کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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5551762 | 1557800 | 2017 | 10 صفحه PDF | دانلود رایگان |
- An active Chikungunya RNA polymerase protein is important for inhibitor screening.
- CHIKV RdRP has strict domain boundaries for bacterial expression and good yield.
- HDX-MS reveals high flexibility in the fingers domain.
- RdRP performs both primed extension and template-independent nucleotide addition.
- Selection of assay components such as detergent is essential to assay development.
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.
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Journal: Antiviral Research - Volume 143, July 2017, Pages 38-47