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

Nanyang Technological University, Tumasik, 00, Singapore
Journal of General Virology (Impact Factor: 3.18). 03/2005; 86(Pt 2):405-12. DOI: 10.1099/vir.0.80411-0
Source: PubMed

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 beta-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.

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Available from: Julien Lescar, Sep 26, 2015
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    • "Moreover, DIII is often recovered in the insoluble inclusion bodies in bacterial cultures, thus requiring a cumbersome solubilization and refolding process to yield DIII proteins that resemble their native conformation [29]. The high level of endotoxins in E. coli-based expression system also raises biosafety concerns and demands an expensive process of purification and validation for their removal to ensure the safety of the final product [10]. Here, we demonstrated that a transient plant expression system provided a rapid production of WNV DIII in N. benthamiana plants. "
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    ABSTRACT: We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μ g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μ g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.
    04/2014; 2014(4):952865. DOI:10.1155/2014/952865
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    • "[18], [19]. The EWNV glycoprotein is composed of three domains (DI, DII and DIII) that are connected by flexible hinge regions [20]; domain III contains the receptor-binding region [21] and most of the type-specific and potentially neutralizing B-cell epitopes [22], [23]. Domain III by itself has been shown to be sufficient to induce a protective immune response [6], [9], [11], [14]. "
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    ABSTRACT: West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.
    PLoS ONE 03/2014; 9(3):e91766. DOI:10.1371/journal.pone.0091766 · 3.23 Impact Factor
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    • "DIII is an immunoglobulin-like structure connected to DI by a single linker peptide and is postulated to have host-cell receptor binding properties. Antibodies with high neutralizing activity have been mapped to DIII epitopes and soluble DIII has been used to block infection of cells with whole virus, both suggesting that DIII contains receptor–ligand epitopes (Abd-Jamil et al., 2008; Chin et al., 2007; Chu et al., 2005; Crill and Roehrig, 2001; Huerta et al., 2008; Roehrig et al., 1998). The DENV2 and TBEV E protein structures only differ noticeably at surface exposed loops. "
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