Bortezomib inhibits ZIKV/DENV by interfering with viral polyprotein cleavage via the ERAD pathway.

Flaviviruses have posed a serious threat to human health in the past decades, and effective therapeutic drugs are lacking; thus, treatment of flavivirus infection is a great challenge. The flavivirus protease NS2B3 is an attractive target for antiviral drug screening. Here, we developed an intracellular Zika virus (ZIKV) NS2AB3 self-cleavage assay to identify inhibitors that interfere with viral polyprotein cleavage and block ZIKV/dengue virus (DENV) replication. Bortezomib was identified as the most potent inhibitor, with a half-maximal effective concentration (EC50) in the nanomolar range. We found that instead of directly inhibiting NS2B3 protease activity, bortezomib dramatically induced the ubiquitination and aggregation of NS3, leading to the attenuation of its protease activity in cells. Two E3 ligases, HRD1 and RNF126, were found to be responsible for NS3 ubiquitination. Our study identifies bortezomib as a potential drug for the treatment of ZIKV/DENV infection and reveals the central role of the ERAD pathway in the inhibition of flaviviruses by bortezomib.