Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities...

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Main Authors: Law, Yee-Song, Wang, Sainan, Tan, Yaw Bia, Shih, Orion, Utt, Age, Goh, Wei Yang, Lian, Bing-Jun, Chen, Ming Wei, Jeng, U-Ser, Merits, Andres, Luo, Dahai
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2022
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Online Access:https://hdl.handle.net/10356/157045
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Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-157045
record_format dspace
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Alphavirus
Nonstructural Protein 2
spellingShingle Science::Biological sciences
Alphavirus
Nonstructural Protein 2
Law, Yee-Song
Wang, Sainan
Tan, Yaw Bia
Shih, Orion
Utt, Age
Goh, Wei Yang
Lian, Bing-Jun
Chen, Ming Wei
Jeng, U-Ser
Merits, Andres
Luo, Dahai
Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
description Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Law, Yee-Song
Wang, Sainan
Tan, Yaw Bia
Shih, Orion
Utt, Age
Goh, Wei Yang
Lian, Bing-Jun
Chen, Ming Wei
Jeng, U-Ser
Merits, Andres
Luo, Dahai
format Article
author Law, Yee-Song
Wang, Sainan
Tan, Yaw Bia
Shih, Orion
Utt, Age
Goh, Wei Yang
Lian, Bing-Jun
Chen, Ming Wei
Jeng, U-Ser
Merits, Andres
Luo, Dahai
author_sort Law, Yee-Song
title Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
title_short Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
title_full Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
title_fullStr Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
title_full_unstemmed Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity
title_sort interdomain flexibility of chikungunya virus nsp2 helicase-protease differentially influences viral rna replication and infectivity
publishDate 2022
url https://hdl.handle.net/10356/157045
_version_ 1759854722735407104
spelling sg-ntu-dr.10356-1570452023-02-28T17:12:03Z Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity Law, Yee-Song Wang, Sainan Tan, Yaw Bia Shih, Orion Utt, Age Goh, Wei Yang Lian, Bing-Jun Chen, Ming Wei Jeng, U-Ser Merits, Andres Luo, Dahai School of Biological Sciences Lee Kong Chian School of Medicine (LKCMedicine) NTU Institute of Structural Biology Science::Biological sciences Alphavirus Nonstructural Protein 2 Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome. Ministry of Education (MOE) Submitted/Accepted version This research was supported by the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2016T22097). Work in the A.M. laboratory was supported by the European Regional Development Fund through the Centre of Excellence in Molecular Cell Engineering, Estonia, 2014-2020.4.01.15-013, and by The Wellcome Trust (200171/Z/15/Z). 2022-05-01T04:57:45Z 2022-05-01T04:57:45Z 2021 Journal Article Law, Y., Wang, S., Tan, Y. B., Shih, O., Utt, A., Goh, W. Y., Lian, B., Chen, M. W., Jeng, U., Merits, A. & Luo, D. (2021). Interdomain flexibility of Chikungunya virus nsP2 helicase-protease differentially influences viral RNA replication and infectivity. Journal of Virology, 95(6), e01470-. https://dx.doi.org/10.1128/JVI.01470-20 0022-538X https://hdl.handle.net/10356/157045 10.1128/JVI.01470-20 33328310 2-s2.0-85102322365 6 95 e01470 en MOE2016T22097 Journal of Virology © 2021 American Society for Microbiology. All rights reserved. This paper was published in Journal of Virology and is made available with permission of American Society for Microbiology. application/pdf