′UTR-GFP-FMDV_3′UTR-poly A sequence with the length of approximately 2.2 kb. Briefly, 500 ng of the plasmid was mixed with 4 µL of the 5× Transcription Optimized Buffer, 0.1 M DTT, 20 U of Recombinant RNasin ® Ribonuclease Inhibitor, 2.5 mM of each rNTP, 15 U of T7 RNA Polymerase, and nuclease-free water to the final volume of 20 µL. The reaction was incubated at 37 °C for 1 h, and the RNA product was analyzed by agarose gel electrophoresis.

Construction of 'Helper Plasmids' for the FMDV Minigenome
The plasmid expressing T7 RNA polymerase, namely pCAGGS_T7, was constructed as described previously [30]. Briefly, the T7 RNA polymerase gene was amplified using DNA isolated from E.coli BL21 (DE3) (kindly provided by Prof. Dr. Wanpen Chaicumpa at Mahidol University, Thailand). The T7 RNA polymerase sequence was purified and cloned into pCAGGS to yield pCAGGS_T7. The sequence encoding the P3 open reading frame (ORF) of FMDV O189 was reverse transcribed using the viral RNA as the template by the action of Superscript III enzyme (Invitrogen, Waltham, MA, USA) with the random The DNA cassette containing T7 promoter and FMDV O189 5 UTR in pKLS1 vector was removed by digesting with the restriction enzymes, NruI and StuI, and placed upstream of FMDV O189 3 UTR, in plasmid pUC57_3 UTR, resulting in the pKLS3 vector. The complete pKLS3 construct is an FMDV minigenome containing the essential FMDV cis-acting elements for viral replication/transcription and translation and a StuI site for foreign gene insertion. (B) The enhanced green fluorescence protein (GFP) gene was inserted into pKLS3 at the StuI site between FMDV 5 and 3 UTRs to generate a minigenome with a transfection reporter pKLS3_GFP.

Construction of 'Helper Plasmids' for the FMDV Minigenome
The plasmid expressing T7 RNA polymerase, namely pCAGGS_T7, was constructed as described previously [30]. Briefly, the T7 RNA polymerase gene was amplified using DNA isolated from E. coli BL21 (DE3) (kindly provided by Prof. Dr. Wanpen Chaicumpa at Mahidol University, Thailand). The T7 RNA polymerase sequence was purified and cloned into pCAGGS to yield pCAGGS_T7. The sequence encoding the P3 open reading frame (ORF) of FMDV O189 was reverse transcribed using the viral RNA as the template by the action of Superscript III enzyme (Invitrogen, Waltham, MA, USA) with the random hexamer to generate the second helper plasmid. The cDNA was then served as the template to amplify the P3 DNA fragment using primers ClaI_P3_F and NheI_P3_R ( Table 3). The FMDV P3 DNA fragment was purified using a gel purification kit (RBC Bioscience, Taipei, Taiwan) and cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), resulting in pGEM_P3. The integrity of the nucleotide sequences in pCAGGS_T7 and pGEM_P3 were verified by DNA sequencing (Macrogen, Seoul, Korea). The fragment of FMDV P3 was double digested with ClaI/NheI restriction enzymes and ligated into the corresponding sites within the pCAGGS vector, resulting in pCAGGS_P3.

