Dengue virus type 2 envelope protein displayed as recombinant phage attachment protein reveals potential cell binding sites.
A method to map the specific site on dengue virus envelope protein (E) that interacts with cells and a neutralizing antibody is developed using serially truncated dengue virus type 2 (DENV-2) E displayed on M13 phages as recombinant E-g3p fusion proteins. Recombinant phages displaying the truncated E consisting of amino acids 297-423 (EB2) and amino acids 379-423 (EB4) were neutralized by DENV-2 patient sera and the DENV-2 E-specific 3H5-1 monoclonal antibodies suggesting that the phages retained the DENV-2 E antigenic properties. The EB4 followed by EB2 recombinant phages bound the most to human monocytes (THP-1), African green monkey kidney (Vero) cells, mosquito (C6/36) cells, ScFv specific against E and C6/36 cell proteins. Two potential cell attachment sites were mapped to loop I (amino acids 297 to 312) and loop II (amino acids 379-385) of the DENV-2 E using the phage-displayed truncated DENV-2 E fragments and by the analysis of the E structure. Loop II was present only in EB4 recombinant phages. There was no competition for binding to C6/36 cell proteins between EB2 and EB4 phages. Loop I and loop II are similar to the sub-complex specific and type-specific neutralizing monoclonal antibody binding sites, respectively. Hence, it is proposed that binding and entry of DENV involves the interaction of loop I to cell surface glycosaminoglycan-motif and a subsequent highly specific interaction involving loop II with other cell proteins. The phage displayed truncated DENV-2 E is a powerful and useful method for the direct determination of DENV-2 E cell binding sites.
Abd-Jamil
Juraina
J
Department of Medical Microbiology, Faculty of Medicine, University Malaya, 50603 Kuala Lumpur, Malaysia.
Cheah
Chen-Yee
CY
AbuBakar
Sazaly
S
D000818:Animals; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002477:Cells / Q000276:immunology / Q000378:metabolism*; D009033:Culicidae / Q000276:immunology / Q000378:metabolism; D003716:Dengue Virus / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D053585:Virus Attachment
C051821:E-glycoprotein, Dengue virus type 2; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins