Figures and data
Four regions with different characteristics in the PEDV genome.
(A) Regions with low SHAPE reactivity and low Shannon entropy, account for 26.40% of the PEDV genome. (B) Regions with low SHAPE reactivity and high Shannon entropy, account for 11.70% of the PEDV genome. (C) Regions with high SHAPE reactivity and high Shannon entropy, account for 26.90% of the PEDV genome. (D) Regions with high SHAPE reactivity and low Shannon entropy, account for 9.67% of the PEDV genome.
SHAPE structural map of the PEDV genome in infected cells.
From top to bottom: SHAPE reactivity, Shannon entropy, base-pairing probabilities, translation reading frames are indicated by arrows, and genomic coordinates are marked at the bottom. Well-folded regions with low SHAPE reactivity and low Shannon entropy are shaded in blue. Potential G4 forming sequences (PQSs) are marked with a black arrow.
Potential RNA motifs with specific structures in high SHAPE-high Shannon regions.
(A) SHAPE reactivity and Shannon entropy of RNA motif (11099-11119 nt) in the PEDV genome. (B) Secondary structure of the RNA motif (11099-11119 nt) predicted by RNAstructure. (C) Structure of CPEB3 nuclease P4 hairpin (PDB: 2M5U). The red section indicates nucleotides that are fully matched with the RNA sequence (11099-11119 nt) in the PEDV genome. (D) SHAPE reactivity and Shannon entropy for the regions containing PQS1. (E) Local secondary structure of the region containing PQS1 predicted with SHAPE reactivity constraints. PQS1 is marked with a blue dashed box. (F) Distribution of PQSs in the PEDV genome.
The G-quadruplex structure, biological functions of PQS1, and antiviral effects of Braco-19.
(A) 1H NMR analysis of PQS1. (B) CD melting profiles of PQS1. (C) Quantitative fluorescence signal using the corrected total cell fluorescence method for EGFP in cells transfected with plasmids containing the empty vector, PQS1 and PQS1mut. (D) Proliferation curve of the PEDV wild type (WT) strain and PQS1 mutant strain. (E) The relative inhibition rates of Braco-19 against AJ1102-WT and AJ1102-PQS1mut. (F) Western blot analysis of the effects of Braco-19 on the viral N protein expression of AJ1102-WT and AJ1102-PQS1mut.
Secondary structure of target regions and antiviral effects of siRNAs.
(A-D) Local secondary structures of the stable single-stranded regions targeted by siRNAs predicted by SHAPE reactivity as a constraint; (A) ss1; (B) ss2; (C) ss3; (D) ss4. (E) qPCR showing the relative abundance of the PEDV RNA genome in infected Vero cells. The four siRNAs targeting the high SHAPE-low Shannon regions and the four siRNAs targeting the duplex regions are labeled ss-1 to ss-4 and ds-1 to ds-4, respectively. si-NC was a control siRNA that did not target any viral or host sequences, and the mock group was not inoculated with virus. (F) TCID50 assays for detecting virus titers. The presented results represent the means and standard deviations of data from three independent experiments. ns: no significant difference. * P < 0.05; ** P < 0.01; *** P < 0.001, Duncan’s multiple comparison test.
Single-stranded RNA regions in viral genomes as the targets for antiviral therapy.
(A) PQSs in the single strands are easier to be induced into G4s by ligands than those in the paired regions. (B) Influence of the secondary structures on the binding efficiency of siRNAs.
