title: Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus.
abstract: Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing the single-chain polyprotein precursor into 10 functional proteins. This vital process relies on the two-component virus-encoded protease complex; nonstructural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated or studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane and purified with the use of fos-choline-14 detergent. The detergent-solubilized protease complex retained autolytic activity and, intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further conducted a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage and provide evidence of the importance of the two distal β-hairpins in the activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon, Thailand 73170.
authors.forename: Opas
authors.initials: O
authors.lastname: Choksupmanee
authors.forename: Kenneth
authors.initials: K
authors.lastname: Hodge
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
authors.forename: Sarin
authors.initials: S
authors.lastname: Chimnaronk
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D017931:DNA Primers; D003716:Dengue Virus / Q000201:enzymology*; D006868:Hydrolysis; D016296:Mutagenesis; D010447:Peptide Hydrolases / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D010447:Peptide Hydrolases