Publication: Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus
2
Issued Date
2012-04-03
Resource Type
ISSN
15204995
00062960
00062960
Other identifier(s)
2-s2.0-84859388866
Rights
Mahidol University
Rights Holder(s)
SCOPUS
Bibliographic Citation
Biochemistry. Vol.51, No.13 (2012), 2840-2851
Suggested Citation
Opas Choksupmanee, Kenneth Hodge, Gerd Katzenmeier, Sarin Chimnaronk Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus. Biochemistry. Vol.51, No.13 (2012), 2840-2851. doi:10.1021/bi2018267 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/13763
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Title
Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus
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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.