Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses
Identifieur interne : 001373 ( Pmc/Corpus ); précédent : 001372; suivant : 001374Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses
Auteurs : Chi-Pang Chuck ; Hak-Fun Chow ; David Chi-Cheong Wan ; Kam-Bo WongSource :
- PLoS ONE [ 1932-6203 ] ; 2011.
Abstract
Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CLpro), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection.
Here, we profiled the substrate specificities of 3CLpro from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CLpro prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CLpro from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CLpro prefers P4-Pro and SARS-CoV 3CLpro prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CLpro with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CLpro with relative activity of 4.3.
The comprehensive substrate specificities of 3CLpro from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.
Url:
DOI: 10.1371/journal.pone.0027228
PubMed: 22073294
PubMed Central: 3206940
Links to Exploration step
PMC:3206940Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses</title><author><name sortKey="Chuck, Chi Pang" sort="Chuck, Chi Pang" uniqKey="Chuck C" first="Chi-Pang" last="Chuck">Chi-Pang Chuck</name><affiliation><nlm:aff id="aff1"><addr-line>School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chow, Hak Fun" sort="Chow, Hak Fun" uniqKey="Chow H" first="Hak-Fun" last="Chow">Hak-Fun Chow</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wan, David Chi Cheong" sort="Wan, David Chi Cheong" uniqKey="Wan D" first="David Chi-Cheong" last="Wan">David Chi-Cheong Wan</name><affiliation><nlm:aff id="aff3"><addr-line>School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wong, Kam Bo" sort="Wong, Kam Bo" uniqKey="Wong K" first="Kam-Bo" last="Wong">Kam-Bo Wong</name><affiliation><nlm:aff id="aff1"><addr-line>School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22073294</idno><idno type="pmc">3206940</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206940</idno><idno type="RBID">PMC:3206940</idno><idno type="doi">10.1371/journal.pone.0027228</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">001373</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001373</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses</title><author><name sortKey="Chuck, Chi Pang" sort="Chuck, Chi Pang" uniqKey="Chuck C" first="Chi-Pang" last="Chuck">Chi-Pang Chuck</name><affiliation><nlm:aff id="aff1"><addr-line>School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chow, Hak Fun" sort="Chow, Hak Fun" uniqKey="Chow H" first="Hak-Fun" last="Chow">Hak-Fun Chow</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wan, David Chi Cheong" sort="Wan, David Chi Cheong" uniqKey="Wan D" first="David Chi-Cheong" last="Wan">David Chi-Cheong Wan</name><affiliation><nlm:aff id="aff3"><addr-line>School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wong, Kam Bo" sort="Wong, Kam Bo" uniqKey="Wong K" first="Kam-Bo" last="Wong">Kam-Bo Wong</name><affiliation><nlm:aff id="aff1"><addr-line>School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL<sup>pro</sup>), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection.</p></sec><sec><title>Methodology/Principal Findings</title><p>Here, we profiled the substrate specificities of 3CL<sup>pro</sup> from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL<sup>pro</sup> prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL<sup>pro</sup> from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL<sup>pro</sup> prefers P4-Pro and SARS-CoV 3CL<sup>pro</sup> prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CL<sup>pro</sup> with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CL<sup>pro</sup> with relative activity of 4.3.</p></sec><sec><title>Conclusions/Significance</title><p>The comprehensive substrate specificities of 3CL<sup>pro</sup> from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Van Der Hoek, L" uniqKey="Van Der Hoek L">L van der Hoek</name></author><author><name sortKey="Pyrc, K" uniqKey="Pyrc K">K Pyrc</name></author><author><name sortKey="Jebbink, Mf" uniqKey="Jebbink M">MF Jebbink</name></author><author><name sortKey="Vermeulen Oost, W" uniqKey="Vermeulen Oost W">W Vermeulen-Oost</name></author><author><name sortKey="Berkhout, Rj" uniqKey="Berkhout R">RJ Berkhout</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hamre, D" uniqKey="Hamre D">D Hamre</name></author><author><name sortKey="Procknow, Jj" uniqKey="Procknow J">JJ Procknow</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Chu, Cm" uniqKey="Chu C">CM Chu</name></author><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author><author><name sortKey="Tsoi, Hw" uniqKey="Tsoi H">HW Tsoi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuiken, T" uniqKey="Kuiken T">T Kuiken</name></author><author><name sortKey="Fouchier, Ra" uniqKey="Fouchier R">RA Fouchier</name></author><author><name sortKey="Schutten, M" uniqKey="Schutten M">M Schutten</name></author><author><name sortKey="Rimmelzwaan, Gf" uniqKey="Rimmelzwaan G">GF Rimmelzwaan</name></author><author><name sortKey="Van Amerongen, G" uniqKey="Van Amerongen G">G van Amerongen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peiris, Js" uniqKey="Peiris J">JS Peiris</name></author><author><name sortKey="Lai, St" uniqKey="Lai S">ST Lai</name></author><author><name sortKey="Poon, Ll" uniqKey="Poon L">LL Poon</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Yam, Ly" uniqKey="Yam L">LY Yam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cheever, Fs" uniqKey="Cheever F">FS Cheever</name></author><author><name sortKey="Daniels, Jb" uniqKey="Daniels J">JB Daniels</name></author><author><name sortKey="Pappenheimer, Am" uniqKey="Pappenheimer A">AM Pappenheimer</name></author><author><name sortKey="Bailey, Ot" uniqKey="Bailey O">OT Bailey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Binn, Ln" uniqKey="Binn L">LN Binn</name></author><author><name sortKey="Lazar, Ec" uniqKey="Lazar E">EC Lazar</name></author><author><name sortKey="Keenan, Kp" uniqKey="Keenan K">KP Keenan</name></author><author><name sortKey="Huxsoll, Dl" uniqKey="Huxsoll D">DL Huxsoll</name></author><author><name sortKey="Marchwicki, Rh" uniqKey="Marchwicki R">RH