Transfection
The plasmids pKLS3_GFP, pCAGGS T7, and pCAGGS_P3, were purified using a Mini-Prep purification kit (GeneMark, Taichung, Taiwan), following the manufacturer's protocol. In the co-transfection experiment, the pKLS3_GFP and pCAGGS_T7 were transfected directly into BHK-21 cells, while pKLS3_GFP, pCAGGS_P3, and pCAGGS_T7 were combined in the tri-transfection protocol. The combinations of plasmids were incubated with Lipofectamine™ 2000 transfection reagent according to the manufacturer's instruction (ThermoFisher Scientific, Waltham, MA, USA) prior to dropping onto overnight seeded BHK-21 cells in 6-well plates. The protocol for co-transfection was optimized by varying the ratio of pKLS3_GFP to pCAGGS_T7 as 1:1, 1:2, and 1:3. In the co-transfection experiment, 10 µL Lipofectamine™ 2000 were incubated in a 250 µL Opti-MEM ® I reduced-serum medium (Invitrogen, Waltham, MA, USA) at room temperature for 5 min. Simultaneously, 1 µg of pKLS3_GFP and 1-3 µg of pCAGGS_T7 were equilibrated in 250 µL Opti-MEM ® I reduced-serum medium.
In the tri-transfection protocol, 1 µg of pKLS3_GFP, 3 µg of pCAGGS_T7, and 1 µg of pCAGGS_P3 were added to a microfuge tube containing 245 µL Opti-MEM ® I reducedserum medium. The diluted DNA and transfection reagent were mixed with each other and incubated at room temperature for 20 min. Subsequently, the mixtures were gently added onto the monolayer of BHK-21 cells prior to incubation at 37 • C with 5% CO 2 for 5 h. Then, the transfection medium was replaced with Opti-MEM ® I reduced-serum medium containing 2% FBS and the cells were incubated at 34 • C with 5% CO 2 . The mixture of pCAGGS_T7 and pCAGGS_P3 plasmids was also included as a negative transfection control. The co-transfection with pKLS3_GFP and pCAGGS_T7, and a single plasmid transfection with pKLS3_GFP were also performed in parallel to compare the expression level. After 24-48 h post-transfection (hpt), the cells were observed for green fluorescent signals using a fluorescence microscope (Olympus, Center Valley, PA, USA).

Inhibition of RNA Dependent RNA Polymerase (RdRp) Activities by Ribavirin
The tri-transfection of pKLS3 and the two helper plasmids was performed on a monolayer of BHK-21 cells in 6-well plates using the FuGENE ® HD transfection reagent (Promega, Madison, WI, USA) per the manufacturer's instructions. Briefly, 1 µg of pKLS3_GFP, 3 µg of pCAGGS_T7, and 1 µg of p CAGGS_P3 were mixed with Opti-MEM ® I reduced-serum medium in the final volume of 150 µL. Fifty microliters of FuGENE ® HD was then added into the plasmids' mixture, and the reaction was incubated at room temperature for 30 min to allow the complex formation. The transfection mixture was gently dripped onto the monolayer of BHK-21 cells prior to incubating the cells at 37 • C with 5% CO 2 for 4 h. Thereafter, the transfection reactions were removed and replaced with either Opti-MEM ® I reduced-serum medium containing 50 µM (EC50) or 160 µM (EC90) (Theerawatanasirikul et al., a manuscript in preparation) of ribavirin (Sigma-Aldrich, Gillingham, UK). Then, the cells were incubated at 37 • C with 5% CO 2 . The fluorescent signals were observed at 24 hpt using a fluorescence microscope (Olympus, Center Valley, PA, USA). The signal intensity and numbers of green fluorescent cells were compared between drug and non-drug treated units. In addition, rupintrivir, a 3C pro inhibitor, was tested at concentrations equal to its EC50 (2 µM) and EC90 (4 µM) (Theerawatanasirikul et al., a manuscript in preparation) to investigate the influence of protease inhibition on the RdRp function.

Molecular Docking of Ribavirin on FMDV RdRp
The interaction of ribavirin and FMDV O189 RdRp (3D pol ) was studied by means of homology modeling exploiting the platform available on the SWISS-MODEL server (https://swissmodel.expasy.org/, accessed on 19 April 2021) [31] to elucidate the binding between ribavirin and O189 RdRp. The three-dimension structure of the reference 3D pol (PDB code ID: 1wne.pdb, [32]) was used as the template for constructing a structural model of O189 3D pol . The quality of the modeled structure was analyzed by using Q-MEAN [33] and Ramachanadran plot [34]. The three-dimension structure of ribavirin (PubChem CID: 37542) was retrieved from the PubChem database for the molecular docking process. The protein structures were prepared as previously described [35]. The molecular docking was performed using PyRx 0.9.8 with Autodock Vina within the environment [36,37]. The docking grid center was randomly docked to the whole RdRp structure, and specified targets to the residues Pro44, Pro169, and M296 [38] with the grid box of x:20, y:20, z:20. The protein-ligand interaction was visualized using UCSF Chimera version 1. 13 Figure 2). The overall analysis from the repeated measure ANOVA showed that log10 of mean titers of all viruses differed from that of non-virus control (p < 0.05). The inclusive titers of O189, Asia1, and A/Lopburi were similar. The analysis of the differences between mean titers at each time point is shown in Table S1. At the sixth time point (24 hpi), the mean titer of O189 was the highest and significantly higher than those of other isolates, with a p value less than 0.05. The titer of O189 was approximately 2 × 10 5 TCID 50 / mL at 24 hpi; therefore, it was selected for further study.

