Article

Chu, J.J. et al. Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein. J. Gen. Virol. 86, 405-412

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.

Download full-text

Full-text

Available from: Julien Lescar
    • "surface receptors (Chen et al., 1996; Chu et al., 2005; Crill & Roehrig, 2001) and with potent neutralizing antibodies (Matsui et al., 2010; Sukupolvi-Petty et al., 2007; Thullier et al., 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Based on the hypothesis that interactions between virions and serum components may influence the outcome of Dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from protein E of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and alpha 2-macroglobulin (α2M) as novel viral interactors. After confirming the specific binding of both SAP and α2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species is actually the receptor-activated form of α2M (α2M*), that α2M* can bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4 > DENV1 ~ DENV2 > DENV3, and that this interaction exhibits a strong avidity effect when multivalent binding is favored (KD 8 x10-8 M for DIIIE2). We also show that α2M* binds to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibits an ED50 of 2.9 x10-8 M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factors interaction network during human DENV infection.
    No preview · Article · Aug 2014 · Journal of General Virology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 μg 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 μg 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.
    Full-text · Article · Apr 2014 · BioMed Research International
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Apr 2014
Show more