title: Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities.
abstract: Chikungunya virus (CHIKV) is an important arboviral infectious agent in tropical and subtropical regions, often causing persistent and debilitating disease. The viral enzyme non-structural protein 4 (nsP4), as RNA-dependent RNA polymerase (RdRP), catalyzes the formation of negative-sense, genomic and subgenomic viral RNAs. Here we report a truncated nsP4 construct that is soluble, stable and purified recombinantly from Escherichia coli. Sequence analyses and homology modelling indicate that all necessary RdRP elements are included. Hydrogen/deuterium exchange with mass spectrometry was used to analyze solvent accessibility and flexibility of subdomains. Fluorophore-conjugated RNA ligands were designed and screened by using fluorescence anisotropy to select a suitable substrate for RdRP assays. Assay trials revealed that nsP4 core domain is conditionally active upon choice of detergent species, and carries out both primed extension and terminal adenylyltransferase activities. The polymerization assay can be further developed to screen for antiviral compounds in vitro.
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Ming Wei
authors.initials: MW
authors.lastname: Chen
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Yaw Bia
authors.initials: YB
authors.lastname: Tan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore; Protein Production Platform, Nanyang Technological University, Singapore.
authors.forename: Bee Ting
authors.initials: BT
authors.lastname: Lim
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore; Protein Production Platform, Nanyang Technological University, Singapore.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cornvik
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Lisa Fong Poh
authors.initials: LFP
authors.lastname: Ng
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore. Electronic address: luodahai@ntu.edu.sg.
authors.forename: Dahai
authors.initials: D
authors.lastname: Luo
mesh_terms: D000998:Antiviral Agents; D020134:Catalytic Domain; D065632:Chikungunya Fever / Q000821:virology; D002646:Chikungunya virus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000235:genetics; D000067776:RNA Recognition Motif Proteins / Q000737:chemistry; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis; D040681:Structural Homology, Protein; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D003902:Detergents; D000067776:RNA Recognition Motif Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C499947:nsP4 protein, Sindbis virus; D009713:Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase
keywords: Chikungunya; Fluorescence anisotropy; HDX-MS; Homology modelling; RdRP