Components of pKLS3 Minigenome
The FMDV minigenome, pKLS3, was constructed based on the essential cis-acting elements for viral replication and gene expression of FMDV O189. These elements included 5′UTR and 3′UTR with the poly A tail. The FMDV O189 5′UTR contained 1053 nucleotides which were arranged in 5′ to 3′ direction as an S-fragment (nucleotides 1-368), a stretch of 14 cytosine residues of the poly C tract, and an l-fragment (nucleotides 383-1053) ( Figure  3A). The uridylylation initiation site for 3B, cre, of FMDV O189 located at nucleotides 558-661 contained a conserved motif AAACA at nucleotides 580-584. The putative secondary structures of FMDV O189 IRES comprised 7 stem-loop structures of 512 nucleotides (residues 542 to 1053), which theoretically form a complex tertiary structure for the ribosome binding. The 3′ UTR of FMDV O189 in the pKLS3 minigenome consisted of 92 nucleotides, which was followed by 48 adenine residues of the poly A tail ( Figure 3B) and 85 nucleotides of the hepatitis delta virus (hdv) ribozyme, respectively. The cloning site for the

Components of pKLS3 Minigenome
The FMDV minigenome, pKLS3, was constructed based on the essential cis-acting elements for viral replication and gene expression of FMDV O189. These elements included 5 UTR and 3 UTR with the poly A tail. The FMDV O189 5 UTR contained 1053 nucleotides which were arranged in 5 to 3 direction as an S-fragment (nucleotides 1-368), a stretch of 14 cytosine residues of the poly C tract, and an l-fragment (nucleotides 383-1053) ( Figure 3A). The uridylylation initiation site for 3B, cre, of FMDV O189 located at nucleotides 558-661 contained a conserved motif AAACA at nucleotides 580-584. The putative secondary structures of FMDV O189 IRES comprised 7 stem-loop structures of 512 nucleotides (residues 542 to 1053), which theoretically form a complex tertiary structure for the ribosome binding. The 3 UTR of FMDV O189 in the pKLS3 minigenome consisted of 92 nucleotides, which was followed by 48 adenine residues of the poly A tail ( Figure 3B) and 85 nucleotides of the hepatitis delta virus (hdv) ribozyme, respectively. The cloning site for the FMDV coding sequence or foreign gene insertion was designed by introducing the recognition site for StuI at the junction between O189 5 and 3 UTRs. All these cisacting elements-5 UTR, 3 UTR, and poly A tail-and the hdv ribozyme sequences were located downstream of the T7 RNA polymerase promoter ( Figure 1A). Replication and transcription processes of the pKLS3 minigenome were driven by the T7 RNA polymerase promoter, while the translation process was initiated at the IRES once the uncapped mRNA was transcribed in the cytoplasm.  The 5′UTR comprised 368 nucleotides of S-fragment that formed a long stem-loop, followed by an l-fragment. The l-fragment was composed of 14 residues of a poly C, pseudoknots (pKs), cre, and IRES elements in a 5′ to 3′ direction. The pseudoknots spanned 175 nucleotides, while cre contained a conserve AAACA motif. The IRES spanned the rest of 5′UTR (located at 542-1053 nucleotides) upstream of the first start codon. (B) The 3′UTR contained 92 nucleotides folding into two stem-loop structures. Downstream the 3′UTR was a long poly A tail containing 48 adenine residues.
The hdv ribozyme was included in the construct to free the transcribed RNA at its 5′ end. Thus, we tested the function of the ribozyme by an in vitro transcription using the closed-circular plasmid as the template. If it functioned properly, the hdv ribozyme was The hdv ribozyme was included in the construct to free the transcribed RNA at its 5 end. Thus, we tested the function of the ribozyme by an in vitro transcription using the closed-circular plasmid as the template. If it functioned properly, the hdv ribozyme was expected to auto-cleave precisely at its 5 end. We found that the RNA product generated by the in vitro transcription was approximately 2.2 kb corresponding to the length of the whole O189 cis-acting elements and the hdv ribozyme presenting in the pKLS3 minigenome ( Figure 4). The result suggests that the hdv ribozyme was able to cut the transcribed RNA.
Viruses 2021, 13, x FOR PEER REVIEW Figure 4. The electrophoretic photograph presenting an in vitro transcribed RNA containi cis-acting elements of the pKLS3 minigenome and a GFP gene. The plasmid pKLS3_GFP w as the plasmid DNA template in an in vitro transcription reaction to test the hdv ribozyme tion. The hdv ribozyme auto-cleaved the transcribed RNA at its 5′ end, resulting in the RN uct containing the cis-acting elements of the pKLS3 minigenome and the GFP gene of appr mately 2.2 kb (lane 1). Lane M is a standard molecular weight DNA marker.