Marchwicki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Poon, Ll" uniqKey="Poon L">LL Poon</name></author><author><name sortKey="Chu, Dk" uniqKey="Chu D">DK Chu</name></author><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author><author><name sortKey="Wong, Ok" uniqKey="Wong O">OK Wong</name></author><author><name sortKey="Ellis, Tm" uniqKey="Ellis T">TM Ellis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Beaudette, Fr" uniqKey="Beaudette F">FR Beaudette</name></author><author><name sortKey="Hudson, Cb" uniqKey="Hudson C">CB Hudson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Doyle, Lp" uniqKey="Doyle L">LP Doyle</name></author><author><name sortKey="Hutchings, Lm" uniqKey="Hutchings L">LM Hutchings</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tyrrell, Da" uniqKey="Tyrrell D">DA Tyrrell</name></author><author><name sortKey="Bynoe, Ml" uniqKey="Bynoe M">ML Bynoe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y Huang</name></author><author><name sortKey="Yuen, Ky" uniqKey="Yuen K">KY Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snijder, Ej" uniqKey="Snijder E">EJ Snijder</name></author><author><name sortKey="Bredenbeek, Pj" uniqKey="Bredenbeek P">PJ Bredenbeek</name></author><author><name sortKey="Dobbe, Jc" uniqKey="Dobbe J">JC Dobbe</name></author><author><name sortKey="Thiel, V" uniqKey="Thiel V">V Thiel</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J Ziebuhr</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorbalenya, Ae" uniqKey="Gorbalenya A">AE Gorbalenya</name></author><author><name sortKey="Snijder, Ej" uniqKey="Snijder E">EJ Snijder</name></author><author><name sortKey="Spaan, Wj" uniqKey="Spaan W">WJ Spaan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gaunt, Er" uniqKey="Gaunt E">ER Gaunt</name></author><author><name sortKey="Hardie, A" uniqKey="Hardie A">A Hardie</name></author><author><name sortKey="Claas, Ec" uniqKey="Claas E">EC Claas</name></author><author><name sortKey="Simmonds, P" uniqKey="Simmonds P">P Simmonds</name></author><author><name sortKey="Templeton, Ke" uniqKey="Templeton K">KE Templeton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dominguez, Sr" uniqKey="Dominguez S">SR Dominguez</name></author><author><name sortKey="Robinson, Cc" uniqKey="Robinson C">CC Robinson</name></author><author><name sortKey="Holmes, Kv" uniqKey="Holmes K">KV Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cavanagh, D" uniqKey="Cavanagh D">D Cavanagh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ignjatovic, J" uniqKey="Ignjatovic J">J Ignjatovic</name></author><author><name sortKey="Sapats, S" uniqKey="Sapats S">S Sapats</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, Cw" uniqKey="Lin C">CW Lin</name></author><author><name sortKey="Tsai, Ch" uniqKey="Tsai C">CH Tsai</name></author><author><name sortKey="Tsai, Fj" uniqKey="Tsai F">FJ Tsai</name></author><author><name sortKey="Chen, Pj" uniqKey="Chen P">PJ Chen</name></author><author><name sortKey="Lai, Cc" uniqKey="Lai C">CC Lai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, H" uniqKey="Yang H">H Yang</name></author><author><name sortKey="Xie, W" uniqKey="Xie W">W Xie</name></author><author><name sortKey="Xue, X" uniqKey="Xue X">X Xue</name></author><author><name sortKey="Yang, K" uniqKey="Yang K">K Yang</name></author><author><name sortKey="Ma, J" uniqKey="Ma J">J Ma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, Sy" uniqKey="Li S">SY Li</name></author><author><name sortKey="Chen, C" uniqKey="Chen C">C Chen</name></author><author><name sortKey="Zhang, Hq" uniqKey="Zhang H">HQ Zhang</name></author><author><name sortKey="Guo, Hy" uniqKey="Guo H">HY Guo</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, Cy" uniqKey="Wu C">CY Wu</name></author><author><name sortKey="Jan, Jt" uniqKey="Jan J">JT Jan</name></author><author><name sortKey="Ma, Sh" uniqKey="Ma S">SH Ma</name></author><author><name sortKey="Kuo, Cj" uniqKey="Kuo C">CJ Kuo</name></author><author><name sortKey="Juan, Hf" uniqKey="Juan H">HF Juan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, L" uniqKey="Chen L">L Chen</name></author><author><name sortKey="Gui, C" uniqKey="Gui C">C Gui</name></author><author><name sortKey="Luo, X" uniqKey="Luo X">X Luo</name></author><author><name sortKey="Yang, Q" uniqKey="Yang Q">Q Yang</name></author><author><name sortKey="Gunther, S" uniqKey="Gunther S">S Gunther</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, K" uniqKey="Fan K">K Fan</name></author><author><name sortKey="Ma, L" uniqKey="Ma L">L Ma</name></author><author><name sortKey="Han, X" uniqKey="Han X">X Han</name></author><author><name sortKey="Liang, H" uniqKey="Liang H">H Liang</name></author><author><name sortKey="Wei, P" uniqKey="Wei P">P Wei</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goetz, Dh" uniqKey="Goetz D">DH Goetz</name></author><author><name sortKey="Choe, Y" uniqKey="Choe Y">Y Choe</name></author><author><name sortKey="Hansell, E" uniqKey="Hansell E">E Hansell</name></author><author><name sortKey="Chen, Yt" uniqKey="Chen Y">YT Chen</name></author><author><name sortKey="Mcdowell, M" uniqKey="Mcdowell M">M McDowell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chuck, Cp" uniqKey="Chuck C">CP Chuck</name></author><author><name sortKey="Chong, Lt" uniqKey="Chong L">LT Chong</name></author><author><name sortKey="Chen, C" uniqKey="Chen C">C Chen</name></author><author><name sortKey="Chow, Hf" uniqKey="Chow H">HF Chow</name></author><author><name sortKey="Wan, Dc" uniqKey="Wan D">DC Wan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hegyi, A" uniqKey="Hegyi A">A Hegyi</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J Ziebuhr</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, S" uniqKey="Lee S">S Lee</name></author><author><name sortKey="Tikhomirova, A" uniqKey="Tikhomirova A">A Tikhomirova</name></author><author><name sortKey="Shalvardjian, N" uniqKey="Shalvardjian N">N Shalvardjian</name></author><author><name sortKey="Chalikian, Tv" uniqKey="Chalikian T">TV Chalikian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kyte, J" uniqKey="Kyte J">J Kyte</name></author><author><name sortKey="Doolittle, Rf" uniqKey="Doolittle R">RF Doolittle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chou, Py" uniqKey="Chou P">PY Chou</name></author><author><name sortKey="Fasman, Gd" uniqKey="Fasman G">GD