Mapping the interactions between the NS4B and NS3 proteins of dengue virus

Jing Zou, Le Tian Lee, Qing Yin Wang, Xuping Xie, Siyan Lu, Yin Hoe Yau, Zhiming Yuan, Susana Geifman Shochat, Congbao Kang, Julien Lescar, Pei-Yong Shi

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Flavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and an in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn α-helix and two short β-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy.

Original languageEnglish (US)
Pages (from-to)3471-3483
Number of pages13
JournalJournal of Virology
Volume89
Issue number7
DOIs
StatePublished - 2015
Externally publishedYes

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Flavivirus
Dengue virus
Dengue Virus
Flaviviridae
RNA helicases
RNA Helicases
multiprotein complexes
Multiprotein Complexes
surface plasmon resonance
Surface Plasmon Resonance
proteins
Sequence Alignment
sequence alignment
Virus Replication
virus replication
Innate Immunity
mutagenesis
Mutagenesis
membrane proteins
Endoplasmic Reticulum

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Mapping the interactions between the NS4B and NS3 proteins of dengue virus. / Zou, Jing; Lee, Le Tian; Wang, Qing Yin; Xie, Xuping; Lu, Siyan; Yau, Yin Hoe; Yuan, Zhiming; Shochat, Susana Geifman; Kang, Congbao; Lescar, Julien; Shi, Pei-Yong.

In: Journal of Virology, Vol. 89, No. 7, 2015, p. 3471-3483.

Research output: Contribution to journalArticle

Zou, J, Lee, LT, Wang, QY, Xie, X, Lu, S, Yau, YH, Yuan, Z, Shochat, SG, Kang, C, Lescar, J & Shi, P-Y 2015, 'Mapping the interactions between the NS4B and NS3 proteins of dengue virus', Journal of Virology, vol. 89, no. 7, pp. 3471-3483. https://doi.org/10.1128/JVI.03454-14
Zou, Jing ; Lee, Le Tian ; Wang, Qing Yin ; Xie, Xuping ; Lu, Siyan ; Yau, Yin Hoe ; Yuan, Zhiming ; Shochat, Susana Geifman ; Kang, Congbao ; Lescar, Julien ; Shi, Pei-Yong. / Mapping the interactions between the NS4B and NS3 proteins of dengue virus. In: Journal of Virology. 2015 ; Vol. 89, No. 7. pp. 3471-3483.
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