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

Figures

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  Figure 1. Construction of recombinant plasmids. (A) 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.

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  Figure 2. The growth kinetics of FMDV type A, A/Lopburi/2012, O/Tai/189/1987, and Asia1/2013 in BHK-21 cells at each time point. The cells were inoculated with each vaccine strain at 0.01 MOI. The viruses were collected for titration at 4-h intervals from 4 to 24 hpi. The bars represent the standard error of mean (SEM).

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  Figure 3. The regions of FMDV O189 UTRs and hdv ribozyme were used as the input sequences to generate the putative RNA secondary structures using mFold (RNA folding form version 2.3). (A) 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.

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  Figure 4. The electrophoretic photograph presenting an in vitro transcribed RNA containing the cis-acting elements of the pKLS3 minigenome and a GFP gene. The plasmid pKLS3_GFP was used as the plasmid DNA template in an in vitro transcription reaction to test the hdv ribozyme function. The hdv ribozyme auto-cleaved the transcribed RNA at its 5′ end, resulting in the RNA product containing the cis-acting elements of the pKLS3 minigenome and the GFP gene of approximately 2.2 kb (lane 1). Lane M is a standard molecular weight DNA marker.

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  Figure 5. Photographs illustrating green fluorescent signals in transfected cells at x100 magnification. (A) The BHK-21 cells were co-transfected with pKLS3_GFP and pCAGGS_T7 at various ratios (pKLS3_GFP: pCAGGS_T7 = 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.

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  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×.

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  Figure 7. Molecular docking of ribavirin onto the three-dimension structure of FMDV O189 RdRp using AutoDock Vina algorithm in PyRx 0.9.8. The ribavirin molecule is displayed as a yellow stick. The backbone of ribavirin interactive residues within the RdRp is light blue. The insets show the binding of ribavirin to the potential residues. For specific docking, ribavirin reacted to Pro44 and Pro169 with the binding affinities of −4.2 kcal/mol (A) and −5.4 kcal/mol (B), respectively. 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.