In vitro synthesis of active recombinant orthoflavivirus nonstructural polyproteins in detergent micelles for biochemical analysis.

The nonstructural (NS) proteins of orthoflaviviruses are cleaved from a precursor polyprotein by both host and viral proteases, subsequently forming a replication complex. However, the biochemical properties underlying polyprotein cleavage and individual NS proteins within the complex remain largely unexplored. Here, we synthesized recombinant dengue virus (DENV-2) polyproteins containing all NS proteins for the biochemical evaluation of the synthesis and cleavage of the polyproteins. The results showed successful synthesis of the full-length polyprotein, which efficiently underwent self-cleavage when the detergent n-dodecyl-β-maltoside (DDM) was added. In addition, the cleaved NS proteins formed a complex in the DDM micelle and possessed the enzymatic activities of NS3 and NS5. Leveraging this in vitro system, we found that polyprotein was cleaved predominantly via cis-intramolecular activity, immediately following polyprotein synthesis. Therefore, the orthoflavivirus polyproteins synthesized here is potentially a valuable tool for the biochemical analysis of orthoflavivirus polyproteins.