Enhancement of MAVS UFMylation and inhibition of lysosomal degradation of MAVS aggregates: New findings on broad-spectrum antiviral mechanism of Anemoside B4.

RNA viruses have evolved various strategies to bypass the mitochondrial antiviral-signaling protein (MAVS) pathway, effectively sidestepping the host's innate immunity. Therefore, searching for MAVS signalosome regulators is crucial for the development of antiviral drugs. Here, we found RNA viral-drived MAVS lysosomal trafficking was inhibited by Anemoside B4 (B4), a triterpenoid saponin from the herbal medicine Pulsatilla chinensis (Bunge) Regel., has significant antiviral activity. We performed biotin-B4 probe tandem proteomic profiling and identified ubiquitin fold modifier 1 (UFM1), one of the newly discovered Ub-like proteins (UBLs), as a key target of B4. Using cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) analysis, and molecular docking analysis we showed that B4 directly bound to UFM1 through Lys34 and Ile57 sites and promoted UFMylation of target proteins. Furthermore, we found that MAVS UFMylation could promote polyubiquitination, K48- and K63-linked ubiquitination, yet preventing K27-linked ubiquitination. MAVS aggregation and innate immunity response were suppressed in virus infected UFM1 KO cells even in the presence of B4. Moreover, the decrease in K27-ubiquitin (Ub) binding on MAVS and lysosomal degradation induced by B4 was also significantly prevented by UFM1 deficiency. Importantly, to verify that MAVS UFMylation is the actual target for B4, cytoprotective and viral titer analysis were executed in cells lacking UFM1 challenged with Enterovirus 71 (EV71) and Influenza A virus (IAV). The findings uncover the new finding broad-spectrum antiviral mechanism of B4, suggesting that UFMylation of MAVS could be an advantageous approach for countering RNA viral infections.