title: Mapping the Interactions between the NS4B and NS3 proteins of dengue virus.
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.
  
  
  OBJECTIVE
  NS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details of how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3-NS4B interaction for antiviral drug discovery.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore State Key Laboratory of Virology, Wuhan Institute of Virology, University of Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zou
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Le Tian
authors.initials: le T
authors.lastname: Lee
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Qing Yin
authors.initials: QY
authors.lastname: Wang
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Xuping
authors.initials: X
authors.lastname: Xie
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Siyan
authors.initials: S
authors.lastname: Lu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Yin Hoe
authors.initials: YH
authors.lastname: Yau
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, University of Chinese Academy of Sciences, Wuhan, China.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Susana
authors.initials: S
authors.lastname: Geifman Shochat
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Congbao
authors.initials: C
authors.lastname: Kang
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore julien@ntu.edu.sg pei_yong.shi@novartis.com.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore julien@ntu.edu.sg pei_yong.shi@novartis.com.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000502:physiology*; D047468:Immunoprecipitation; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080107:NS4B protein, flavivirus