title: Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains.
abstract: The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Hall
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Si En
authors.initials: SE
authors.lastname: Tan
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Marseille, France.
authors.forename: Barbara
authors.initials: B
authors.lastname: Selisko
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Rachael
authors.initials: R
authors.lastname: Slade
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Jody
authors.initials: J
authors.lastname: Hobson-Peters
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Megan
authors.initials: M
authors.lastname: Hughes
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Jason Y
authors.initials: JY
authors.lastname: Leung
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Balmori-Melian
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hall-Mendelin
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Kim B
authors.initials: KB
authors.lastname: Pham
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: David C
authors.initials: DC
authors.lastname: Clark
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Prow
authors.affiliation: Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Khromykh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D001667:Binding, Competitive; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011995:Recombination, Genetic; D017361:Viral Nonstructural Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014902:West Nile virus / Q000201:enzymology / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; D012324:RNA-Dependent RNA Polymerase