title: A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.
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.
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore; Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Tingjin Sherryl
authors.initials: TS
authors.lastname: Soh
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore.
authors.forename: Kitti Wing Ki
authors.initials: KW
authors.lastname: Chan
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Wint Wint
authors.initials: WW
authors.lastname: Phoo
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore.
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore.
authors.forename: Moon Y F
authors.initials: MY
authors.lastname: Tay
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Tobias C
authors.initials: TC
authors.lastname: Cornvik
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore; UPMC UMRS CR7-CNRS ERL 8255-INSERM U1135 Centre d'Immunologie et des Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine Pierre et Marie Curie, Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Dahai
authors.initials: D
authors.lastname: Luo
mesh_terms: D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000737:chemistry* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
chemical_list: C582223:NS5 protein, dengue virus; D017361:Viral Nonstructural Proteins