Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target

Figures

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  Figure 1. Identification of the G4-forming sequence in the ASFV genome.A, both strands of the ASFV genome were subjected to bioinformatic analysis by using QGRS. Each blue and red dot corresponds to a G4-forming sequence located in the sense strand (+) and anti-sense strand (−), respectively. The G-score was plotted against the genome length. B, the schematic map of E165R's G4 distribution, and the genome of eight representative ASFV strains were aligned with Jalview. C, the conserved G4 motif was created by WebLogo software. ASFV, African swine fever virus; G4, G-quadruplex.

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  Figure 2. Characterization of G4 formation of E165R G4 DNA.A, oligonucleotides of G4-WT DNA and G4-Mut were heated at 9 °C for 3 min and then slowly cooled to 25 °C. The sample was subjected to native PAGE analysis in which the buffer contained 100 mM K+, or to denatured PAGE containing 7 M urea. Then, the gel was imaged with a GE AI600 imaging system. B, human telomere sequence (Tel22) was used as a positive control for E165R G4 DNA. C, G4-WT, c-myc, Tel22, and G4-Mut were annealed in the presence of K+ or the presence of Li+, and then NMM was added. The fluorescence of NMM was recorded using a SpectraMax i3X multifunctional microplate reader, the excitation wavelength was set to 393 nm, and the emission spectrum was collected in the range of 450 nm to 700 nm. D, G4-DNA was annealed in the presence and absence of 100 mM k+, followed by CD scanning. G4-Mut was used as a negative control, while c-myc and Tel22 served as positive control. G4, G-quadruplex; NMM, N-methyl mesoporphyrin IX.

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  Figure 3. G4 ligands stabilize the G4 structure formed in G4-DNA of E165R.A–D, the G4-DNA of E165R or F21T was annealed in the presence of 100 mM k+, followed by the addition of different concentrations of ligand (0 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM) at 25 °C for 30 min. The 6-carboxyfluorescein fluorescence was recorded, and the normalized 6-carboxyfluorescein fluorescence was plotted against the various temperatures. The dotted line indicates the middle of the maximum fluorescence and represents the Tm of the G4 structure. G4, G-quadruplex.

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  Figure 4. G4 ligands inhibit DNA synthesis at the G4 sites.A, schematic illustration of the Taq polymerase termination assay, where the 5′-6-carboxyfluorescein-labeled primer was complementary to the template DNA harboring the G4 motif and then extended by Taq polymerase in the presence of different ligands. The extension product was run in denatured PAGE with 7M urea, and the gel was imaged with an Amersham Imager 600. The black arrow indicates the premature product (pp) caused by G4, while the white arrow indicates the full-length product (FL). % pp was calculated by intensity (PP)/[intensity (PP)+intensity (FL)]. B, the template with the G4-Mut counterpart was subjected to a Taq polymerase assay as shown in Figure 4A. G4, G-quadruplex.

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  Figure 5. Characterization of RNA G4 formation.A, the RNA oligonucleotide corresponding to the G4 DNA of E165R was annealed in 100 mM K+ and then incubated with ThT. The excitation and emission wavelengths were 425 nm and 495 nm, respectively. The changed fluorescence of ThT plotted to different oligonucleotides was calculated by FI/FI0, where FI and FI0 indicated sample and background fluorescence, respectively. B, the fluorescence spectrum of ThT as shown in Figure 5A. C, fluorescence spectrum of NMM in the presence of RNA-G4-WT or RNA-G4-Mut annealed in 100 mM K+. D, CD spectral analysis for rG4 in the presence and absence of 100 mM k+. The Tel22R was used as a positive control in Figure 5, A–D. E, FAM-labeled RNA-G4-WT and G4-Mut were transfected into 293T cells, BG4 antibody (red) was applied to probe G4 formation, and the nuclei were stained with DAPI. Colocalization of G4-WT and BG4 is indicated by the white arrow. The scale bar on the original image was 10 μm, and the scale bar on the enlarged image was 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; G4, G-quadruplex; NMM, N-methyl mesoporphyrin IX.

