Engineering Dengue virus NS3 Protease for structural studies

Abstract

Dengue virus (DENV) NS3 protease (NS3pro) is essential for DENV replication. The atomic structure of DENV 2 WT NS3pro in an open conformation has been solved. Obtaining the atomic structure of DENV NS3pro in a closed conformation would be valuable for gaining insight into specific active site residues and for developing anti-viral inhibitors targeting NS3pro. In this study, the roles of a hydrophobic, four amino acid turn (HT29-32) which protrudes from the protein surface was explored with regard to the protein¿s biophysical characteristics, ability to crystallize and ability to bind lipid for all four dengue virus serotypes. Compared to the wild-type (WT) NS3pro, HT29-32 mutants either retain or change structural and functional characteristics depending on the virus serotype. Crystallization experiments for DENV 2 HT29-32 mutants yielded no crystals, suggesting that the turn is critical for crystal formation. In addition, the mutations reduced the protein¿s ability to associate with lipid.