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Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyl transferase and RNA-dependant RNA polymerase domains

¹ University of Queensland, Australia;
² Centre National de la Recherche Scientifique, Marseille, France

³ E-mail: roy.hall{at}uq.edu.au

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 about 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 an in vitro assay, and suggests that this region represents a potential target for the design of RdRp inhibitors. Using a series of synthetic peptides we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 amino acid 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 the antibody recognises 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.

Received 4 June 2009; accepted 25 August 2009.