Integrated Transcriptomics Reveals a SHEV ORF3-Mediated circRNA Network That Disrupts Riboflavin Metabolism and Activates the ko05212 Pathway

Figures

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  Figure 1. Volcano plot showing differentially expressed circRNAs. Detailed analysis revealed distinct expression patterns for key metabolism-related molecules.

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  Figure 2. Expression Landscape of m6A Methylation Regulators in SHEV ORF3-Expressing HepG2 Cells. The heatmap displays the relative expression levels (row Z-score normalized FPKM) of key m6A writers, erasers, and readers. Red indicates upregulation, and blue indicates downregulation. Note the distinct downregulation trend of the m6A eraser FTO (indicated by the blue shade in the ORF3 group), suggesting a potential global alteration in m6A modification status.

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  Figure 3. Bubble Plot of KEGG Pathway Enrichment Analysis for Host Genes of Differentially Expressed circRNAs. The y-axis lists the top 18 significantly enriched KEGG pathways, while the x-axis represents the enrichment factor. The size of each bubble corresponds to the number of differentially expressed genes enriched in that pathway. The color of the bubbles indicates the significance level based on the p-value (red indicates high significance; green indicates low significance). The results demonstrate that both the Riboflavin metabolism and Pancreatic cancer pathways are significantly enriched.

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  Figure 4. Expression Heatmap of Key circRNAs Involved in the Riboflavin Metabolism Pathway. Each column represents an individual sample, while each row corresponds to a specific circRNA. The color scale indicates relative expression levels normalized by Z-score, where red denotes high expression and blue denotes low expression. The results reveal that, compared to the control group, hsa_circ_0077855 (derived from ENPP3) exhibits a distinct high-expression pattern (red) in the ORF3 group, whereas hsa_circ_0130715 (derived from ENPP1) displays a significant low-expression pattern (blue). This distinct reversal in expression profiles further corroborates the targeted remodeling of the riboflavin metabolism network by the ORF3 protein.

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  Figure 5. Sankey Diagram of the SHEV ORF3-Mediated circRNA-miRNA-mRNA Ternary Interaction Network. The diagram illustrates the regulatory pathways flowing from circRNAs (left) to miRNAs (middle), and finally targeting mRNAs (right). The width of the connecting bands represents the strength of the predicted binding interactions. The red bands specifically highlight the hsa_circ_0077855/miR-181a/ENPP3 axis, demonstrating that ORF3 relieves the miRNA-mediated repression of the metabolic gene ENPP3 by upregulating the circRNA.

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  Figure 6. AlphaFold 3 molecular docking simulation. Specific quantitative model evaluation metrics revealed that the interface predicted Template Modeling score (ipTM) for the complex was only 0.08, and the predicted Template Modeling score (pTM) was 0.11. According to AlphaFold 3 scoring criteria, an extremely low ipTM score (<0.2) typically indicates the lack of a stable interaction interface between the two molecules or the failure to form a stable binary complex.