Functional Evaluation of pKLS3 as An FMDV Minigenome
To test the function of pKLS3 in transcription and translation processes, the G was introduced into pKLS3 at the StuI site as a reporter gene to generate pKL ( Figure 1B). In addition, pCAGGS_T7 was an indispensable helper plasmid, as it s intracellular T7 RNA polymerase in trans to transcribe the first strand RN pKLS3_GFP and pCAGGS_T7 were directly co-transfected into BHK-21 cells at d plasmid ratios (pKLS3_GFP: pCAGGS_T7 = 1:1, 1:2, and 1:3). The green fluoresc were visualized in all transfection conditions at 24 hpt ( Figure 5A). The GFP exp level was higher when increasing the amount of pCAGGS_T7. The highest fluo signals were detected at the pKLS3_GFP to pCAGGS_T7 ratio of 1 to 3, sugges optimal transfection condition ( Figure 5A). Increasing the amount of pCAGGS_T to 4 µg did not improve the transfection efficiency in terms of fluorescent inten

Functional Evaluation of pKLS3 as An FMDV Minigenome
To test the function of pKLS3 in transcription and translation processes, the GFP gene was introduced into pKLS3 at the StuI site as a reporter gene to generate pKLS3_GFP ( Figure 1B). In addition, pCAGGS_T7 was an indispensable helper plasmid, as it supplied intracellular T7 RNA polymerase in trans to transcribe the first strand RNA. The pKLS3_GFP and pCAGGS_T7 were directly co-transfected into BHK-21 cells at different plasmid ratios (pKLS3_GFP: pCAGGS_T7 = 1:1, 1:2, and 1:3). The green fluorescent cells were visualized in all transfection conditions at 24 hpt ( Figure 5A). The GFP expression level was higher when increasing the amount of pCAGGS_T7. The highest fluorescent signals were detected at the pKLS3_GFP to pCAGGS_T7 ratio of 1 to 3, suggesting the optimal transfection condition ( Figure 5A). Increasing the amount of pCAGGS_T7 from 3 to 4 µg did not improve the transfection efficiency in terms of fluorescent intensity and numbers of GFP positive cells. The results indicate that RNA molecules containing the GFP ORF were successfully translated into the GFP proteins clearly detected in transfected cells. However, the efficiency of GFP expression defined by the number GFP positive cells was less than 50%.

