Dimerization of Flavivirus NS4B Protein
| العنوان: | Dimerization of Flavivirus NS4B Protein |
|---|---|
| المؤلفون: | CongBao Kang, Julien Lescar, Le Tian Lee, Zhiming Yuan, Pei Yong Shi, Aline Reynaud, Ramya Chandrasekaran, Lijian Yap, Qing Yin Wang, Xuping Xie, Hongping Dong, Jing Zou |
| المساهمون: | School of Biological Sciences |
| المصدر: | Journal of Virology. 88:3379-3391 |
| بيانات النشر: | American Society for Microbiology, 2014. |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | viruses, Amino Acid Motifs, Immunology, Mutagenesis (molecular biology technique), Viral Nonstructural Proteins, Biology, Dengue virus, medicine.disease_cause, Microbiology, Dengue, Replication factor C, Virology, medicine, Humans, Replicon, Endoplasmic reticulum, RNA, Dengue Virus, Genome Replication and Regulation of Viral Gene Expression, Science::Biological sciences [DRNTU], Membrane protein, Biochemistry, Viral replication, Insect Science, Dimerization, West Nile virus, West Nile Fever |
| الوصف: | 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. IMPORTANCE The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans -complementation enhances our understanding of NS4B in flavivirus replication. |
| وصف الملف: | application/pdf |
| تدمد: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.02782-13 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::84b87bd639c99d707f994544f8e9dd08 https://doi.org/10.1128/jvi.02782-13 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....84b87bd639c99d707f994544f8e9dd08 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10985514 0022538X |
|---|---|
| DOI: | 10.1128/jvi.02782-13 |