In this study, infectious clone of EV-A71 was constructed either with or without the enhanced green fluorescence protein (EGFP) reporter gene. These constructs are required to understand the mechanism of antiviral resistance, as well as to characterize the mechanism of action of an antiviral. In this study, EV-A71 infectious cDNA clone is utilized to characterize the degree of mismatch tolerance of the vivo-MO-1 against EV-A71. Desired mutations are introduced into vivo-MO-1 targeted region through site-directed mutagenesis. EV-A71_EGFP is used to study the mechanism of action of vivo-MOs against EV-A71 infection.
4.1.1 Construction and characterization of the enterovirus A71 cDNA clone 4.1.1.1 Amplification and cloning of the full-length enterovirus A71 genome
EV-A71 viral RNA was extracted using QIAamp viral RNA mini kit and subjected to cDNA synthesis. As shown in the agarose gel electrophoresis in Figure 4.1A, the full-length EV-A71 genome size of approximately 7.5 kb was successfully amplified using iProof high fidelity DNA polymerase. The PCR product was then gel purified and subjected to A-tailing using GoTaq DNA polymerase. The product with single 3’
adenosine (A) was then cloned into pCR-XL-TOPO plasmid vector and the recombinant plasmid was then transformed into E. coli TOP10. The recombinant plasmid was then linearized by EagI. The electrophoresed intact recombinant plasmid pCR-TOPO-XL-EV-A71 is shown in Figure 4.1B.
4.1.1.2 Characterization of enterovirus A71 infectious cDNA clone
The schematic illustration of EV-A71 infectious cDNA clone in pCR-XL-TOPO vector is shown in Figure 4.2. The EV-A71 genome was cloned downstream of the SP6 promoter. Upstream of the SP6 promoter was a MluI restriction site and downstream of the poly(A) tail was an EagI restriction site. Infectious EV-A71 genomic RNA was synthesized using SP6 RNA polymerase. The in vitro transcribed RNA contains an additional G residue at the 5’ end and CGGCC residues at the 3’ end. As depicted in Figure 4.1C, the size of the in vitro transcribed RNA was approximately 7.5 kb.
Smearing after the expected band was observed, which was likely due to some RNA degradation occurring during in vitro transcription or incomplete denaturation. The presence of non-viral residues at the 5’ and 3’ ends were found to reduce viral replication efficacy. This correlated well with the results demonstrated in this study. RD cells transfected with the purified viral RNA developed CPE faster than the RD cells transfected with in vitro transcribed RNA. The CPE was usually observed 48 to 72 hours post-transfection of the in vitro transcribed RNA. Characterization of the plaque morphology and the replication kinetics of the cDNA-derived EV-A71 and the wild-type virus are shown in Figure 4.3. There were no differences in the plaque morphology and the replication kinetics between the viruses produced from the infectious cDNA clone and the wild-type virus.
4.1.2 Construction and characterization of the enterovirus A71 enhanced green fluorescence protein (EGFP) reporter virus
4.1.2.1 Amplification and cloning of full-length enterovirus A71 EGFP genome
In order to avoid alteration of the EV-A71 genome through addition of restriction enzyme cutting sites, overlapping extension PCR was used to fuse the EGFP gene into the EV-A71 genome. The recombinant plasmid pCR-XL-TOPO-EV-A71 and
pEGFP-N1 (Clonetech, USA) were used as the backbone for EV-A71_EGFP construction. The primers were designed with at least 15 nucleotides overlap. A total of four DNA fragments were fused together. Fragment 1 (808 bp) covered the EV-A71 5’UTR which was flanked by the SP6 promoter and part of the EGFP gene. Fragment 2 (777 bp) covered the EGFP gene and was flanked by part of the EV-A71 5’UTR and VP4.
Fragment 3 (3037 bp) covered the EV-A71 P1 region which was flanked by part of the EGFP gene and the EV-A71 2A gene. Fragment 4 (3783 bp) covered the entire EV-A71 P2 and P3 regions and was flanked by part of the EV-A71 2A region and a poly(A) tail.
The fragments were then fused fragment-by-fragment using overlapping extension PCR (Figure 4.4A). The final full-length EV-A71 genome with EGFP gene (8.2 kb) was then gel purified and cloned into a pCR-XL-TOPO vector. All the PCRs were performed using Q5 high fidelity DNA polymerase, which has > 100X fidelity compared to normal Taq polymerase.