Enhancement of the GFP Expression Level by pCAGGS_P3
pCAGGS_P3 consisted of an ORF encoding for a P3 polyprotein of FMDV O189. Once translated, the P3 was processed to four mature proteins, including 3A, three copies of 3B (VPg), 3C (protease), and 3D (RdRp). This plasmid DNA was used as a helper vector  = 1:1, 1:2, and 1:3). The GFP expression level was enhanced by the increased amount of the plasmid carrying T7 RNA polymerase, and the highest signal was observed when pCAGGS_T7 was three times pKLS3. (B) Tri-transfection with pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 dramatically elevated the GFP expression level compared to co-transfection with pKLS3_GFP and pCAGGS_T7. No detectable signal was observed in the single plasmid, pKLS3_GFP, transfected cells. (C) BHK-21 cells were tri-transfected with various ratios of pKLS3_GFP: pCAGGS_T7: pCAGGS_P3 (1:3:1, 1:3:2, and 1:3:3) and the optimum ratio was 1:3:1 as it produced the highest fluorescent signal.

Enhancement of the GFP Expression Level by pCAGGS_P3
pCAGGS_P3 consisted of an ORF encoding for a P3 polyprotein of FMDV O189. Once translated, the P3 was processed to four mature proteins, including 3A, three copies of 3B (VPg), 3C (protease), and 3D (RdRp). This plasmid DNA was used as a helper vector to supply these proteins in trans. We expected that pCAGGS_P3 would expand the numbers of the transcripts produced by T7 RNA polymerase, serving as the templates for protein synthesis. The results demonstrated that the tri-transfection with pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 markedly increased both fluorescent signal intensity and numbers of the GFP expressing cells compared to the co-transfection with pKLS3_GFP and pCAGGS_T7 ( Figure 5B). Additionally, the positive signal was not detected in culture wells transfected with pKLS3_GFP alone. We also found that increasing the pCAGGS_P3 ratio reduced the level of GFP expression ( Figure 5C). Collectively, the results indicate that the optimal plasmid concentrations in the tri-transfection protocol were 1 µg of pKLS3_GFP, 3 µg of pCAGGS_T7, and 1 µg of pCAGGS_P3.

pKLS3_GFP as a Tool to Screen Antiviral Drug Targeting FMDV RdRp
The previous results showed that pCAGGS_T7 and pCAGGS_P3 could function in trans on the pKLS3 minigenome to drive transcription of the GFP gene. We then examined the application of this minigenome for antiviral drug screening. Ribavirin is a synthetic purine nucleoside (guanosine) analog with a broad-spectrum antiviral activity. It is effective against a large panel of RNA viruses, including picornaviruses, such as poliovirus, FMDV, and enterovirus 71. Inhibition of viral infection occurs through a direct interaction between RdRp and ribavirin-5 -triphosphate. The misincorporation of ribavirin into the viral genome results in increased viral mutation rates, leading to the extinction of the virus population [39]. In this study, the effect of ribavirin to inhibit the function of RdRp in the pKLS3 minigenome system was determined. In addition, we also investigated whether a non-RdRp inhibitor, rupintrivir, would influence our pKLS3_GFP minigenome system. Rupintrivir is a synthetic compound targeting the 3C pro of picornaviruses, such as enterovirus 71 and human rhinovirus [40]. BHK-21 cells transfected with pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 were treated with either low (EC50) or high (EC90) doses of either ribavirin or rupintrivir. The cytotoxic effect of the tested antiviral compounds on BHK-21 cells was also determined, and the 50% cytotoxicity concentration (CC50) of ribavirin and rupintrivir were higher than 500 µM [35]. Thus, the drug concentrations used in this study had no impact on cell viability.
The results showed that ribavirin almost completely inhibited the GFP expression as determined by markedly decreased numbers of GFP-positive cells ( Figure 6). Rupintrivir, a 3C pro inhibitor, which inhibits P3 processing, also decreased the GFP expression. However, the effect of the protease inhibitor was much less pronounced than that of ribavirin. The increased doses of ribavirin and rupintrivir from EC50 to EC90 did not decrease the fluorescent signals much. Please note that the EC50 and EC90 were performed using wild-type FMDV (Theerawatanasirikul et al., a manuscript in preparation). This finding indicates that our FMDV minigenome system using a combination of pKLS3_GFP and the two helper plasmids is a valuable screening tool for antiviral drugs targeting RdRp and may be 3C pro of FMDV. Additionally, this platform can be applied to other picornaviruses. Figure 6. Application of the FMDV minigenome system composing of pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 in cell-based antiviral drug detection. BHK-21 cells were transfected with the three plasmids, then treated with antiviral agents, including ribavirin and rupintrivir, at EC50 and EC90 doses. Ribavirin inhibited FMDV RdRp leading to dramatically decreased GFP expression level, while rupintrivir significantly decreased the number of the GFP positive cells. The microscopic magnification was 100×.

