Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein.
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.
Chu
J J H
JJH
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
Rajamanonmani
R
R
School of Biological Sciences, Nanyang Technological University, 1, Nanyang Walk, Singapore 637616, Singapore.
Li
J
J
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
Bhuvanakantham
R
R
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
Lescar
J
J
School of Biological Sciences, Nanyang Technological University, 1, Nanyang Walk, Singapore 637616, Singapore.
Ng
M-L
ML
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D009033:Culicidae; D003716:Dengue Virus / Q000187:drug effects; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D012995:Solubility; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects*; D014902:West Nile virus / Q000187:drug effects* / Q000276:immunology
D000956:Antigens, Viral; D007106:Immune Sera; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C065696:glycoprotein E, Flavivirus