Dimerization of flavivirus NS4B protein

Jing Zou, Xuping Xie, Le Tian Lee, Ramya Chandrasekaran, Aline Reynaud, Lijian Yap, Qing Yin Wang, Hongping Dong, Congbao Kang, Zhiming Yuan, Julien Lescar, Pei-Yong Shi

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3379-3391
Number of pages13
JournalJournal of Virology
Volume88
Issue number6
DOIs
StatePublished - 2014
Externally publishedYes

Fingerprint

Flavivirus
dimerization
Dimerization
Dengue virus
Dengue Virus
RNA
viral proteins
Viral Proteins
lethal genes
mutants
Proteins
proteins
Amino Acids
replicon
West Nile virus
Replicon
amino acids
Micelles
micelles
light scattering

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Zou, J., Xie, X., Lee, L. T., Chandrasekaran, R., Reynaud, A., Yap, L., ... Shi, P-Y. (2014). Dimerization of flavivirus NS4B protein. Journal of Virology, 88(6), 3379-3391. https://doi.org/10.1128/JVI.02782-13

Dimerization of flavivirus NS4B protein. / Zou, Jing; Xie, Xuping; Lee, Le Tian; Chandrasekaran, Ramya; Reynaud, Aline; Yap, Lijian; Wang, Qing Yin; Dong, Hongping; Kang, Congbao; Yuan, Zhiming; Lescar, Julien; Shi, Pei-Yong.

In: Journal of Virology, Vol. 88, No. 6, 2014, p. 3379-3391.

Research output: Contribution to journalArticle

Zou, J, Xie, X, Lee, LT, Chandrasekaran, R, Reynaud, A, Yap, L, Wang, QY, Dong, H, Kang, C, Yuan, Z, Lescar, J & Shi, P-Y 2014, 'Dimerization of flavivirus NS4B protein', Journal of Virology, vol. 88, no. 6, pp. 3379-3391. https://doi.org/10.1128/JVI.02782-13
Zou J, Xie X, Lee LT, Chandrasekaran R, Reynaud A, Yap L et al. Dimerization of flavivirus NS4B protein. Journal of Virology. 2014;88(6):3379-3391. https://doi.org/10.1128/JVI.02782-13
Zou, Jing ; Xie, Xuping ; Lee, Le Tian ; Chandrasekaran, Ramya ; Reynaud, Aline ; Yap, Lijian ; Wang, Qing Yin ; Dong, Hongping ; Kang, Congbao ; Yuan, Zhiming ; Lescar, Julien ; Shi, Pei-Yong. / Dimerization of flavivirus NS4B protein. In: Journal of Virology. 2014 ; Vol. 88, No. 6. pp. 3379-3391.
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