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  Figure 6. G4 stabilizers repress EGFP reporter expression.A, schematic illustration of the reporter plasmid, where G4-WT and its counterpart G4-Mut were cloned into the coding sequence of EGFP. B, cells were pretreated with different ligands and then transfected with the reporter constructs, and EGFP was detected using a confocal microscope. C and D, quantified analysis of EGFP with flow cytometry. DMSO-treated samples were used as the negative control. All experiments were performed in triplicate. Data were analyzed by One-way ANOVA with Dunnett's multiple comparisons test, ∗∗∗ p < 0.001. ns, not significant. The scale bar was 20 μm. DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; G4, G-quadruplex.

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  Figure 7. G4 stabilizers suppress E165R expression.A, schematic representation of the plasmids used, in which the full-length E165R and the mutated counterpart where G was substituted with A to disturb G4 formation were inserted into the p3 × FLAG-CMV-10 vector. The verified plasmids were transfected into 293T cells and then cultured in medium supplemented with different concentrations of ligands for 24 h. The collected cells were lysed in radio immunoprecipitation assay lysis buffer and then subjected to Western blot analysis. B, the protein level of E165R-G4-WT and E165R-G4-Mut. C, the protein level of E165R-G4-WT and E165R-G4-Mutwhen treated with various concentrations of PDS (0 μM, 1 μM, 2 μM, 4 μM, and 8 μM). D, the protein level of E165R when treated with various concentrations of TMPyP4 (0 μM, 1 μM, 2 μM, 4 μM, and 8 μM). E, the protein level of E165R when treated with various concentrations of TMPyP2 (0 μM, 1 μM, 2 μM, 4 μM, and 8 μM). F and G, Q-PCR determined the RNA level of E165R-G4-WT and its mutated type after adding various ligands. All experiments were performed in triplicate. Data were analyzed by One-way ANOVA with Dunnett's multiple comparisons test, ∗∗p < 0.01, ∗∗∗∗ p <…

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  Figure 8. Effects of G4 ligands on the proliferation of African swine fever virus in different cells.A, cytotoxicity of the four G4 ligands in Vero cells. B, cytotoxicity of the four G4 ligands in 293T cells. C, effects of the four G4 ligands on the proliferation of African swine fever virus in Vero cells. Vero cells were pretreated with the indicated G4 ligands and then infected with African swine fever virus (ASFV-GFP) at an MOI of 0.5, followed by the replacement of the medium containing the corresponding G4 ligands. After 36 h of infection, fluorescence was observed under a fluorescence microscope. D, effects of the four G4 ligands on ASFV proliferation in 293T cells. E, the inhibitory effect of the four G4 ligands on ASFV proliferation in Vero cells was determined by TCID50 assay. F, the inhibitory effect of the four G4 ligands in 293T cells was determined by TCID50 assay. All experiments were performed in triplicate. Data were analyzed by One-way ANOVA with Dunnett's multiple comparisons test, ∗p < 0.05, ∗∗p < 0.01. The scale bar was 200 μm. ASFV, African swine fever virus; G4, G-quadruplex; MOI, multiplicity of infection; TCID50, median tissue culture infectious dose.

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  Figure 9. PDS affects the genome copy number and protein synthesis of ASFV. Vero cells were pretreated with different concentrations of PDS, infected with the ASFV at an MOI of 0.5, and then replaced with medium containing PDS (5 μM, 10 μM, 20 μM, and 30 μM). After 36 h of infection, the cells were harvested. A, Q-PCR determined the viral genome copy number, all experiments were performed in triplicate. Data were analyzed by One-way ANOVA with Dunnett's multiple comparisons test,∗∗∗∗p < 0.0001. B, Western blotting was used to assess the viral protein (p54 and p72) levels, and β-actin was used as a loading control. C, vero cells that were infected with ASFV at an MOI of 0.5 were treated with 20 μM PDS, at different hpi, the RNA was extracted and subjected to RT-qPCR analysis, while the proteins were harvested and subjected to Western blotting (D). The data represent the mean (±SD) of three independent experiments. Significant differences compared to the control are analyzed by unpaired two-tailed Student’s t test., ns, not significant. ASFV, African swine fever virus; MOI, multiplicity of infection; PDS, pyridostatin; q-PCR, quantitative PCR.

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