Molecular Docking of Ribavirin on FMDV RdRp
The model of FMDV O189 RdRp was developed, and the quality of the structural modeling was adequate for the molecular docking analysis to confirm whether ribavirin could bind to the active site on RdRp of FMDV. The amino acid sequence identity between the reference and O189 RdRp was 98.94%. The structural quality determined by QMEAN was scored at −0.86, while the Ramachandran plot was favored at 94.23%. The results of randomized docking showed that the ribavirin molecule preferentially buried in the pocket containing the residues Pro169 and Met296 with a binding affinity of −5.4 and −6.4 kcal/mol, respectively (Figure 7). The residues Pro44, Pro169, and Met296, were selected as specific docking targets according to the previous study [38]. In their study, M296I, P44S, and P169S substitutions were observed in the viral populations after being consecutively passaged in the media with ribavirin. For specific docking, ribavirin mainly reacted to Pro44 via π-sigma (−4.2 kcal/mol) and Pro169 via van der Waals (−5.4 kcal/mol) interactions. The possible interlinkages and binding affinities of ribavirin on FMDV O189 RdRp are depicted in Figure 7. Figure 6. Application of the FMDV minigenome system composing of pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 in cell-based antiviral drug detection. BHK-21 cells were transfected with the three plasmids, then treated with antiviral agents, including ribavirin and rupintrivir, at EC50 and EC90 doses. Ribavirin inhibited FMDV RdRp leading to dramatically decreased GFP expression level, while rupintrivir significantly decreased the number of the GFP positive cells. The microscopic magnification was 100×.

Molecular Docking of Ribavirin on FMDV RdRp
The model of FMDV O189 RdRp was developed, and the quality of the structural modeling was adequate for the molecular docking analysis to confirm whether ribavirin could bind to the active site on RdRp of FMDV. The amino acid sequence identity between the reference and O189 RdRp was 98.94%. The structural quality determined by QMEAN was scored at −0.86, while the Ramachandran plot was favored at 94.23%. The results of randomized docking showed that the ribavirin molecule preferentially buried in the pocket containing the residues Pro169 and Met296 with a binding affinity of −5.4 and −6.4 kcal/mol, respectively (Figure 7). The residues Pro44, Pro169, and Met296, were selected as specific docking targets according to the previous study [38]. In their study, M296I, P44S, and P169S substitutions were observed in the viral populations after being consecutively passaged in the media with ribavirin. For specific docking, ribavirin mainly reacted to Pro44 via π-sigma (−4.2 kcal/mol) and Pro169 via van der Waals (−5.4 kcal/mol) interactions. The possible interlinkages and binding affinities of ribavirin on FMDV O189 RdRp are depicted in Figure 7.

