Zika NS2B is a crucial factor recruiting NS3 to the ER and activating its protease activity.

Zika virus (ZIKV) is an emergent flavivirus associated with severe neurological disorders. ZIKV NS3 protein is a viral protease that cleaves the ZIKV polyprotein precursor into individual viral proteins. In this study, we found that ZIKV NS3 by itself exhibited mitochondrial localization, which was quite different from its endoplasmic reticulum (ER) localization in ZIKV-infected cells. We screened viral proteins and identified NS2B as the bona fide recruiter of NS3 to the ER. The NS2B C-terminal tail interacted with NS3 protease domain to retain NS3 on the ER. β-Sheet motifs that formed between NS2B and the NS3 protease domain played important roles in their interaction, while mutation in the β-strand of NS2B attenuated NS2B-NS3 interaction and impaired the ability of NS3 protease to cleave the polyprotein precursor into multiple viral proteins. Consequently, NS2B mutations led to severe inhibition of ZIKV replication and production due to insufficient NS3 protease activity. In summary, our study reveals the critical role of NS2B in NS3 recruitment and protease function and provides mechanistic insight into ZIKV replication.