Fasman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xue, X" uniqKey="Xue X">X Xue</name></author><author><name sortKey="Yu, H" uniqKey="Yu H">H Yu</name></author><author><name sortKey="Yang, H" uniqKey="Yang H">H Yang</name></author><author><name sortKey="Xue, F" uniqKey="Xue F">F Xue</name></author><author><name sortKey="Wu, Z" uniqKey="Wu Z">Z Wu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Anand, K" uniqKey="Anand K">K Anand</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J Ziebuhr</name></author><author><name sortKey="Wadhwani, P" uniqKey="Wadhwani P">P Wadhwani</name></author><author><name sortKey="Mesters, Jr" uniqKey="Mesters J">JR Mesters</name></author><author><name sortKey="Hilgenfeld, R" uniqKey="Hilgenfeld R">R Hilgenfeld</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, Q" uniqKey="Zhao Q">Q Zhao</name></author><author><name sortKey="Li, S" uniqKey="Li S">S Li</name></author><author><name sortKey="Xue, F" uniqKey="Xue F">F Xue</name></author><author><name sortKey="Zou, Y" uniqKey="Zou Y">Y Zou</name></author><author><name sortKey="Chen, C" uniqKey="Chen C">C Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jenkins, Gm" uniqKey="Jenkins G">GM Jenkins</name></author><author><name sortKey="Rambaut, A" uniqKey="Rambaut A">A Rambaut</name></author><author><name sortKey="Pybus, Og" uniqKey="Pybus O">OG Pybus</name></author><author><name sortKey="Holmes, Ec" uniqKey="Holmes E">EC Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lai, Mm" uniqKey="Lai M">MM Lai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pasternak, Ao" uniqKey="Pasternak A">AO Pasternak</name></author><author><name sortKey="Spaan, Wj" uniqKey="Spaan W">WJ Spaan</name></author><author><name sortKey="Snijder, Ej" uniqKey="Snijder E">EJ Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Duffy, S" uniqKey="Duffy S">S Duffy</name></author><author><name sortKey="Shackelton, La" uniqKey="Shackelton L">LA Shackelton</name></author><author><name sortKey="Holmes, Ec" uniqKey="Holmes E">EC Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, L" uniqKey="Zhu L">L Zhu</name></author><author><name sortKey="George, S" uniqKey="George S">S George</name></author><author><name sortKey="Schmidt, Mf" uniqKey="Schmidt M">MF Schmidt</name></author><author><name sortKey="Al Gharabli, Si" uniqKey="Al Gharabli S">SI Al-Gharabli</name></author><author><name sortKey="Rademann, J" uniqKey="Rademann J">J Rademann</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22073294</article-id><article-id pub-id-type="pmc">3206940</article-id><article-id pub-id-type="publisher-id">PONE-D-11-15731</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0027228</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Enzymes</subject><subj-group><subject>Enzyme Kinetics</subject></subj-group></subj-group></subj-group><subj-group><subject>Microbiology</subject><subj-group><subject>Virology</subject><subj-group><subject>Viral Enzymes</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Medicine</subject><subj-group><subject>Infectious Diseases</subject><subj-group><subject>Viral Diseases</subject><subj-group><subject>SARS</subject></subj-group></subj-group></subj-group><subj-group><subject>Pulmonology</subject><subj-group><subject>Respiratory Infections</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses</article-title><alt-title alt-title-type="running-head">Substrate Specificities of 3C-Like Proteases</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chuck</surname><given-names>Chi-Pang</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chow</surname><given-names>Hak-Fun</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wan</surname><given-names>David Chi-Cheong</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wong</surname><given-names>Kam-Bo</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, China</addr-line></aff><aff id="aff3"><label>3</label><addr-line>School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Poon</surname><given-names>Leo L. M.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">University of Hong Kong, Hong Kong</aff><author-notes><corresp id="cor1">* E-mail: <email>kbwong@cuhk.edu.hk</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: C-PC DC-CW K-BW. Performed the experiments: C-PC. Analyzed the data: C-PC K-BW. Contributed reagents/materials/analysis tools: H-FC DC-CW. Wrote the paper: C-PC K-BW.</p></fn></author-notes><pub-date pub-type="collection"><year>2011</year></pub-date><pub-date pub-type="epub"><day>2</day><month>11</month><year>2011</year></pub-date><volume>6</volume><issue>11</issue><elocation-id>e27228</elocation-id><history><date date-type="received"><day>10</day><month>8</month><year>2011</year></date><date date-type="accepted"><day>12</day><month>10</month><year>2011</year></date></history><permissions><copyright-statement>Chuck et al.</copyright-statement><copyright-year>2011</copyright-year><license xlink:href="https://creativecommons.org/publicdomain/zero/1.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.</license-p></license></permissions><abstract><sec><title>Background</title><p>Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL<sup>pro</sup>), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection.</p></sec><sec><title>Methodology/Principal Findings</title><p>Here, we profiled the substrate specificities of 3CL<sup>pro</sup> from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL<sup>pro</sup> prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL<sup>pro</sup> from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL<sup>pro</sup> prefers P4-Pro and SARS-CoV 3CL<sup>pro</sup> prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CL<sup>pro</sup> with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CL<sup>pro</sup> with relative activity of 4.3.</p></sec><sec><title>Conclusions/Significance</title><p>The comprehensive substrate specificities of 3CL<sup>pro</sup> from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.