Abstract
Flaviviruses are small (50 nm) positive-strand RNA viruses that contain a lipid-bilayer membrane. Forty species of the flavivirus family have been associated with human diseases and most of them are transmitted to their vertebrate hosts by infected mosquitoes or ticks. The flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV), contains a single-stranded RNA genome of approximately 11 kb in size. The virion contains three structural proteins - a 12 kD nucleocapsid or core protein (C), a 8 kD non-glycosylated membrane protein (M), and a 53 kD glycosylated envelope protein (E), as well as seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The E protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. Structural elements of the E protein are also involved in viral attachment, fusion and penetration. The domain III of the dengue virus envelope protein (DENV-ED3) is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses. In order to provide the structural basis for immunologic protection and for vaccine design effective against DENV, here we report the 1H, 15N and 13C resonance assignments of the 109 residue DENV-ED3. We also determined the solution structure of the domain III of the JEV E protein. In addition, we have identified the neutralizing epitopes of the JEV domain III to its monoclonal antibody (mAb E3.3) by comparing NMR chemical shifts of the free (14 kD) and the antibody-bound (178 kD) forms. Our results provide a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses. Finally, virus infects the host cells through binding to the cellular receptors of proteins, carbohydrates or lipids, often in complex cell surface matrix structures. It has been reported that flaviviruses had evolved to use GAGs as part of their strategy to invade host cells. Thus, the heparin binding activity of JEV may play an important role in its function on cellular surfaces. In the present study, we create mutants by substituting basic residues (K279, K286, R288, K290) with alanine in the putative heparin-binding regions to identify which of the basic residue contribute to the heparin binding of E279 to E297. In addition, the structural information will be used for the rational design of vaccines effective against flaviviruses and for the development of glycosaminoglycan-derived inhibitors against them.