RNA G‐Quadruplexes in the Porcine Deltacoronavirus Genome: Structural Regulation and Therapeutic Targeting for Antiviral Strategies

Figures

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  Figure 1. Bioinformatic analysis of potential RG4 sequences in PDCoV genome. (A) Venn diagram showing overlapping predictions of PQSs by QGRS mapper and G4Hunter. (B) Positional distribution and prediction scores of identified PQSs in the PDCoV genome. (C) Two G‐quadruplex layers are arranged in a stacked configuration, forming a noncanonical secondary structure known as a G‐tetrad. (D) Sequence conservation analysis of G‐rich regions capable of forming RG4 structures.

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  Figure 2. In vitro confirmation and characterization of PDCoV RG4 formation. (A) Native PAGE analysis of Nsp2‐PQS‐WT and Mut. (B) Native PAGE analysis of M‐PQS‐WT and Mut. (C) Denaturing PAGE analysis of Nsp2‐PQS‐WT and Mut. (D) Denaturing PAGE analysis of M‐PQS‐WT and Mut. (E, F) CD spectra of Nsp2‐PQS‐WT/Mut and M‐PQS‐WT/Mut in 100 mM KCl. (G) NMM fluorescence spectra of Nsp2‐PQS‐WT and Mut. (H) ThT fluorescence spectra of Nsp2‐PQS‐WT and Mut. (I) ThT fluorescence spectra of Nsp2‐PQS‐WT and Mut under KCl or LiCl conditions. (J) NMM fluorescence spectra of M‐PQS‐WT and Mut. (K) ThT fluorescence spectra of M‐PQS‐WT and Mut. (L) ThT fluorescence spectra of M‐PQS‐WT and Mut under KCl or LiCl conditions.

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  Figure 3. Binding of G4 ligands to Nsp2 and M RG4s. (A, B) Chemical structures of TMPyP4 and PDS. (C, D) Fluorescence polarization binding curves of TMPyP4 and PDS with Nsp2‐PQS‐WT/Mut under KCl or LiCl conditions. (E, F) Fluorescence polarization binding curves of TMPyP4 and PDS with M‐PQS‐WT/Mut under KCl or LiCl conditions. FAM‐labeled RNA oligonucleotides were used at a fixed concentration of 10 nM, and ligand concentrations ranged from 0 to 200 μM. Binding curves were fitted using a one‐site–specific binding model to calculate K D values.

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  Figure 4. RG4 structures formed in the Nsp2/M genes of PDCoV in living cells. (A) Confocal immunofluorescence images showing colocalization of Nsp2‐ and M‐WT/Mut RNA (green) with BG4 antibody (red) in LLC‐PK1 cells. Nuclei were counterstained with DAPI (blue). White boxes indicate representative FAM‐RNA/BG4 colocalization foci in the merged images, shown as yellow puncta, and the selected regions are enlarged on the right. Scale bar: 5 μm. (B) Quantification of FAM‐RNA/BG4 colocalization foci. ∗∗∗ p ≤ 0.001.

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  Figure 5. G4 ligands TMPyP4 and PDS suppress PDCoV replication. (A) Cytotoxicity of TMPyP4 and PDS in LLC‐PK1 cells were evaluated using a CCK‐8 assay. (B) LLC‐PK1 cells were incubated with varying concentrations of TMPyP4 or PDS prior to infection with PDCoV at an MOI of 0.1. After 12 h, total cell lysates were harvested, and PDCoV N protein levels were analyzed by WB. (C) Cell samples treated with TMPyP4 or PDS were collected for extraction, and PDCoV replication was quantified using qPCR. (D) The inhibitory effect of two ligands on PDCoV was determined by TCID50 assay. (E) Immunofluorescence was used to assess PDCoV proliferation after 24 h of treatment with specified doses of PDS or TMPyP4 (green: viral protein; blue: DAPI‐stained nuclei).

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  Figure 6. Posttranscriptional regulation of Nsp2 expression by RG4. (A) Schematic of Nsp2 full‐length ORF or RG4 mutant constructs cloned into the MCS of pEGFP‐N1. Bold letters indicate RG4‐forming sequences; red letters denote mutations. (B) Nsp2‐EGFP protein levels (left) and mRNA (right) expression in cells following transfection with either pEGFP‐Nsp2‐WT or Mut plasmids. (C) The positions of the concentration and grayscale value numbers in the figure are as closely aligned with the band positions as possible. (D) Protein levels in cells treated with increasing concentrations of TMPyP4 (left) or PDS (right) for 24 h. (E) qPCR quantification of pEGFP‐Nsp2‐WT (left) or Mut (right) mRNA levels in cells treated with G4 ligands (20 μM) for 24 h. (F) Schematic model illustrating RG4 regulation during PDCoV infection.