</p></sec></abstract><counts><page-count count="6"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>A number of coronaviruses (CoVs) have been identified as causative agents of respiratory tract and gastroenteritis diseases in mammals and birds <xref rid="pone.0027228-vanderHoek1" ref-type="bibr">[1]</xref>, <xref rid="pone.0027228-Hamre1" ref-type="bibr">[2]</xref>, <xref rid="pone.0027228-Woo1" ref-type="bibr">[3]</xref>, <xref rid="pone.0027228-Kuiken1" ref-type="bibr">[4]</xref>, <xref rid="pone.0027228-Peiris1" ref-type="bibr">[5]</xref>, <xref rid="pone.0027228-Cheever1" ref-type="bibr">[6]</xref>, <xref rid="pone.0027228-Binn1" ref-type="bibr">[7]</xref>, <xref rid="pone.0027228-Poon1" ref-type="bibr">[8]</xref>, <xref rid="pone.0027228-Beaudette1" ref-type="bibr">[9]</xref>, <xref rid="pone.0027228-Doyle1" ref-type="bibr">[10]</xref>, <xref rid="pone.0027228-Tyrrell1" ref-type="bibr">[11]</xref>. Sequence analysis suggests that these coronaviral strains can be classified into three main groups – alphacoronavirus (group 1), betacoronavirus (group 2), and gamacoronavirus (group 3) <xref rid="pone.0027228-Woo2" ref-type="bibr">[12]</xref>. The sequence of severe acute respiratory syndrome coronavirus (SARS-CoV), discovered in 2003, was found to be diverse from any existing groups of CoVs. The group 2 CoVs are then further divided into 2a and 2b sub-groups, with the original group 2 CoVs assigned to group 2a and SARS-CoV to group 2b <xref rid="pone.0027228-Snijder1" ref-type="bibr">[13]</xref>, <xref rid="pone.0027228-Gorbalenya1" ref-type="bibr">[14]</xref>. Most of coronaviral strains are group 1 and 2a members. They include the four human coronaviruses (HCoVs) strains, NL63, 229E, OC43 and HKU1, that associate with up to 5% of total respiratory tract disease cases <xref rid="pone.0027228-Gaunt1" ref-type="bibr">[15]</xref>, <xref rid="pone.0027228-Dominguez1" ref-type="bibr">[16]</xref>. The most infamous strain in group 3 is infectious bronchitis virus (IBV), which can cause lethal infections in birds <xref rid="pone.0027228-Cavanagh1" ref-type="bibr">[17]</xref>, <xref rid="pone.0027228-Ignjatovic1" ref-type="bibr">[18]</xref>.</p><p>3C-like protease (3CL<sup>pro</sup>), which is also named main protease, is responsible for the processing of the viral polyproteins into at least 15 non-structural proteins, most of which are constituents of the viral replication and transcription complex. The cleavage process can be acted in cis and in trans <xref rid="pone.0027228-Lin1" ref-type="bibr">[19]</xref>. This enzyme is a good drug target for anti-coronaviral infection, as inhibiting the autocleavage process can inhibit viral replication and reduce virus-induced cytopathic effects on host cells <xref rid="pone.0027228-Yang1" ref-type="bibr">[20]</xref>, <xref rid="pone.0027228-Li1" ref-type="bibr">[21]</xref>, <xref rid="pone.0027228-Wu1" ref-type="bibr">[22]</xref>, <xref rid="pone.0027228-Chen1" ref-type="bibr">[23]</xref>. A detailed knowledge of substrate specificity of 3CL<sup>pro</sup> is helpful in the rational design of inhibitors. Substrate specificity of SARS-CoV 3CL<sup>pro</sup> was extensively investigated after the outbreak of SARS in 2003. Fan <italic>et al</italic>. measured the protease activity against 34 single-substituted variants at P5 to P1' positions, while Goetz <italic>et al</italic>. profiled the specificity at P4 to P1 positions by using a fully degenerated library of tetrapeptide mixtures <xref rid="pone.0027228-Fan1" ref-type="bibr">[24]</xref>, <xref rid="pone.0027228-Goetz1" ref-type="bibr">[25]</xref>. Chuck <italic>et al</italic>. profiled the substrate preference of SARS-CoV 3CL<sup>pro</sup> by measuring the activity of 3CL<sup>pro</sup> against substrate variants with single substitutions at P5 to P3' positions <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>.</p><p>On the other hand, reports describing the substrate specificities of 3CL<sup>pro</sup> in group 1, 2a, and 3 are scarce. Only the activity of 3CL<sup>pro</sup> from HCoV-229E (group 1), transmissible gastroenteritis coronavirus (group 1) and mouse hepatitis virus (group 2a) against three to four of their own autocleavage sequences have been measured by Hegyi <italic>et al</italic>. <xref rid="pone.0027228-Hegyi1" ref-type="bibr">[27]</xref>. Comprehensive study on substrate specificities of group 1, 2a and 3 3CL<sup>pro</sup> is lacking. Here, we profiled the substrate specificities of selected 3CL<sup>pro</sup> from group 1, 2a, 2b and 3 CoVs. Activities of 3CL<sup>pro</sup> from HCoV-NL63 (group 1), HCoV-OC43 (group 2a), SARS-CoV (group 2b) and IBV (group 3) against a substrate library of 19×8 variants were measured by fluorescence resonance energy transfer (FRET) assay <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. Similarities and differences in substrate specificities among different 3CL<sup>pro</sup> are discussed.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>Profiling substrate specificities of 3CL<sup>pro</sup> from group 1, 2a, 2b, and 3 CoVs</title><p>We have previously created a 19×8 substrate library by performing saturation mutagenesis at P5 to P3' positions on the wild type (WT) sequence (SAVLQ↓SGF), which corresponds to the autocleavage sequence at the N-terminus of SARS-CoV 3CL<sup>pro</sup><xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. The values of k<sub>obs</sub>/[3CL<sup>pro</sup>] of the proteases against this WT sequence were 443±11, 124±13, 180±5 and 174±19 mM<sup>-1</sup> min<sup>-1</sup> for HCoV-NL63 (group 1), HCoV-OC43 (group 2a), SARS-CoV (group 2b), and IBV (group 3), respectively. That all proteases can cleave the WT sequence efficiently justifies that we can use our substrate library to profile the substrate specificities of 3CL<sup>pro</sup> from other groups of CoVs. Based on the FRET assay we developed, we measured the activities of 3CL<sup>pro</sup> from HCoV-NL63, HCoV-OC43, SARS-CoV and IBV against the 19×8 substrate variants (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, <xref ref-type="supplementary-material" rid="pone.0027228.s002">Table S1</xref>) <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. To identify the structural basis of substrate preferences for different CoVs, the protease activities were correlated with side chain volume <xref rid="pone.0027228-Lee1" ref-type="bibr">[28]</xref>, hydrophobicity <xref rid="pone.0027228-Kyte1" ref-type="bibr">[29]</xref>, and α-helix and β-sheet propensities <xref rid="pone.0027228-Chou1" ref-type="bibr">[30]</xref> as described <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. The correlations were quantified in terms of correlation coefficients and p-values (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>, <xref ref-type="supplementary-material" rid="pone.0027228.s003">Table S2</xref>).