Denque Virus Receptor Interactions

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DESCRIPTION (provided by applicant): The resurgence of dengue virus infection is a major public health threat to the tropical developing world; there is no effective treatment or vaccine. The primary hypothesis underlying our research program is that interactions between dengue virus envelope protein and receptors on target cells represents a pivotal mechanism of pathogenicity, and that understanding this interaction will enable us to develop effective therapeutic strategies based on inhibition of virus-target cell binding. The project includes three complementary aims; preliminary data are included to document the feasibility of all 3 aims. A distinctive heparan sulfate proteoglycan is a conserved dengue virus receptor expressed by cells derived from multiple organs and species. The glycosaminoglycan component of the proteoglycan embodies the virus envelope protein binding activity. The primary goal of Aim 1 is to fully characterize this glycosaminoglycan, using envelope protein affinity chromatography, followed by depolymerization and oligosaccharide sequencing. We will also attempt to identify the proteoglycan core protein. The dengue virus envelope protein accounts for cell-binding activity. The goal of Aim 2 is to identify the specific envelope protein region and amino acids responsible for cell-binding. We will use site-directed mutagenesis to generate mutant envelope proteins, and identify mutations that result in loss of binding. To confirm the biological significance of mutations, recombinant viruses incorporating mutations will be generated, and assessed for loss of cell binding. To confirm (initial data) that envelope proteins from clinical isolates bind the same receptor as laboratory dengue virus strains, we will also compare the binding profiles of envelope proteins from non-passaged dengue virus clinical isolates with laboratory-virus derived envelope proteins. Soluble glycosaminoglycans, and the polysulfonate Suramin prevent dengue virus envelope protein binding to target cells, and prevent infection; chemically persulfated glycosaminoglycans are particularly potent inhibitors. The goal of Aim 3 is to synthesize sulfated/sialylated molecules, based on these molecules, and to test their activity in inhibiting envelope protein binding and dengue virus infectivity.