Dimerization of flavivirus NS4B protein
Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replic...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2015
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| Online Access: | https://hdl.handle.net/10356/100386 http://hdl.handle.net/10220/25720 |
| _version_ | 1826114084384276480 |
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| author | Dong, Hongping Reynaud, Aline Zou, Jing Xie, Xuping Lee, Le Tian Chandrasekaran, Ramya Yap, Lijian Wang, Qing-Yin Kang, Congbao Yuan, Zhiming Lescar, Julien Shi, Pei-Yong |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Dong, Hongping Reynaud, Aline Zou, Jing Xie, Xuping Lee, Le Tian Chandrasekaran, Ramya Yap, Lijian Wang, Qing-Yin Kang, Congbao Yuan, Zhiming Lescar, Julien Shi, Pei-Yong |
| author_sort | Dong, Hongping |
| collection | NTU |
| description | Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. |
| first_indexed | 2024-10-01T03:33:43Z |
| format | Journal Article |
| id | ntu-10356/100386 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:33:43Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | ntu-10356/1003862023-02-28T17:05:07Z Dimerization of flavivirus NS4B protein Dong, Hongping Reynaud, Aline Zou, Jing Xie, Xuping Lee, Le Tian Chandrasekaran, Ramya Yap, Lijian Wang, Qing-Yin Kang, Congbao Yuan, Zhiming Lescar, Julien Shi, Pei-Yong School of Biological Sciences DRNTU::Science::Biological sciences Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. Published version 2015-06-02T02:40:32Z 2019-12-06T20:21:31Z 2015-06-02T02:40:32Z 2019-12-06T20:21:31Z 2014 2014 Journal Article Zou, J., Xie, X., Lee, L. T., Chandrasekaran, R., Reynaud, A., Yap, L., et al. (2014). Dimerization of flavivirus NS4B protein. Journal of virology, 88(6), 3379-3391. 0022-538X https://hdl.handle.net/10356/100386 http://hdl.handle.net/10220/25720 10.1128/JVI.02782-13 24390334 en Journal of virology © 2014 American Society for Microbiology. This paper was published in Journal of Virology and is made available as an electronic reprint (preprint) with permission of American Society for Microbiology. The paper can be found at the following official DOI: [http://dx.doi.org/10.1128/JVI.02782-13]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law. 13 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Dong, Hongping Reynaud, Aline Zou, Jing Xie, Xuping Lee, Le Tian Chandrasekaran, Ramya Yap, Lijian Wang, Qing-Yin Kang, Congbao Yuan, Zhiming Lescar, Julien Shi, Pei-Yong Dimerization of flavivirus NS4B protein |
| title | Dimerization of flavivirus NS4B protein |
| title_full | Dimerization of flavivirus NS4B protein |
| title_fullStr | Dimerization of flavivirus NS4B protein |
| title_full_unstemmed | Dimerization of flavivirus NS4B protein |
| title_short | Dimerization of flavivirus NS4B protein |
| title_sort | dimerization of flavivirus ns4b protein |
| topic | DRNTU::Science::Biological sciences |
| url | https://hdl.handle.net/10356/100386 http://hdl.handle.net/10220/25720 |
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