</p><fig id="pone-0027228-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.g001</object-id><label>Figure 1</label><caption><title>Substrate specificity of 3CL<sup>pro</sup> at P5 to P3' positions.</title><p>Relative protease activity of 3CL<sup>pro</sup> from HCoV-NL63 (circles, group 1), HCoV-OC43 (squares, group 2a), SARS-CoV (diamond, group 2b) and IBV (triples, group 3) against 19×8 of substrate variants were measured by FRET assay. Relative activities that are significantly (p-value<0.001) higher than the rest are represented as filled symbols.</p></caption><graphic xlink:href="pone.0027228.g001"></graphic></fig><fig id="pone-0027228-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.g002</object-id><label>Figure 2</label><caption><title>Correlation between 3CL<sup>pro</sup> activities and structural properties of substituting residues.</title><p>The relative protease activities of 3CL<sup>pro</sup> from HCoV-NL63 (shaded, group 1), HCoV-OC43 (white, group 2a), SARS-CoV (black, group 2b) and IBV (grey, group 3), were correlated with structural properties of substituting residue properties, including side chain volume <xref rid="pone.0027228-Lee1" ref-type="bibr">[28]</xref>, hydrophobicity <xref rid="pone.0027228-Kyte1" ref-type="bibr">[29]</xref> and α-helix and β-sheet propensities <xref rid="pone.0027228-Chou1" ref-type="bibr">[30]</xref>. Correlation coefficients of +/−0.56 and +/−0.44 correspond to p-values of 0.01 and 0.05 respectively.</p></caption><graphic xlink:href="pone.0027228.g002"></graphic></fig></sec><sec id="s2b"><title>Differences in substrate specificities among 3CL<sup>pro</sup></title><p>We then tested if the relative activities of 3CL<sup>pro</sup> from any CoV strains were significantly different from the other by analysis of variance. Substitutions that resulted in significantly higher relative activities (p<0.001) were indicated as filled symbol in <xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>. IBV 3CL<sup>pro</sup> (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, triangles) was the most efficient in cleaving A4P and A4F with relative activities of 1.09±0.24 and 0.58±0.14, respectively, while SARS 3CL<sup>pro</sup> (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, diamonds) preferred A4V with relative activity of 1.39±0.19. HCoV-OC43 3CL<sup>pro</sup> (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, squares) appeared to be the most versatile in accepting substitutions at P1 and P2 positions, and could cleave Q1H, Q1M, L2M and L2C, significantly better than 3CL<sup>pro</sup> from other strains. No significant differences were observed for other substitutions, suggesting that 3CL<sup>pro</sup> from different CoVs shares many similarities in substrate preferences.</p></sec><sec id="s2c"><title>Substrate preferences that are common to all 3CL<sup>pro</sup></title><p>The most preferred P1 residue is Gln (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>), which forms hydrogen-bonds with the side-chain of an invariant His residue and the backbone carbonyl group of an invariant Phe residue (His-163 and Phe-140 in SARS-CoV 3CL<sup>pro</sup>) in the P1 binding pocket. Interestingly, our results showed that 3CL<sup>pro</sup> from all groups of CoVs can cleave His at P1 position reasonably well. The relative activities for 3CL<sup>pro</sup> from HCoV-NL63, HCoV-OC43, SARS-CoV, and IBV were 0.26±0.08, 0.47±0.08, 0.19±0.03 and 0.25±0.12, respectively (<xref ref-type="supplementary-material" rid="pone.0027228.s002">Table S1</xref>). Consistent with this observation, His is found natively at P1 positions in the polyproteins from group 1 and 2a CoVs (<xref ref-type="supplementary-material" rid="pone.0027228.s004">Table S3</xref>). Taken together, the ability to cleave His at P1 position is a conserved property for all 3CL<sup>pro</sup>. Moreover, we showed that all 3CL<sup>pro</sup> can cleave Q1M, albeit at an even lower rate, and all other substitutions resulted in undetected activity.</p><p>The protease activities correlate positively with the hydrophobicity of substituting residues at P2 position (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>). In fact, among the P2 variants, only L2M, L2C, L2F, L2I and L2V were cleavable, suggesting that P2 position favors hydrophobic residues. However, substitution with β-branched residues, Val or Ile, led to >10-folds decreases in the activity (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, <xref ref-type="supplementary-material" rid="pone.0027228.s002">Table S1</xref>). Considering that Leu, Val and Ile share similar hydrophobicity and side chain volume, the large differences in activities suggest that β-branched residues are not preferred in all 3CL<sup>pro</sup>, probably due to steric clashes with the P2 binding pocket. Taken together, P2 position prefers hydrophobic residues without β-branch, and the most preferred residue is Leu.</p><p>At P3 position, the protease activities on Arg/Lys-substituting variants were 5 to 14 fold higher than that on Asp/Glu-substituting variants (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, <xref ref-type="supplementary-material" rid="pone.0027228.s002">Table S1</xref>). This observation suggests that P3 position prefers positively charged residues over negatively charged one. In the active site of 3CL<sup>pro</sup>, there is no substrate-binding pocket for P3 residue. Molecular modeling showed that there is an invariant Glu residue (Glu-166 in SARS-CoV 3CL<sup>pro</sup>) in the active site of 3CL<sup>pro</sup> that may form favorable charge-charge interactions with a positively charged residue at the P3 position, which may explain why Arg/Lys are favored over Asp/Glu at this position (<xref ref-type="supplementary-material" rid="pone.0027228.s001">Figure S1</xref>). Moreover, no cleavage was observed for substrate containing Pro-substitution at P3 position.