G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets

Figures

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  Figure 1. Identification and characterization of G4 formation in vitro. (A) A G-tetrad with four guanines connected by eight Hoogsteen hydrogen bonds stabilized by K+ and an intramolecular G-quadruplex (G4) composed of three stacking G-tetrads. (B) Schematic representation of G-rich sequences at the upstream region of OriL within the PRV genome, OriL-S was located at the sense strand, while OriL-A was located at the antisense strand. (C,D) Circular dichroism (CD) spectra of wild-type (WT) or mutated-type (Mut) oligonucleotides of OriL-S (5 µM) and OriL-A (5 µM), which were annealed in a buffer with 50 mM LiAsO4 and 100 mM KCl. (E,F) Wild-type (WT), mutated-type (Mut), and WT complementary with antisense oligonucleotides (as) of OriL-S and OriL-A were subjected to native gel analysis (representative image), the corresponding illustration of G4, liner, and double-stranded DNA was directed by an arrow. (G,H) WT and Mut oligonucleotides of OriL-S and OriL-A were electrophoresed on denaturing gel with 7 M urea (representative image). All the oligos were labeled with FAM at the 5′-end, and the marker (M) was synthesized with the indicated length but did not form a secondary structure.

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  Figure 2. PhenDC3 binds and stabilizes G4 formed in OriL-S and OriL-A. (A,B) Fluorescence melting curve of OriL-S and OriL-A in the presence of different concentrations of PhenDC3 (horizontal arrow); the T1/2 indicates the temperature required for reaching the mid-value between the minimum and maximum fluorescence in FRET melting, and the T1/2 was plotted against concentrations of PhenDC3 (bottom). (C,D) Synthesized single-stranded template DNA were annealed with FAM-labeled primers and were extended by Taq polymerase in the absence or in the presence of PhenDC3. Extended products were subjected to denaturing gel analysis (representative image). Premature termination (PT) stopped by G4 and full-length products (FL) were denoted by a filled and open triangle, respectively. P indicates primer used in Taq polymerase stop assay, marker (M) was made by the same extension–reaction with a template without the G-core and its 3′ flanking sequence. The ratio of premature termination (PT) over full-length (FL) products in three independent experiments is demonstrated on the right of the panel.

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  Figure 3. Effect of the G4-stabilizer PhenDC3 on PRV proliferation in PK-15 cells. (A) Treating PK-15 cells with increasing concentrations of PhenDC3 for 48 h, the cell viability was assessed by a CCK-8 assay. (B) PK-15 cells were pretreated with different concentrations of PhenDC3 (1 µM–50 µM) and then infected with PRV HN1201 at an MOI of 1; after incubating with PhenDC3 for 24 h, the viral titer was determined by TCID50 assay. (C) PK-15 cells were treated as in (B); the expression level of gE was determined by western blot analysis (representative image). (D) PK-15 cells were pretreated with different concentrations of PhenDC3 (1 µM–50 µM), and then infected with PRV-GFP (MOI = 0.1); after incubating with different concentrations of PhenDC3 for 24 h, the GFP-positive cells were detected with fluorescence microscopy (top) and quantitatively analyzed by flow cytometry (bottom). (E) PK-15 cells were untreated or treated with 25 µM and 50 µM PhenDC3 for 1 h, then the cell nuclei were probed with primary antibody Lamin B1 together with Alexa Fluor® 555 conjugated goat anti-rabbit IgG (H + L). The cells were imaged with a Zeiss LSM 800 Confocal microscopy, λexc = 405 nm was used for imaging…

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  Figure 4. Compound PhenDC3 inhibited PRV DNA replication in PK-15 cells. (A) For the pretreatment assay, PK-15 cells were pretreated with increasing concentrations of PhenDC3 (1 μM–50 µM). After removing the PhenDC3, cells were infected with PRV-GFP (MOI = 0.1) for 1 h at 37 °C. Viral inoculums were washed to remove the unabsorbed virus, then the maintenance medium (1% FBS/DMEM) was added and cultured for 24 h at 37 °C. The GFP-positive cells were quantified by flow cytometry. (B) For the viral attachment assay, PK-15 cells were treated with different concentrations of PhenDC3 concurrently with PRV-GFP (MOI = 0.1) at 4 °C for 1 h; the excess viral inoculums were removed and replaced with a maintenance medium (1% FBS/DMEM), then shifted to 37 °C and incubated for 24 h. The GFP-positive cells were quantified by flow cytometry. (C) For the viral entry assay, PK-15 cells were infected with PRV-GFP (MOI = 0.1) for 1 h at 4 °C. Viral inoculums were washed three times to remove the unabsorbed virus. Different concentrations of PhenDC3 were added and incubated at 37 °C for 1 h to allow viral internalization. The culture was replaced with a maintenance medium (1% FBS/DMEM) and the cells were…