Coupled Binding and Folding of NS2B/NS3 Protease and Linker Effects Revealed by Topology-Based Modeling.

Orthoflavivirus, such as West Nile Virus (WNV), dengue virus (DENV), and ZIKA virus (ZIKV), are globally distributed pathogens that pose substantial threats to human health. Currently, there are still no effective antiviral drugs for WNV or ZIKV. Despite the availability of two licensed DENV vaccines, their use remains limited due to potential risks, highlighting an urgent need for antiviral drug development. The highly conserved orthoflavivirus protease NS2B/NS3 is required for viral replication, making it a promising antiflavivirus target. A major challenge, however, is the highly charged active site of this enzyme, which requires charged chemical matters with low bioavailability. An alternative and more attractive strategy is to target potential allosteric sites or folding intermediate states of the protease. In this work, we employ the topology-based coarse-grained Go̅ modeling to explore the coupled binding and folding pathways of WNV NS2B/NS3 protease and study the effects of the widely used experimental construct with a G4SG4 linker between NS2B and NS3 on stability and folding. Our results provide a holistic conformational landscape of protease binding and folding, including several key intermediate states. We find that the presence of the G4SG4 linker alters the folding pathways and destabilizes the NS2B C-terminus. The latter is consistent with experimental observations that the G4SG4-linked protease has lower activity and adopts an open state without the substrate in crystal structures. Together, these findings provide, for the first time, a complete picture of the binding and folding of the NS2B/NS3 protease and identify important folding intermediate states that could be targeted for allosteric antiviral drug development.