Fig 1.

Principle and design of the NS1-specific artificial SUMO ligase (ASL). NS1 contains two well-defined globular domains in its structure, the RNA binding domain (RBD) (amino acid residues 1 to 73) and the effector domain (ED) (amino acid residues 86 to 204), separated by an unstructured flexible linker (amino acid residues 74 to 85, represented by a wiggly blue line). Both domains are capable of forming dimers, thus allowing full-length NS1 to form dimers (A) and multimers. As the main SUMOylation site in NS1 is located near its C-terminal end, and the dimeric structures formed by the RBD alone are relatively unstable, we envisioned that a fusion protein consisting of NS1's RBD and linker region fused to the full-length SUMO-conjugating enzyme Ubc9 (RBD∼Ubc9) (B) would be “charged” by the E1 SUMO-activating enzyme (SAE2/1) present in the cell (C); form transient NS1:RBD∼Ubc9 heterodimers, thus positioning Ubc9 in close proximity to K219 in NS1 (D); and enhance NS1 SUMOylation (E). Thus, the RBD∼Ubc9 fusion would act as an ASL for NS1. Although it is likely that NS1 might subsequently be de-SUMOylated by the SUMO-specific isopeptidases present in the cell (referred to as SENPs) (F), the enhancing effect mediated by the RBD∼Ubc9 fusion would still increase the steady-state concentration of SUMOylated NS1 in the cell. ASL* represents the SUMO-charged form of the ASL, in which SUMO is attached to the active site in Ubc9, and ASL represents the uncharged form of the ASL.