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.

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A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

Journal of General Virology ◽

10.1099/vir.0.2008/003731-0 ◽

2008 ◽

Vol 89 (12) ◽

pp. 3063-3072 ◽

Cited By ~ 20

Author(s):

Jody Hobson-Peters ◽

Philip Toye ◽

Melissa D. Sánchez ◽

Katharine N. Bossart ◽

Lin-Fa Wang ◽

...

Keyword(s):

West Nile Virus ◽

Fusion Protein ◽

Synthetic Peptides ◽

Mammalian Cells ◽

Protein Glycosylation ◽

The West ◽

West Nile ◽

High Sequence Identity ◽

Recombinant Peptide ◽

E Protein

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

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A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: A case study using monoclonal antibodies against the West Nile virus E protein

Molecular Immunology ◽

10.1016/j.molimm.2008.07.020 ◽

2008 ◽

Vol 46 (1) ◽

pp. 125-134 ◽

Cited By ~ 20

Author(s):

Galina F. Denisova ◽

Dimitri A. Denisov ◽

Jeffrey Yeung ◽

Mark B. Loeb ◽

Michael S. Diamond ◽

...

Keyword(s):

Monoclonal Antibodies ◽

West Nile Virus ◽

Epitope Prediction ◽

Computer Algorithm ◽

The West ◽

West Nile ◽

E Protein ◽

Antibody Epitope

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Evidence for novel binding sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen

Biochemical Journal ◽

10.1042/bj2890445 ◽

1993 ◽

Vol 289 (2) ◽

pp. 445-451 ◽

Cited By ~ 15

Author(s):

L V Parise ◽

B Steiner ◽

L Nannizzi ◽

A B Criss ◽

D R Phillips

Keyword(s):

Monoclonal Antibodies ◽

Binding Sites ◽

Synthetic Peptides ◽

Full Length ◽

Platelet Glycoprotein ◽

Binding Domains ◽

Distinct Mechanism ◽

Fibrinogen Binding ◽

Platelet Binding ◽

The Individual

The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen.

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