</p><p>The protease activities correlate negatively with side chain volume, and positively with the hydrophobicity of substituting residues at P4 position (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>). The correlations with hydrophobicity were more evident (with correlation coefficients >0.89) when only small residues (Ala, Asn, Asp, Cys, Gly, Ser, and Thr) with side chain volumes <70 Å<sup>3</sup> (<xref ref-type="fig" rid="pone-0027228-g003">Figure 3</xref>) were included in the analysis. This result suggests that as long as the side chain can fit into the P4 binding pocket, the protease activity is directly proportional to the hydrophobicity of the substituting residues. On the other hand, charged residues like Lys, Arg, His, Asp and Glu were not cleavable, presumably due to the unfavorable burial of charges in the hydrophobic P4 pocket.</p><fig id="pone-0027228-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.g003</object-id><label>Figure 3</label><caption><title>All 3CL<sup>pro</sup> prefer small hydrophobic residues at P4 position.</title><p>All 3CL<sup>pro</sup> activities are highly correlated to hydrophobicity of residues with side chain volumes of <70 Å<sup>3</sup> (filled symbols). The correlation coefficients and the corresponding p-values are indicated.</p></caption><graphic xlink:href="pone.0027228.g003"></graphic></fig><p>In general, the activities of 3CL<sup>pro</sup> correlate positively with the hydrophobicity and β-sheet propensity of substituting residues at P5 position (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>). The correlations are significant (p<0.05) for group 2a, 2b, and 3 CoVs, but are weaker for group 1 CoV. Like the P3 position, there is no substrate-binding pocket for P5 residue. In the crystal structure of SARS-CoV 3CL<sup>pro</sup> in complex with a peptide substrate, the P5 residue adopts an extended β-strand conformation to avoid clashing of P5-P6 residues with the protease <xref rid="pone.0027228-Xue1" ref-type="bibr">[31]</xref>. Residues with high β-sheet propensity may stabilize the extended conformation at P5 and improve enzyme-substrate interaction. As shown in <xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, a number of substitutions at P5 position resulted in a substrate better than the WT sequence (i.e. with relative activity >1). Consistent with the suggestion that P5 position favors residues with high hydrophobicity and β-sheet propensity, Val-substitution consistently yielded substrates with higher than WT activities for all 3CL<sup>pro</sup>. On the other hand, negatively charged residues (Asp/Glu) were not favored at P5 position, with significantly lower activities (0.16 to 0.50).</p><p>At P1' position, the protease activities correlate negatively with side chain volume of substituting residues (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>). In fact, the relative activities for substrates with the smallest residues (Gly, Ala, Ser, and Cys) at P1' position were in the range of 0.64 to 1.40, which were consistently higher than those for other larger residues (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>). At P2' position, all variants, except G2'P, could be cleaved with relative activities of 0.17 to 1.04 (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>). The protease activities also correlate negatively with the side chain volume (<xref ref-type="fig" rid="pone-0027228-g002">Figure 2</xref>), but the difference in the protease activities was relatively small (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>). At P3' position, no obvious substrate preference was observed.</p></sec><sec id="s2d"><title>The effect of combining multiple favorable substitutions</title><p>Our profiling analysis showed that all CoV 3CL<sup>pro</sup> prefer P5-Val and P3-Arg (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>). To test if we can combine two favorable substitutions to create a more active substrate, we have created a doubly-substituted substrate variant ‘<underline>V</underline>A<underline>R</underline>LQ↓SGF’. The protease activities of HCoV-NL63, HCoV-OC43, SARS-CoV and IBV against the doubly-substituted sequence were 1.70±0.07, 1.87±0.17, 1.70±0.12 and 3.24±0.37, respectively (<xref ref-type="table" rid="pone-0027228-t001">Table 1</xref>). The results suggest that the increase in activity is additive, and the sequence ‘<underline>V</underline>A<underline>R</underline>LQ↓SGF’ can represent a good broad-spectrum substrate for all 3CL<sup>pro</sup>.</p><table-wrap id="pone-0027228-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.t001</object-id><label>Table 1</label><caption><title>3CL<sup>pro</sup> activities against doubly- and triply-substituted substrate variants. WT substrate was substituted at P3 to P5 positions to generate doubly- and triply-substituted variants. The relative activities of 3CL<sup>pro</sup> on these substrate variants are reported.</title></caption><alternatives><graphic id="pone-0027228-t001-1" xlink:href="pone.0027228.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Variant sequence</td><td align="left" rowspan="1" colspan="1">HCoV-NL63</td><td align="left" rowspan="1" colspan="1">HCoV-OC43</td><td align="left" rowspan="1" colspan="1">SARS-CoV</td><td align="left" rowspan="1" colspan="1">IBV</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><underline>V</underline>AVLQ↓SGF</td><td align="left" rowspan="1" colspan="1">1.23±0.40</td><td align="left" rowspan="1" colspan="1">1.55±0.30</td><td align="left" rowspan="1" colspan="1">1.80±0.31</td><td align="left" rowspan="1" colspan="1">1.58±0.27</td></tr><tr><td align="left" rowspan="1" colspan="1">SA<underline>R</underline>LQ↓SGF</td><td align="left" rowspan="1" colspan="1">1.14±0.24</td><td align="left" rowspan="1" colspan="1">1.36±0.17</td><td align="left" rowspan="1" colspan="1">0.97±0.12</td><td align="left" rowspan="1" colspan="1">1.72±0.22</td></tr><tr><td align="left" rowspan="1" colspan="1"><underline>V</underline>A<underline>R</underline>LQ↓SGF</td><td align="left" rowspan="1" colspan="1">1.70±0.07</td><td align="left" rowspan="1" colspan="1">1.87±0.17</td><td align="left" rowspan="1" colspan="1">1.70±0.17</td><td align="left" rowspan="1" colspan="1">3.24±0.37</td></tr><tr><td align="left" rowspan="1" colspan="1">S<underline>P</underline>VLQ↓SGF</td><td align="left" rowspan="1" colspan="1">0.06±0.01</td><td align="left" rowspan="1" colspan="1">0.29±0.07</td><td align="left" rowspan="1" colspan="1">0.61±0.10</td><td align="left" rowspan="1" colspan="1">1.09±0.24</td></tr><tr><td