title: Prediction and biochemical analysis of putative cleavage sites of the 3C-like protease of Middle East respiratory syndrome coronavirus.
abstract: Coronavirus 3C-like protease (3CLpro) is responsible for the cleavage of coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature non-structural proteins (nsps) of nsp4-16. The nsp5 of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as 3CLpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp1a/1ab awaits further analysis. Here, we proposed a method for predicting coronaviral 3CLpro cleavage sites which balances the prediction accuracy and false positive outcomes. By applying this method to MERS-CoV, the 11 canonical cleavage sites were readily identified and verified by the biochemical assays. The Michaelis constant of the canonical cleavage sites of MERS-CoV showed that the substrate specificity of MERS-CoV 3CLpro is relatively conserved. Interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by MERS-CoV nsp5. These results pave the way for identification and functional characterization of new nsp products of coronaviruses.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Andong
authors.initials: A
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Cong
authors.initials: C
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Xingyu
authors.initials: X
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Shan
authors.initials: S
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Ceyang
authors.initials: C
authors.lastname: Su
authors.affiliation: School of Basic Medical Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China. Electronic address: chenyu@whu.edu.cn.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, PR China; School of Basic Medical Sciences, Wuhan University, Wuhan, PR China. Electronic address: dguo@whu.edu.cn.
authors.forename: Deyin
authors.initials: D
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D000086782:Coronavirus 3C Proteases; D018352:Coronavirus Infections / Q000821:virology*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases
keywords: 3C-like protease; Canonical cleavage sites; MERS-CoV; Michaelis constants; Non-canonical cleavage sites