Biochemical characterization of an unlinked flavivirus membrane-associated NS2B-NS3pro

Abstract

Flaviviruses such as Dengue virus (DENV) remain major global health threats, with no licensed antivirals targeting their viral proteases. The non-structural protein (NS) 2B-3 serine protease (NS2B-NS3pro), essential for viral replication, is a validated drug target. However, structural studies of membrane-associated full-length NS2B-NS3pro remain elusive, and no such structures have been solved to date.

Here, we expressed, purified, and characterized a BRIL domain-inserted unlinked full-length NS2B-NS3pro construct from DENV serotype 4 (DENV4). The construct retained catalytic activity and was complexed with BAG2, a conformation-specific anti-BRIL Fab, to improve stability and facilitate structural analysis. Analytical Gel Filtration Chromatography (or SEC) analysis revealed that both NS2B-NS3-BRIL, with and without BAG2, formed large protein–detergent complexes. This was confirmed by negative-stain electron microscopy showing monodisperse particles suitable for cryogenic-EM (cryo-EM).

This platform supports high-resolution structural studies of NS2B-NS3pro in a membrane-mimetic context. Ongoing cryo-EM analysis—including BAG2 co-complexing and inhibitor soaking—aims to capture conformationally relevant states for structure-guided antiviral screening.