Abstract
Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses’ genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV’s attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.
Highlights
Pseudorabies virus (PRV), a subfamily of Alphaherpesvirinae, infects its natural host, the pig, and infects other animals, including wild boars, rodents, ruminants, and carnivores [1,2], which threatens the swine industry
The PRV strain hSD-1/2019 was isolated from a patient with acute encephalitis [9], providing direct evidence that PRV has the potential for cross-species transmission from animals to humans
Viral Titration Vero cells were seeded at a density of 2 × 105 cells per well in 12-well tissue cultureplates, pretreated with different concentrations of PhenDC3 (1 μM–50 μM) for 1 h, and infected with PRV HN1201 at a multiplicity of infection (MOI) of 1 plaque-forming unit (PFU)/cell
Summary
Pseudorabies virus (PRV), a subfamily of Alphaherpesvirinae, infects its natural host, the pig, and infects other animals, including wild boars, rodents, ruminants, and carnivores [1,2], which threatens the swine industry. We reported a conserved G-rich sequence located in the EP0 promoter able to form G4, which negatively regulated EP0 expression in the presence of the G4 stabilizer PDS [26]. We identified two conserved G4-forming sequences at the upstream region of OriL in the PRV genome, which were located in the sense and antisense strands. Multiple biophysical and biochemistry experiments demonstrated that the two G-rich sequences could fold into a parallel G4 structure and be stabilized by PhenDC3 (a G4-targeting compound). The addition of PhenDC3 into PRV-infected cells inhibited DNA replication in cells. These results indicate that the G4 upstream of OriL may serve as new therapeutic targets against PRV
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