align="left" rowspan="1" colspan="1"><underline>VPR</underline>LQ↓SGF</td><td align="left" rowspan="1" colspan="1">0.15±0.04</td><td align="left" rowspan="1" colspan="1">0.91±0.12</td><td align="left" rowspan="1" colspan="1">0.99±0.12</td><td align="left" rowspan="1" colspan="1">4.33±0.98</td></tr><tr><td align="left" rowspan="1" colspan="1">S<underline>V</underline>VLQ↓SGF</td><td align="left" rowspan="1" colspan="1">0.76±0.10</td><td align="left" rowspan="1" colspan="1">0.59±0.07</td><td align="left" rowspan="1" colspan="1">1.39±0.19</td><td align="left" rowspan="1" colspan="1">0.59±0.09</td></tr><tr><td align="left" rowspan="1" colspan="1"><underline>VV</underline>VLQ↓SGF</td><td align="left" rowspan="1" colspan="1">1.23±0.06</td><td align="left" rowspan="1" colspan="1">0.60±0.05</td><td align="left" rowspan="1" colspan="1">1.97±0.19</td><td align="left" rowspan="1" colspan="1">0.86±0.05</td></tr><tr><td align="left" rowspan="1" colspan="1"><underline>VVR</underline>LQ↓SGF</td><td align="left" rowspan="1" colspan="1">1.63±0.07</td><td align="left" rowspan="1" colspan="1">0.55±0.04</td><td align="left" rowspan="1" colspan="1">2.50±0.51</td><td align="left" rowspan="1" colspan="1">2.19±0.13</td></tr></tbody></table></alternatives></table-wrap><p>On the other hand, our profiling analysis suggests that 3CL<sup>pro</sup> from SARS-CoV and IBV have different substrate preferences at P4 position – SARS-CoV prefers P4-Val (relative activity = 1.09±0.24) while IBV prefers P4-Pro (relative activity = 1.39±0.10) (<xref ref-type="fig" rid="pone-0027228-g001">Figure 1</xref>, <xref ref-type="supplementary-material" rid="pone.0027228.s002">Table S1</xref>). To see if we can exploit this distinct substrate preference at P4 position to create a substrate more specific for IBV 3CL<sup>pro</sup>, we have created the triply-substituted variant ‘<underline>VPR</underline>LQ↓SGF’. The protease activity of IBV 3CL<sup>pro</sup> against this sequence was boosted to 4.33±0.98, while that of the other strains were significantly reduced, demonstrating that this substrate sequence can represent a specific substrate-sequence for IBV 3CL<sup>pro</sup> (<xref ref-type="table" rid="pone-0027228-t001">Table 1</xref>). Similarly, the protease activity of SARS-CoV 3CL<sup>pro</sup> against the triply-substituted sequence ‘<underline>VVR</underline>LQ↓SGF’ was boosted to 2.50±0.51, while that of the other strains were reduced (<xref ref-type="table" rid="pone-0027228-t001">Table 1</xref>). Taken together, these results suggest that one can combine the substrate preference profiled in this study to create a better substrate sequences.</p></sec></sec><sec id="s3"><title>Discussion</title><p>This study provides the first comprehensive profiling of substrate specificities of 3CL<sup>pro</sup> from group 1, 2a, and 3 CoVs. We showed that the substrate specificities of these 3CL<sup>pro</sup> share many similarities to those of 3CL<sup>pro</sup> from SARS-CoV (group 2b) reported previously by us <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. <xref ref-type="table" rid="pone-0027228-t002">Table 2</xref> summarizes the substrate specificities that are common to all 3CL<sup>pro</sup>. Although the substrate specificities for 3CL<sup>pro</sup> from different groups of CoVs share a number of similarities, unique substrate preferences were identified in this study. In particular, we showed that only IBV 3CL<sup>pro</sup>, but not other proteases, prefers P4-Pro (<xref ref-type="fig" rid="pone-0027228-g003">Figure 3</xref>).</p><table-wrap id="pone-0027228-t002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.t002</object-id><label>Table 2</label><caption><title>Summary of substrate specificities that are common among all 3CL<sup>pro</sup>.</title></caption><alternatives><graphic id="pone-0027228-t002-2" xlink:href="pone.0027228.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Position</td><td align="left" rowspan="1" colspan="1">Substrate preferences</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">P5</td><td align="left" rowspan="1" colspan="1">No strong preference</td></tr><tr><td align="left" rowspan="1" colspan="1">P4</td><td align="left" rowspan="1" colspan="1">Small hydrophobic residues</td></tr><tr><td align="left" rowspan="1" colspan="1">P3</td><td align="left" rowspan="1" colspan="1">Positively charged residues</td></tr><tr><td align="left" rowspan="1" colspan="1">P2</td><td align="left" rowspan="1" colspan="1">High hydrophobicity and absence of β-branch</td></tr><tr><td align="left" rowspan="1" colspan="1">P1</td><td align="left" rowspan="1" colspan="1">Gln</td></tr><tr><td align="left" rowspan="1" colspan="1">P1'</td><td align="left" rowspan="1" colspan="1">Small residues</td></tr><tr><td align="left" rowspan="1" colspan="1">P2'</td><td align="left" rowspan="1" colspan="1">Small residues</td></tr><tr><td align="left" rowspan="1" colspan="1">P3'</td><td align="left" rowspan="1" colspan="1">No strong preference</td></tr></tbody></table></alternatives></table-wrap><p>To understand the structural basis of this unique substrate preference, we compared the structures of IBV 3CL<sup>pro</sup> with other coronaviral 3CL<sup>pro</sup>. We noticed that strand-11 of IBV 3CL<sup>pro</sup> is positioned further away from the P4 and P5 substrate-binding site compared to other 3CL<sup>pro</sup> (<xref ref-type="fig" rid="pone-0027228-g004">Figure 4</xref>) <xref rid="pone.0027228-Xue1" ref-type="bibr">[31]</xref>, <xref rid="pone.0027228-Anand1" ref-type="bibr">[32]</xref>, <xref rid="pone.0027228-Zhao1" ref-type="bibr">[33]</xref>. This results in a wider substrate-binding pocket in IBV 3CL<sup>pro</sup>. We further docked the substrate variant A4P into the substrate-binding pocket of IBV 3CL<sup>pro</sup>. Due to the cyclic structure of Pro residue, the backbone Ø dihedral angle of the P4 residue is restrained to ca. −60°, which causes the substrate peptide to bend towards the strand-11 of 3CL<sup>pro</sup>. Such conformation of substrate is much better accommodated by IBV 3CL<sup>pro</sup>, which has a wider substrate-binding pocket near the P4 and P5 positions. This observation justifies why only IBV 3CL<sup>pro</sup> cleaves P4-Pro efficiently.</p><fig id="pone-0027228-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0027228.g004</object-id><label>Figure 4</label><caption><title>IBV 3CL<sup>pro</sup> has a wider substrate-binding pocket to accommodate substrate containing P4-Pro.