4.1.2.2 Characterization of enterovirus A71 EGFP-expressing infectious cDNA clone The schematic illustration of EV-A71_EGFP-expressing infectious cDNA clone is depicted in Figure 4.5. The EGFP gene was fused between 5’UTR and VP4 followed by a 2A cleavage site (with amino acid sequence of –AITTL-). After viral polyprotein synthesis, the EGFP which was fused with VP4 will be cleaved off by EV-A71 2A proteases to produce a functional EGFP. The EV-A71_EGFP-expressing infectious cDNA clone was flanked by restriction sites BstB1 upstream of the SP6 promoter sequence and AgeI located downstream of the poly(A) tail. The in vitro transcribed RNA contains an additional G residue at the 5’ end and CCGG residues at the 3’ end.
The in vitro transcribed RNA was about 8 kb in length (Figure 4.4B). The GFP signal could be detected 24 hours transfection and the CPE was observed 72 days post-transfection. The replication kinetics and the plaque morphology were determined using the P1 stock. As shown in Figure 4.6, EV-A71 expressing EGFP has a smaller plaque
size with a diameter of 1 mm and less well-defined edges compared to wild type EV-A71 with an average 1.5 mm diameter. EV-EV-A71_EGFP infectious virus had slower replication kinetics compared to the wild type EV-A71, which could be a result of inefficiency in RNA genome packaging.
Figure 4.1: Agarose gel electrophoresis of full-length EV-A71 genome. Agarose gel electrophoresis images of (A) full-length EV-A71genomic PCR product (lane 1), (B) purified plasmid pCR-TOPO-XL-EV-A71 from E.coli TOP10 (lane 2) and (C) SP6 in vitro transcribed RNA (lane 3). Lane M1 contains VC 1 kb DNA ladder and lane M2 contains 0.5-10 kb RNA ladder. The sizes of the DNA and RNA ladders are indicated as base pairs (bp) and bases (b).
Figure 4.2: Schematic illustration of EV-A71 infectious cDNA clone in pCR-XL-TOPO. EV-A71 genomic cDNA was cloned downstream of a SP6 RNA polymerase promoter. The in vitro transcribed positive-sense RNA carried an additional G residue at the 5’ end and a poly(A)50 tail followed by additional CGGCC residues at the 3’ end. The arrow indicates the transcription start site.
Figure 4.3: Replication kinetics of the EV-A71 infectious cDNA clone. Phenotypic characterization of wild-type EV-A71 and clone-derived EV-A71 based on the (A) plaque morphology after 48 hours post-infection and (B) replication kinetics in RD cells. At each time point, titers are the average of two biological replicates; error bars represent the standard deviation of the mean.
Figure 4.4: Agarose gel electrophoresis of overlapping PCR DNA fragments and in vitro transcribed RNA. Agarose gel electrophoresis images of (A) overlapping extension PCR products and (B) SP6 in vitro transcribed RNA. Lane M1, 1 kb DNA ladder; Lane 1, fragment 1; lane 2, overlapped product of fragments 1 and 2; lane 3, overlapped product of fragments 1, 2 and 3; lane 4, full-length EV-A71_EGFP; lane M2, 0.5-10 kb RNA ladder; and lane 6, in vitro transcribed RNA.
Figure 4.5: Schematic illustration of EV-A71_EGFP-expressing cDNA clone in pCR-XL-TOPO. The EV-A71_EGFP genome was located downstream of bacteriophage SP6 RNA polymerase promoter sequence and upstream of a poly(A)50 tail. The EGFP gene was fused in between the EV-A71 5’UTR and VP4 gene, followed by a 2A cleavage site using overlapping extension PCR. The positive-sense RNA derived from the SP6 RNA polymerase consists of an additional G residue at the 5’ end and CCGG residues at the 3’ end. The arrow indicates the transcription start site.
Figure 4.6: Characterization of EV-A71_EGFP-expressing clone. (A) RD cells were infected with rescued EV-A71_EGFP at a MOI of 0.1. The viral-induced CPE and the EGFP expression was observed 24 hours post-infection.
The EGFP signal (green) was detected using a fluorescence microscope at an excitation wavelength of 488 nm. Phenotypic characterization of EV-A71 and EV-A71_EGFP based on the (B) plaque formation size after 72 hours post-infection and (C) replication kinetics in RD cells. At each time point, titers are the average of two biological replicates; error bars represent the standard deviation of the mean.
4.2 Inhibition of enterovirus A71 infections by a novel antiviral peptide derived