Discussion
Viral replicon systems have been developed previously by a number of research groups to study the replication of RNA viruses. Replicons or minigenomes are defined as self-replicating but non-infectious RNAs. Thus, the FMDV minigenome provides an opportunity for molecular biology research related to FMDV replication, transcription, and translation, which has normally been restricted to high biosecurity and containment facilities. Here, we report the development of an FMDV minigenome, pKLS3, which is a DNAbased vector containing the minimum cis-acting elements essential for transcription and translation of FMDV. Because this platform utilizes T7 promoter to facilitate the transcription of the first-strand RNA, the pCAGGS_T7 plasmid expressing the T7 RNA polymerase is a crucial component in this system. Our minigenome possessed a hdv ribozyme downstream from the poly A tail, which auto-cleaved at its 5′end, leading to the transcription termination of the T7 RNA polymerase. Another component, pCAGGS_P3, is not mandatory; however, it acts as the transcription enhancer to increase the yield of the transcripts for protein synthesis by cap-independent translation. The ribosome assembled at the IRES located within the 5′UTR scans through the ribonucleotide sequence and starts reading at the first or second ATG to perform the translation process. Although various FMDV replicons or minigenomes were established, all required in vitro transcription and RNA transfection [23][24][25][26]41]. In addition, these minigenomes also contain the P2 sequence, which was absent in our construct. Thus, pKLS3 was the smallest FMDV minigenome thus far.
For pKLS3 to function as the minigenome, all cis-acting components for the transcription and translation processes, including 5′UTR and 3′UTR, should be intact and work In the random docking, ribavirin was preferentially buried in the deep pocket in which Met296 resided with the binding affinity of −6.4 kcal/mol (C). Notes: white, blue, and red sticks represent hydrogen, nitrogen, and oxygen, respectively.

