Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities
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 an...
| Main Authors: | , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
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2017
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| Online Access: | https://hdl.handle.net/10356/86660 http://hdl.handle.net/10220/44135 |
| _version_ | 1811691209725313024 |
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| author | Chen, Ming Wei Tan, Yaw Bia Zheng, Jie Zhao, Yongqian Lim, Bee Ting Cornvik, Tobias Lescar, Julien Ng, Lisa Fong Poh Luo, Dahai |
| author2 | Lee Kong Chian School of Medicine (LKCMedicine) |
| author_facet | Lee Kong Chian School of Medicine (LKCMedicine) Chen, Ming Wei Tan, Yaw Bia Zheng, Jie Zhao, Yongqian Lim, Bee Ting Cornvik, Tobias Lescar, Julien Ng, Lisa Fong Poh Luo, Dahai |
| author_sort | Chen, Ming Wei |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T06:16:15Z |
| format | Journal Article |
| id | ntu-10356/86660 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T06:16:15Z |
| publishDate | 2017 |
| record_format | dspace |
| spelling | ntu-10356/866602020-11-01T05:23:01Z Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities Chen, Ming Wei Tan, Yaw Bia Zheng, Jie Zhao, Yongqian Lim, Bee Ting Cornvik, Tobias Lescar, Julien Ng, Lisa Fong Poh Luo, Dahai Lee Kong Chian School of Medicine (LKCMedicine) School of Biological Sciences Protein Production Platform Chikungunya RdRP 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. MOE (Min. of Education, S’pore) Accepted version 2017-12-13T02:39:07Z 2019-12-06T16:26:48Z 2017-12-13T02:39:07Z 2019-12-06T16:26:48Z 2017 Journal Article Chen, M. W., Tan, Y. B., Zheng, J., Zhao, Y., Lim, B. T., Cornvik, T., et al. (2017). Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities. Antiviral Research, 143, 38-47. 0166-3542 https://hdl.handle.net/10356/86660 http://hdl.handle.net/10220/44135 10.1016/j.antiviral.2017.04.001 en Antiviral Research © 2017 Elsevier. This is the author created version of a work that has been peer reviewed and accepted for publication by Antiviral Research, Elsevier. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1016/j.antiviral.2017.04.001]. 34 p. application/pdf |
| spellingShingle | Chikungunya RdRP Chen, Ming Wei Tan, Yaw Bia Zheng, Jie Zhao, Yongqian Lim, Bee Ting Cornvik, Tobias Lescar, Julien Ng, Lisa Fong Poh Luo, Dahai Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title | Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title_full | Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title_fullStr | Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title_full_unstemmed | Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title_short | Chikungunya virus nsP4 RNA-dependent RNA polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities |
| title_sort | chikungunya virus nsp4 rna dependent rna polymerase core domain displays detergent sensitive primer extension and terminal adenylyltransferase activities |
| topic | Chikungunya RdRP |
| url | https://hdl.handle.net/10356/86660 http://hdl.handle.net/10220/44135 |
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