title: Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate.
abstract: An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.
authors.affiliation: Division of Virology, Defense Research and Development Establishment, Jhansi Road, Gwalior 474002, India. p.pat@rediffmail.com
authors.forename: Priyabrata
authors.initials: P
authors.lastname: Pattnaik
authors.forename: J Pradeep
authors.initials: JP
authors.lastname: Babu
authors.forename: Shailendra Kumar
authors.initials: SK
authors.lastname: Verma
authors.forename: Vijay
authors.initials: V
authors.lastname: Tak
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003716:Dengue Virus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D006497:Heparan Sulfate; D008958:Models, Molecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D006497:Heparan Sulfate; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins