A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

Yongqian Zhao, Tingjin Sherryl Soh, Jie Zheng, Kitti Wing Ki Chan, Wint Wint Phoo, Chin Chin Lee, Moon Y F Tay, Kunchithapadam Swaminathan, Tobias C. Cornvik, Siew Pheng Lim, Pei-Yong Shi, Julien Lescar, Subhash G. Vasudevan, Dahai Luo

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numbere1004682
Pages (from-to)1-27
Number of pages27
JournalPLoS Pathogens
Volume11
Issue number3
DOIs
StatePublished - Mar 1 2015
Externally publishedYes

Fingerprint

RNA Replicase
Virus Replication
Flavivirus
Japanese Encephalitis Virus
Methyltransferases
Dengue Virus
Proteins
Viral Nonstructural Proteins
Molecular Conformation
Deuterium
Thumb
Site-Directed Mutagenesis
Guanosine Triphosphate
Antiviral Agents
Hydrogen
Mass Spectrometry
RNA
Temperature
Membranes
dengue virus NS5 protein

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

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 C.; Lim, Siew Pheng; Shi, Pei-Yong; Lescar, Julien; Vasudevan, Subhash G.; Luo, Dahai.

In: PLoS Pathogens, Vol. 11, No. 3, e1004682, 01.03.2015, p. 1-27.

Research output: Contribution to journalArticle

Zhao, Y, Soh, TS, Zheng, J, Chan, KWK, Phoo, WW, Lee, CC, Tay, MYF, Swaminathan, K, Cornvik, TC, Lim, SP, Shi, P-Y, Lescar, J, Vasudevan, SG & Luo, D 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, vol. 11, no. 3, e1004682, pp. 1-27. https://doi.org/10.1371/journal.ppat.1004682
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 C. ; 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. In: PLoS Pathogens. 2015 ; Vol. 11, No. 3. pp. 1-27.
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