title: Investigation of Dengue Fever Virus Envelope Glycoprotein Carbohydrate-Ligand Recognition Events Essential for Mammalian Cell Infection
Creators: name: McAtamney, Sarah
Publisher: name: Griffith University
Year: 2009
Subjects: subject: Dengue Fever
subject: Dengue Fever virus
subject: DENV
subject: mosquito-borne flavivirus
subject: carbohydrate rocognition domain
subject: CRD
subject: mammalian cell surface glycans
subject: surface glycans
subject: glycolipid
subject: Paragloboside
subject: DENV-2 EGP DIII
subject: DENV-2
subject: mammalian cell infection

description: Dengue Fever virus (DENV) is a very old mosquito-borne flavivirus that has made a modern worldwide re-emergence as a result of population movement and growth, urbanisation and lapse of vector control. The World Health Organisation estimates that 2.5 billion people, or two-fifths of the world’s population are at risk from DENV, which can cause serious illness and in its severe forms, death. Despite the humanitarian and economic burden that DENV and its flaviviral relatives create, there are no chemotherapeutic drugs available and vaccine development is challenging. Mammalian host cell infection by DENV is mediated by the Envelope glycoprotein (EGP), which covers the entire exposed surface of the mature virus particle and is comprised of three exposed protein domains (DI, DII and DIII) and a transmembrane anchor. While significant effort has been invested to better understand how DIII of EGP participates in receptor mediated endocytosis of DENV into host cells, including the site and structure of the receptor binding site, or carbohydrate recognition domain (CRD), and the structure of ligands involved remain undefined. A recent study of mammalian cell surface glycans involved in DENV infection by Dr Kazuya Hidari and co-workers identified DENV inhibition by the glycolipid Paragloboside, which includes the tetrasaccharide Lacto-N-neotetraose (nLc4)1. This thesis reports an investigation of DENV-2 EGP DIII ligand specificity and characterisation of the DIII CRD involved in mammalian cell infection. To achieve this, soluble and high level expression of DENV-2 ThNH-7/93 EGP DIII was established from Pichia pastoris (P. pastoris) yeast and the recombinant DIII was successfully purified to near homogeneity by single step affinity chromatography. The biological activity of DIII was assessed by DENV permissible cell based assays and the recombinant protein was shown to have retained its wildtype host cell receptor binding activity. Recombinant DIII protein was utilised to successfully establish glycan microarray and saturation transfer difference nuclear magnetic resonance (STD NMR) methodologies, Confidential – not to be copied ii which are useful in the study of EGP ligand specificity. Investigation of nLc4 ligand binding to EGP confirmed that this tetrasaccharide binds to the CRD DIII, involving each of its carbohydrate moieties. Epitope mapping by STD NMR spectroscopy also revealed that the H-1 proton of the N-acetyl-D-glucosamine (GlcNAc) makes closest contact with DIII via its N-acetyl group. Screening of carbohydrate libraries with DENV-2 and a multivalent DIII complex identified additional EGP specificity to several novel binding ligands that share a GlcNAc moiety at the first or second non-reducing cytoplasmexposed positions.