Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein

Abstract

The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of b-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.

Immunization of Flavivirus West Nile Recombinant Envelope Domain III Protein Induced Specific Immune Response and Protection against West Nile Virus Infection

“…Crystallography data on the ectodomain of the flavivirus E protein revealed three distinct domains (DI, DII, and DIII) (3). The high antagonistic effect of recombinant WNV E DIII protein in blocking WNV infection in both mammalian and mosquito cells strongly suggested that WNV E DIII protein functions as the receptor-binding domain (4) and is responsible for the recognition and attachment to the cellular receptor (5)(6)(7). Importantly, a majority of the neutralizing epitopes have been mapped to the domain III region of the flavivirus E protein (8 -11).…”

“…The soluble WNV E DIII protein was further purified and concentrated as previously described in Ref. 4. Cleavage of the thioredoxin tag was conducted using enterokinase (Invitrogen Life Technologies) following the manufacturer's recommendations.…”

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

“…DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2,(6)(7)(8)(9)(10)(11)(12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6,9,13).…”

Autochthonous case of dengue in France, October 2013

“…The DIII has been shown with DENV to be involved in virus attachment to Vero cells in culture (Crill and Roehrig, 2001). These binding characteristics have been confirmed using expressed DIII (Chu et al, 2005). The DI contains the E glycoprotein molecular hinge.…”