Exploiting functional regions in the viral RNA genome as druggable entities

Figures

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  Figure 1.. Four regions with different characteristics in the porcine epidemic diarrhea virus (PEDV) genome. (A) Schematic of SHAPE-MaP for probing the RNA structure of the PEDV genome in situ. (B) Well-folded regions (low SHAPE reactivity and low Shannon entropy; 26.40% of genome). These regions represent stably folded RNA structures with minimal conformational flexibility, serving as structural scaffolds or functional elements in viral replication. (C) Dynamic structured regions (low SHAPE reactivity and high Shannon entropy; 11.70% of genome). These conformationally plastic domains mediate regulatory switches between alternative secondary structures during infection. (D) Dynamic unpaired regions (high SHAPE reactivity and high Shannon entropy; 26.90% of genome). These regions are prone to form non-canonical nucleic acid structures (e.g., G-quadruplexes), which can be stabilized by small-molecule ligands to inhibit viral replication. (E) Persistent unpaired regions (high SHAPE reactivity and low Shannon entropy; 9.67% of genome). These regions are more accessible for siRNA binding, facilitating recruitment of Argonaute proteins and Dicer to form the RNA-induced silencing complex (RISC)…

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  Figure 1—figure supplement 1.. Genome-wide SHAPE-MaP analysis of porcine epidemic diarrhea virus (PEDV). (A) Heat scatter plot of the SHAPE reactivities across two biological replicates for the ex vivo datasets. (B) Reverse transcription mutation rates (median indicated by line, average indicated by ‘.’) for two ex vivo biological replicates: NAI-treated (modified) vs. the DMSO-treated (unmodified). A significant increase in the reverse transcription mutation rate was observed in the NAI-treated group compared to the DMSO-treated group, suggesting that NAIs are effective at modifying viral genomic RNA ex vivo. (C) SHAPE secondary structure modeling of the PEDV 5’ UTR. ***p<0.001 by equal variance unpaired Student’s t-test.

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  Figure 2.. SHAPE structural map of the porcine epidemic diarrhea virus (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.

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  Figure 2—figure supplement 1.. Folding characteristics of known functional regions and coding areas in the porcine epidemic diarrhea virus (PEDV) genome. The length of ORF1ab is 20,345 nt, accounting for 72.5% of the total length of the PEDV genome (28,044 nt) (a major contributor to the global median). The blue dashed line and the orange dashed line represent the global median SHAPE reactivity and the global median Shannon entropy, respectively.

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  Figure 2—figure supplement 2.. Well-folded structures 1–8 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 3.. Well-folded structures 9–15 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 4.. Well-folded structures 10–21 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 5.. Well-folded structures 22–29 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 6.. Well-folded structures 30–37 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 7.. Well-folded structures 38–42 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 8.. Well-folded structures 43–50 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint.Green shading indicates high accessibility windows.

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  Figure 2—figure supplement 9.. Well-folded structures 51–54 in porcine epidemic diarrhea virus (PEDV) genome. Secondary structure predicted by SHAPE reactivity as a constraint. Green shading indicates high accessibility windows.