Discussion
Viral replicon systems have been developed previously by a number of research groups to study the replication of RNA viruses. Replicons or minigenomes are defined as self-replicating but non-infectious RNAs. Thus, the FMDV minigenome provides an opportunity for molecular biology research related to FMDV replication, transcription, and translation, which has normally been restricted to high biosecurity and containment facilities. Here, we report the development of an FMDV minigenome, pKLS3, which is a DNA-based vector containing the minimum cis-acting elements essential for transcription and translation of FMDV. Because this platform utilizes T7 promoter to facilitate the transcription of the first-strand RNA, the pCAGGS_T7 plasmid expressing the T7 RNA polymerase is a crucial component in this system. Our minigenome possessed a hdv ribozyme downstream from the poly A tail, which auto-cleaved at its 5 end, leading to the transcription termination of the T7 RNA polymerase. Another component, pCAGGS_P3, is not mandatory; however, it acts as the transcription enhancer to increase the yield of the transcripts for protein synthesis by cap-independent translation. The ribosome assembled at the IRES located within the 5 UTR scans through the ribonucleotide sequence and starts reading at the first or second ATG to perform the translation process. Although various FMDV replicons or minigenomes were established, all required in vitro transcription and RNA transfection [23][24][25][26]41]. In addition, these minigenomes also contain the P2 sequence, which was absent in our construct. Thus, pKLS3 was the smallest FMDV minigenome thus far.
For pKLS3 to function as the minigenome, all cis-acting components for the transcription and translation processes, including 5 UTR and 3 UTR, should be intact and work properly. In fact, 5 UTR and 3 UTR interacted together and with other viral and host proteins to form a replication complex. Although the exact component of the replication complex has not been elucidated yet, evidence showed that the cellular poly C binding protein 2 (PCBP2) bound the IRES and interacted with the other host proteins, such as poly-A binding protein (PABP) that attached to the 3 UTR [26]. Translation efficiency also depends on the characteristics of 5 UTR, such as the length, the sequences upstream of the start codon, and the secondary structure, particularly IRES [42]. In addition, the 5 and 3 UTRs contain binding sites for regulatory proteins [43] to facilitate both cap-dependent and cap-independent translation initiations through RNA interactions and helicase-mediated remodeling of RNA structures [44].
Although the helper plasmid containing the FMDV P3 polyprotein, pCAGGS_P3, was not a necessary component for the role of pKLS3 minigenome in the first strand RNA synthesis, it markedly enhanced the GFP expression. In addition to supply the RdRp for the efficient transcription of uncapped RNA produced by T7 RNA polymerase, the P3 also encodes a protease, 3C pro . FMDV 3C pro is responsible for shutting off cellular transcription of host mRNA by cleaving the nuclear histone H3 and inhibiting host protein translation [45]. As cellular transcription occurs in the nucleus, 3C pro provided in trans mostly interferes with the host protein synthesis by inhibiting cap-dependent translation initiation [46]. FMDV 3C pro plays a significant role by cleaving eIF4AI and eIF4GI, the translation initiation factors required by capped mRNA for binding with the 40S ribosomal subunit and unwinding secondary structures of RNA [47]. Thus, the 3C pro facilitated the translation of uncapped mRNA generated by our pKLS3 minigenome system by hijacking the cleaved eIF4GI for binding with its IRES.
The length of the poly A tail was highly important for the function of the pKLS3 minigenome. We found that the length of poly A tail was significantly related to the level of the GFP expression. One of our constructs containing 19 adenine residues failed to express a detectable level of the green fluorescent signal. Previous studies have shown that 3 UTR and the length of poly A tail of RNA viruses are responsible for viral genome replication [14,16]. In the poliovirus, the 10-fold increase in negative-strand RNA synthesis occurred by increasing the numbers of adenine from 12 to 13 residues [48]. In addition, increasing the poly A tail length from 15 to 20 residues has been shown to improve the viral copy number of duck hepatitis A virus type 1 [14]. In the hepatitis C virus, the poly A tail with 50 adenine residues demonstrated enhanced translation efficiency, depending upon its IRES [49]. A recent study related to mRNA vectors revealed that long poly A tails were essential for RNA stabilities, as they were naturally degraded in the cytoplasm [50]. In general, 30 adenine residues are needed for a functional mRNA, and the longer poly A tail, the higher mRNA stability [51]. These findings were consistent with our study, showing that the GFP expression in transfected cells was detected when the length of the poly A tail increased from 19 to 48 residues.
Recently, the mRNA vaccine has become one of the most robust technologies to produce vaccines and biomedical therapeutics for emerging infectious diseases and cancers due to its short manufacturing lead time and scalability. The transcribed mRNAs possess the same major advantages as DNA vaccines but lack the adverse effect of DNA integration into the host genome [52]. In addition, the process is more rapid and economical to produce than conventional vaccine production, as the viral or protein purification steps are not required. Since the final products do not contain any infectious agents as well as the byproducts from mammalian cell culture, the mRNAs are considered safe. Mostly, mRNAs used for vaccines and drug therapies are generated by in vitro transcription using mRNA expression vectors carrying genes encoding the specific antigens [52,53]. In addition to the antigens, the cis-acting elements on the mRNA molecules should be properly designed for highly efficient protein synthesis in cells. Previously, the lacZ gene flanked by 5 and 3 UTRs from Xenopus laevis β -globin was generated and used as an mRNA vaccine to immunize mice [52]. Furthermore, both 5 and 3 UTRs could be optimized. For example, human endogenous genes were explored to design a platform for SARS-CoV-2 mRNA vaccine production [54]. In their study, the comparison between eukaryotes and noneukaryotes UTRs revealed that the IRESs of encephalomyocarditis virus (EMCV) and FMDV possessed high binding affinities to ribosomal subunits and supported ribosomal retaining and recycling for more efficient translation. However, mRNA production is relatively challenging to maintain high quality and stability throughout the process.
Thus, the development of nucleic acid vaccines and therapeutics are mainly based on DNA molecules. Generally, a plasmid DNA, once delivered into the cells, still requires sequential numbers of intracellular events for DNA translocation from the cell periphery to the nucleus to initiate the transcription. The transcribed mRNA is then transported back to the cytoplasm and consecutively translated into a protein. Since plasmid DNA does not easily pass through the nuclear membranes, only a few DNA molecules can enter the nucleus even in actively dividing cells [55].
We have shown here for the first time the establishment of a DNA-based pKLS3 minigenome system and its application for foreign protein expression and antiviral drug screening. Although pKLS3 and the helper plasmids are DNA, both transcription and translation processes for efficient protein synthesis occur solely in the cytoplasmic compartment, such as the mRNA vaccine. Additionally, handling, storing, and transport of this minigenome system are more convenient than dealing with RNAs. Unlike some DNA and viral vector vaccines, they do not integrate into the host chromosome. With these properties, the pKLS3 minigenome could be one of the attractive candidate vectors for modern biopharma development.

Patents
pKLS3 vector, its associated products, and the developmental process are protected under the Patent Application Number: 1901006625.