{"doi":"10.17504/protocols.io.yxmvm9dw9l3p/v1","title":"Zika NS2B-3-4A Protein Construct cleavage pattern assay v1","abstract":"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","journal":null,"year":2025,"id":572381,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9473,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":823651,"name":"Adeeba H. Dhalech","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:57:23.653298Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}