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.