Zika NS2B-3-4A Protein Construct cleavage pattern assay v1

Cis cleavage pattern by viral protease is analyzed using an expression plasmid pT7CFE1-NHA-Chis-target of interest. For flaviviruses (ZIka/DENV) the plasmid has been designed to include the cofactor domain of NS2B, NS2B-3 cleavage site, protease and helicase domains of NS3, and first 49 residues of NS4A to include the NS3-4A cleavage site (1). The plasmid lacks transmembrane domains. For enteroviruses (EVD68/EVA71), the plasmid includes the cleavage of structural protein VP1 from VP12A by the enteroviral 2A protease (2). Protein synthesis is carried out using a Rabbit reticulocyte lysate (TNT‱ Quick Coupled Transcription/Translation Systems; Promega) and 1 µg/50 µL of plasmid template via a coupled transcription/translation system in presence of S-35 labeled L-Methionine (EasyTag; PerkinElmer) (3) and output is quantified using SDS gels. D.A. Constant, R. Mateo, C.M. Nagamine, & K. Kirkegaard, Targeting intramolecular proteinase NS2B/3 cleavages for trans-dominant inhibition of dengue virus, Proc. Natl. Acad. Sci. U.S.A. 115 (40) 10136-10141,https://doi.org/10.1073/pnas.1805195115(2018). DohertyJS,KirkegaardK.2023.Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds. J Virol97:e00928-23.https://doi.org/10.1128/jvi.00928-23 https://www.promega.com/products/protein-expression/cell-free-protein-expression/tnt-quick-coupled-transcription_translation-system/?catNum=L1170#protocols