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

2008 ◽  
Vol 89 (12) ◽  
pp. 3063-3072 ◽  
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.

scholarly journals Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

2009 ◽  
Vol 90 (12) ◽  
pp. 2912-2922 ◽  
Author(s):  
Roy A. Hall ◽  
Si En Tan ◽  
Barbara Selisko ◽  
Rachael Slade ◽  
Jody Hobson-Peters ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Structural Model ◽  
Full Length ◽  
Physical Interaction ◽  
Rdrp Activity ◽  
The West ◽  
West Nile

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.

scholarly journals A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice

2010 ◽  
Vol 7 (1) ◽  
Author(s):  
Gunther Spohn ◽  
Gary T Jennings ◽  
Byron EE Martina ◽  
Iris Keller ◽  
Markus Beck ◽  
...  
Keyword(s):  
West Nile Virus ◽  
The West ◽  
West Nile ◽  
E Protein ◽  
Lethal Infection ◽  
Domain Iii

scholarly journals A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions

2018 ◽  
Vol 4 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Leslie Goo ◽  
Kari Debbink ◽  
Nurgun Kose ◽  
Gopal Sapparapu ◽  
Michael P. Doyle ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
West Nile Virus ◽  
Human Monoclonal Antibody ◽  
The West ◽  
West Nile ◽  
E Protein ◽  
Antibody Targeting

scholarly journals N-Linked Glycosylation of West Nile Virus Envelope Proteins Influences Particle Assembly and Infectivity

2005 ◽  
Vol 79 (21) ◽  
pp. 13262-13274 ◽  
Author(s):  
Sheri L. Hanna ◽  
Theodore C. Pierson ◽  
Melissa D. Sanchez ◽  
Asim A. Ahmed ◽  
Mariam M. Murtadha ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Envelope Proteins ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
E Proteins ◽  
Virus Envelope ◽  
West Nile ◽  
Particle Assembly ◽  
E Protein ◽  
The Impact

ABSTRACT West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.

Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

2014 ◽  
Vol 56 (5) ◽  
pp. 459-469 ◽  
Author(s):  
Indulis Cielens ◽  
Ludmila Jackevica ◽  
Arnis Strods ◽  
Andris Kazaks ◽  
Velta Ose ◽  
...  
Keyword(s):  
West Nile Virus ◽  
The West ◽  
West Nile ◽  
E Protein ◽  
Domain Iii ◽  
Rna Phage

scholarly journals Vaccination of Mice Using the West Nile Virus E-Protein in a DNA Prime-Protein Boost Strategy Stimulates Cell-Mediated Immunity and Protects Mice against a Lethal Challenge

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e87837 ◽  
Author(s):  
Marina De Filette ◽  
Silke Soehle ◽  
Sebastian Ulbert ◽  
Justin Richner ◽  
Michael S. Diamond ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cell Mediated Immunity ◽  
The West ◽  
West Nile ◽  
Lethal Challenge ◽  
E Protein ◽  
Protein Boost

scholarly journals Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence

2012 ◽  
Vol 93 (9) ◽  
pp. 1965-1975 ◽  
Author(s):  
Y. X. Setoh ◽  
N. A. Prow ◽  
J. Hobson-Peters ◽  
M. Lobigs ◽  
P. R. Young ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Viral Particle ◽  
Cell Surface Expression ◽  
Antigenic Structure ◽  
West Nile ◽  
E Protein

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNVNY99) and a weakly virulent Australian subtype (WNVKUN). Five amino acid differences occur in WNVNY99 compared with WNVKUN (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNVNY99 prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNVKUN prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNVKUN prM to the same level as that of WNVNY99, and enhanced the secretion of WNVKUN prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNVKUN infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

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