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

Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

Characterization of RNA G-quadruplexes in porcine epidemic diarrhea virus genome and the antiviral activity of G-quadruplex ligands.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there are still no anti-PEDV drugs with accurate targets. G-quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA, and have attracted great attention as potential targets for antiviral strategy. In this study, we reported two putative G4-forming sequences (PQS) in S and Nsp5 genes of PEDV genome based on bioinformatic analysis, and identified that S-PQS and Nsp5-PQS were enabled to fold into G4 structure by using circular dichroism spectroscopy and fluorescence turn-on assay. Furthermore, we verified that both S-PQS and Nsp5-PQS PQS could form G4 structure in live cells by immunofluorescence microscopy. In addition, G4-specific compounds, such as TMPyP4 and PDS, could significantly inhibit transcription, translation and proliferation of PEDV in vitro. Importantly, these compounds exert antiviral activity at the post-entry step of PEDV infection cycle, by inhibiting viral genome replication and protein expression. Lastly, we demonstrated that TMPyP4 can inhibit reporter gene expression by targeting G4 structure in Nsp5. Taken together, these findings not only reinforce the presence of viral G-quadruplex sequences in PEDV genome but also provide new insights into developing novel antiviral drugs targeting PEDV RNA G-quadruplexes.

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

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.

Identification and characterization of G-quadruplex formation within the EP0 promoter of pseudorabies virus.

EP0 is an important early gene that modulates the life cycle of pseudorabies virus (PRV). A guanine-rich sequence overlapping with three Sp1 binding sites is located upstream of the transcription start site (TSS) in the EP0 promoter. Using native polyacrylamide gel electrophoresis (PAGE) and circular dichroism (CD), we verified that the G-rich region in the EP0 promoter forms an intramolecular parallel G-quadruplex (G4) in the presence of K+ ions. Further dimethyl sulphate (DMS) footprinting and Taq polymerase stop assays indicates the potential polymorphic folding of G4. In addition, a small chemical ligand, pyridostatin (PDS), promotes and stabilizes the formation of G4. Interestingly, based on the results of electrophoretic mobility shift assays (EMSA), the Sp1 protein bound to G4-bearing DNA with more affinity than DNA lacking the G4 structure. According to the luciferase reporter assay, G4 negatively regulates the EP0 promoter activity. These results demonstrate that Sp1 and G4 cooperate to regulate EP0 promoter activity.