A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication
Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransfera...
| Main Authors: | , , , , , , , , , , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
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2015
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/107252 http://hdl.handle.net/10220/25565 |
| _version_ | 1811676871522254848 |
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| author | Zhao, Yongqian Soh, Tingjin Sherryl Zheng, Jie Chan, Kitti Wing Ki Phoo, Wint Wint Lee, Chin Chin Tay, Moon Y. F. Swaminathan, Kunchithapadam Cornvik, Tobias Carl Lim, Siew Pheng Shi, Pei-Yong Lescar, Julien Vasudevan, Subhash G. Luo, Dahai |
| author2 | Rey, Félix A. |
| author_facet | Rey, Félix A. Zhao, Yongqian Soh, Tingjin Sherryl Zheng, Jie Chan, Kitti Wing Ki Phoo, Wint Wint Lee, Chin Chin Tay, Moon Y. F. Swaminathan, Kunchithapadam Cornvik, Tobias Carl Lim, Siew Pheng Shi, Pei-Yong Lescar, Julien Vasudevan, Subhash G. Luo, Dahai |
| author_sort | Zhao, Yongqian |
| collection | NTU |
| description | Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5. |
| first_indexed | 2024-10-01T02:28:21Z |
| format | Journal Article |
| id | ntu-10356/107252 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T02:28:21Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | ntu-10356/1072522022-02-16T16:29:43Z A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication Zhao, Yongqian Soh, Tingjin Sherryl Zheng, Jie Chan, Kitti Wing Ki Phoo, Wint Wint Lee, Chin Chin Tay, Moon Y. F. Swaminathan, Kunchithapadam Cornvik, Tobias Carl Lim, Siew Pheng Shi, Pei-Yong Lescar, Julien Vasudevan, Subhash G. Luo, Dahai Rey, Félix A. Lee Kong Chian School of Medicine (LKCMedicine) School of Biological Sciences DRNTU::Science::Biological sciences::Microbiology::Bacteria Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5. NMRC (Natl Medical Research Council, S’pore) Published version 2015-05-18T02:22:50Z 2019-12-06T22:27:25Z 2015-05-18T02:22:50Z 2019-12-06T22:27:25Z 2015 2015 Journal Article Zhao, Y., Soh, T. S., Zheng, J., Chan, K. W. K., Phoo, W. W., Lee, C. C., et al. (2015). A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication. PLOS pathogens, 11(3). 1553-7374 https://hdl.handle.net/10356/107252 http://hdl.handle.net/10220/25565 10.1371/journal.ppat.1004682 25775415 en PLOS pathogens © 2015 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 27 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences::Microbiology::Bacteria Zhao, Yongqian Soh, Tingjin Sherryl Zheng, Jie Chan, Kitti Wing Ki Phoo, Wint Wint Lee, Chin Chin Tay, Moon Y. F. Swaminathan, Kunchithapadam Cornvik, Tobias Carl Lim, Siew Pheng Shi, Pei-Yong Lescar, Julien Vasudevan, Subhash G. Luo, Dahai A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title | A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title_full | A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title_fullStr | A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title_full_unstemmed | A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title_short | A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication |
| title_sort | crystal structure of the dengue virus ns5 protein reveals a novel inter domain interface essential for protein flexibility and virus replication |
| topic | DRNTU::Science::Biological sciences::Microbiology::Bacteria |
| url | https://hdl.handle.net/10356/107252 http://hdl.handle.net/10220/25565 |
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