Tandemly Repeated G-Quadruplex Structures in the Pseudorabies Virus Genome: Implications for Epiberberine-Based Antiviral Therapy

Figures

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  Figure 1. Identification of multiple G4-forming sequences in the PRV genome. (A) Schematic representation of the PRV genome. The arrow indicates the transcription direction. (B) The genomic sequences of three representative PRV variants, HN1201(KP722022.1), HSD-1/2019 (MT468550.1), and JS-2012 (KP257591.1), were retrieved from NCBI. The potential G-quadruplex-forming sequences (PQS) were analyzed using the QGRS Mapper tool, and the guanine bases involved in the formation of the G-quadruplex are denoted in pink.

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  Figure 2. Characterization of G4 formation in R1-PQS and R2-PQS regions. (A) Wild-type (WT) and mutant (Mut) oligonucleotides corresponding to the R1-PQS and R2-PQS regions were analyzed by CD spectroscopy. (B) WT and Mut oligonucleotides were incubated with Thioflavin T (THT), and fluorescence was measured to assess G4 formation. (C) WT and Mut oligonucleotides were incubated with N-methylmesoporphyrin IX (NMM), and fluorescence was recorded to confirm G4 structure formation.

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  Figure 3. Epiberberine binding and stabilization of G-quadruplexes formed by R1-PQS and R2-PQS. (A) Molecular structure of EPI. (B,C) The telomeric sequence (TA[Q]) was used as a positive control. G4 formation was assessed by fluorescence after incubation of R1-PQS, R2-PQS, and TA[Q] with EPI. (D) The structural changes of R1-PQS and R2-PQS upon EPI binding were analyzed by CD spectroscopy. (E) The thermal stability of R1-PQS and R2-PQS with EPI binding was evaluated by CD melting curves, with the melting temperature (Tm) determined by fitting the data to a sigmoidal curve using the Boltzmann function.

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  Figure 4. R1-PQS and R2-PQS form G-quadruplexes in cells. (A) FAM-labeled R1-PQS, R1-PQS-Mut, R2-PQS, and R2-PQS-Mut oligonucleotides were transfected into 293T cells and stained with BG4 antibody (red) to detect G4 structures. Co-localization of G4 structures and BG4 is shown in yellow. White boxes indicate regions of co-localization, while red boxes highlight areas shown at higher magnification in the insets. (B) PK-15 cells infected with PRV-HN1201 were stained with BG4 antibody (red) to detect G4 structures and anti-UL42 antibody (green) to detect the UL42 protein. DAPI (blue) was used to stain the cell nuclei. Red boxes indicate regions that are enlarged in the insets. Scale bar: 10 µm. Images were captured by fluorescence microscopy.

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  Figure 5. EPI inhibits DNA replication by targeting G4 structures formed by R1-PQS and R2-PQS. (A) Schematic of the Taq polymerase stop assay showing premature termination products (red arrows) due to G4-induced steric hindrance. Green arrows indicate the full-length products synthesized by Taq polymerase. (B) Primer extension with telomeric sequence (positive control) and varying concentrations of EPI to assess its effect on DNA replication. (C,D) Positive control using PDS, a G4 stabilizer, in the Taq polymerase stop assay. (E) Primer extension assays on wild-type and mutant R1-PQS and R2-PQS sequences with varying EPI concentrations. (F) PCR amplification of the PRV-HN1201 genome to assess G4 formation in the R1-PQS and R2-PQS regions upon EPI treatment. In (B,E), “−” indicates the absence of both K⁺ and EPI. In (C,D), “−” indicates the absence of both K⁺ and PDS. “+” indicates the presence of K⁺.

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  Figure 6. EPI inhibits PRV proliferation in cells. (A) PK-15 and Vero cells were treated with varying concentrations of EPI for 24, 36, or 48 h, and cell viability was measured using the CCK-8 assay. (B) PK-15 and Vero cells were pre-treated with different concentrations of EPI, followed by infection with PRV-GFP (MOI = 0.1). The GFP-positive cells were visualized using a fluorescence microscope. (C) The fluorescence intensity from panel (B) was quantitatively analyzed by flow cytometry. (D) Berberine and PDS, as positive control G4 stabilizers, were used to assess their inhibitory effects on PRV-GFP replication in PK-15 cells, as determined by flow cytometry. (E) PK-15 and Vero cells were infected with PRV-HN1201 (MOI = 0.1) and treated with varying concentrations of EPI. After 24 h, cellular DNA was extracted, and viral DNA levels were quantified by qPCR. (F) PK-15 cells were pre-treated with different concentrations of EPI and then infected with PRV-HN1201 at an MOI of 0.1 or 1. After 24 h of incubation with EPI, viral titers were determined using the TCID50 method. (G) PK-15 and Vero cells were treated as described in panel (E), and PRV gB protein expression was assessed by Western…

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  Figure 7. EPI impedes PRV replication in cells. (A–D) EPI inhibits PRV-HN1201 at different stages of the viral life cycle, including adsorption, entry, replication, and release, as assessed by qPCR. (E) Flow cytometry analysis of PRV-GFP infection in PK-15 cells treated with EPI or ACV. *** p < 0.001, ns: not significant.

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  Figure 8. EPI inhibits PRV replication in mice. (A) Schematic diagram of the experimental procedure for treating PRV-infected mice with EPI. (B) Survival rates of mice in each group. Survival rates were calculated using the following formula: Survival rate = (Number of surviving mice/Total number of mice) × 100%. (C) Viral load in tissues from each group of mice. On day 3 post-PRV challenge, brain, heart, lung, and kidney tissues were collected, and viral DNA was extracted. The viral copy numbers in each tissue were determined by qPCR and compared to the DMSO-treated control group. (D) Histopathological examination of brain, heart, lung, and kidney tissues from mice; arrows indicate pathological lesions. Sections were stained with H&E, and the scale bar represents 100 μm. 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, *** p < 0.001, ns: not significant.