Flavokawain B inhibits ER-associated protein dislocation and reduces infectious Zika virus production.

Flavokawain B (FB), a chalcone from kava, induces endoplasmic reticulum (ER) stress and inhibits protein neddylation, but its effect on ER protein quality control is unknown. Here we identify inhibition of ER-associated degradation (ERAD) substrate dislocation as a cellular activity of FB. In a live-cell dislocation-dependent reconstituted GFP (drGFP) assay, FB inhibited dislocation of the luminal ERAD substrate null Hong Kong α1-antitrypsin (NHK) and of the membrane substrate CD3δ, with apparent IC50 values of 4.26 and 9.79 μM. FB also reduced NHK ubiquitination and stabilized NHK in a concentration-dependent manner. Unlike bortezomib, FB did not stabilize the short-lived cytosolic ubiquitin-proteasome system reporter GFPu, measured by live-cell imaging and immunoblotting, arguing against generalized proteasome blockade as the primary cause of NHK stabilization. In a temperature-series cellular thermal shift assay (CETSA), FB increased the heat-resistant soluble fraction of both HRD1 and gp78, suggesting that FB perturbs an ERAD-associated protein environment involving these ligases. Because HRD1-mediated ERAD sustains flaviviral protein homeostasis, we asked whether this phenotype extends to virus production: FB reduced infectious Zika virus (ZIKV) output to approximately 40% of control at 5 μM and to below 20% at 7.5-10 μM, without a proportional loss of cell viability. FB is therefore a chemical probe of ERAD dislocation with an associated antiviral phenotype.