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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/13763
Title: Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus
Authors: Opas Choksupmanee
Kenneth Hodge
Gerd Katzenmeier
Sarin Chimnaronk
Mahidol University
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 3-Apr-2012
Citation: Biochemistry. Vol.51, No.13 (2012), 2840-2851
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. © 2012 American Chemical Society.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84859388866&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/13763
ISSN: 15204995
00062960
Appears in Collections:Scopus 2011-2015

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