Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains.
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.
Hall
Roy A
RA
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Tan
Si En
SE
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Selisko
Barbara
B
Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Marseille, France.
Slade
Rachael
R
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Hobson-Peters
Jody
J
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Canard
Bruno
B
Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Marseille, France.
Hughes
Megan
M
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Leung
Jason Y
JY
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Balmori-Melian
Ezequiel
E
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Hall-Mendelin
Sonja
S
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Pham
Kim B
KB
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Clark
David C
DC
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Prow
Natalie A
NA
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
Khromykh
Alexander A
AA
Centre for Infectious Desease Research, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.
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
D000911:Antibodies, Monoclonal; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; D012324:RNA-Dependent RNA Polymerase