Hedgehog pathway inhibitor HhAntag suppresses virus infection via the GLI-S1PR axis

The interplay between various signaling pathways, including tumor development, immune response, and viral infection, suggests potential mutual regulation within biological systems. To explore this, we screened 85 inhibitors targeting the Notch, Hedgehog, and Wnt signaling pathways to identify the potential antiviral candidates. Using two reporter viruses (VSV-GFP and DENV-Luc), we identified novel inhibitors with antiviral properties. Notably, the Hedgehog pathway inhibitor HhAntag exhibited broad-spectrum antiviral activity, significantly reducing the replication of viruses such as VSV, DENV, ZIKV, and SFTSV. The inhibitory effects of HhAntag were consistent with the downregulation of its target protein, GLI1; while overexpression of GLI1 promoted viral infection. HhAntag did not interfere with viral attachment, entry, or early transcription but specifically inhibited viral protein translation. Additionally, RNA-seq analysis revealed reduced expression of sphingosine-1-phosphate (S1P) signaling pathway receptors, S1PR1 and S1PR5, following HhAntag treatment. HhAntag suppresses virus infection via the GLI-S1PR axis. This study revealed the interplay between tumor-associated Hedgehog (Hh) pathway and viral infection and highlights the potential of HhAntag as a broad-spectrum antiviral drug.