</title><p>The structure of IBV 3CL<sup>pro</sup> (PDB: 2Q6D, yellow cartoon and white surface) is superimposed with 3CL<sup>pro</sup> from HCoV-229E (PDB: 1P9S, light blue), HCoV-HKU1 (PDB: 3D23, light green), and SARS-CoV (PDB: 2Q6G, pink) <xref rid="pone.0027228-Xue1" ref-type="bibr">[31]</xref>, <xref rid="pone.0027228-Anand1" ref-type="bibr">[32]</xref>, <xref rid="pone.0027228-Zhao1" ref-type="bibr">[33]</xref>. The structure of WT substrate (magenta) is derived from crystal structure of SARS-CoV 3CL<sup>pro</sup> in complex with the autocleavage sequence (TSAVLQ↓SGFRKM) (PDB: 2Q6G) <xref rid="pone.0027228-Xue1" ref-type="bibr">[31]</xref>. The structure of the A4P substrate variant (cyan) was modeled based on the crystal structure of IBV 3CL<sup>pro</sup> in complex with its own autocleavage sequence (PDB: 2Q6D) <xref rid="pone.0027228-Xue1" ref-type="bibr">[31]</xref>. Note that strand-11 of IBV 3CL<sup>pro</sup> is positioned further away from P4 to P5 positions, resulting in a wider substrate-binding pocket.</p></caption><graphic xlink:href="pone.0027228.g004"></graphic></fig><p>Similarities in substrate specificity suggest that it is feasible to create a broad-spectrum inhibitor that targets all 3CL<sup>pro</sup>. A broad-spectrum inhibitor is desirable for a first line defense against coronaviral infection because CoVs are capable of generating novel strains with high virulence through high frequency of mutations and recombination <xref rid="pone.0027228-Jenkins1" ref-type="bibr">[34]</xref>, <xref rid="pone.0027228-Lai1" ref-type="bibr">[35]</xref>, <xref rid="pone.0027228-Pasternak1" ref-type="bibr">[36]</xref>, <xref rid="pone.0027228-Duffy1" ref-type="bibr">[37]</xref>.. Based on the autocleavage sequence of SARS-CoV 3CL<sup>pro</sup> (i.e. AVLQ↓), Rao and co-workers designed broad-spectrum peptidomimetic inhibitors that can inhibit 3CL<sup>pro</sup> from different groups of CoVs <xref rid="pone.0027228-Yang1" ref-type="bibr">[20]</xref>. Their results are consistent with our observation that the autocleavage sequence of SARS-CoV 3CL<sup>pro</sup> can be well cleaved by all 3CL<sup>pro</sup>. The substrate preferences profiled in this study will provide a rational basis to improve the broad-spectrum 3CL<sup>pro</sup> inhibitors. For example, by combining favorable substitutions at P3 to P5 positions, we identified a substrate sequence ‘VARLQ↓SGF’ that can be cleaved with high relative activities by 3CL<sup>pro</sup> from all groups of CoVs (<xref ref-type="table" rid="pone-0027228-t001">Table 1</xref>). This substrate sequence may serve as a good starting point of the design of broad-spectrum peptidomimetic inhibitors for 3CL<sup>pro</sup>.</p><p>Although it is generally accepted that substrate specificity provides insights into the design of peptidomimetic protease inhibitors, there are exceptions to the dogma that good peptidomimetic inhibitors should be derived from good substrate sequences. For example, Hilgenfeld and co-workers showed that the P2 position of peptide aldehyde inhibitors can accommodate aspartate or serine, which are poor substrates for SARS-CoV 3CL<sup>pro</sup><xref rid="pone.0027228-Zhu1" ref-type="bibr">[38]</xref>.</p><p>In the FRET assay developed by us, all 3CL<sup>pro</sup> can efficiently cleave the WT sequence of ‘SAVLQ↓SGF’ with activity of 120–440 mM<sup>−1</sup> min<sup>−1</sup>, and the activity can be further improved by 1.7 to 3.2 fold using the substrate sequence of ‘VARLQ↓SGF’. Because the substrate sequences can be cleaved by all 3CL<sup>pro</sup> with high efficiency, one could use the FRET assay to screen for broad-spectrum inhibitors targeting 3CL<sup>pro</sup> from all groups of CoVs.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and Methods</title><sec id="s4a"><title>Cloning, Expression and Purification of 3CL<sup>pro</sup> and the Substrate Library</title><p>Cloning, expression and purification of SARS-CoV 3CL<sup>pro</sup> were described previously <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. Codon-optimized DNA sequences encoding HCoV-NL63 (GenBank AY567487) and HCoV-OC43 (GenBank AAX85666), and IBV (GenBank M95169) 3CL<sup>pro</sup> were purchased from Mr. Gene (<ext-link ext-link-type="uri" xlink:href="http://mrgene.com">http://mrgene.com</ext-link>). The coding sequences of 3CL<sup>pro</sup> from HCoV-NL63, HCoV-OC43 and IBV were sub-cloned and expressed in <italic>E. coli</italic> strain BL21 (DE3) pLysS as fusion proteins with N-terminal tags of poly-histidine-small ubiquitin-related modifier (His<sub>6</sub>-SUMO) or poly-histidine-maltose binding protein (His<sub>6</sub>-MBP). Protein expression was induced by addition of 0.1 mM of isopropyl β-D-1-thiogalactopyranoside. After overnight incubation at 25°C, cells were harvested by centrifugation and resuspended in buffer A (20 mM Tris, pH 7.8, 150 mM NaCl and 1 mM tris(2-carboxyethyl)phosphine) with 30 mM imidazole and disrupted by sonication. Soluble fraction was subject to immobilized metal ion affinity chromatography for purification as described for SARS-CoV 3CL<sup>pro</sup><xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. The His<sub>6</sub>-SUMO or His<sub>6</sub>-MBP tags were removed by protease digestion using sentrin-specific protease 1 or factor Xa, respectively, followed by immobilized metal ion affinity chromatography. Native 3CL<sup>pro</sup> were finally purified by G75 size exclusion column and stored in buffer A. Elution profiles of size exclusion chromatography indicated that all 3CL<sup>pro</sup> purified were dimeric.</p><p>The construction, expression and purification of the substrate library were described previously <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. In brief, the WT substrate sequence ‘TSAVLQ↓SGFRKM’ was inserted between the cyan fluorescent protein and the yellow fluorescent protein to create the substrate protein. Saturation mutagenesis was performed at each of the P5 to P3' positions to generate a substrate library of 19×8 variants.</p></sec><sec id="s4b"><title>FRET assay for 3CL<sup>pro</sup> activity measurement</title><p>The protease activity of 3CL<sup>pro</sup> was measured by the FRET assay we developed previously <xref rid="pone.0027228-Chuck1" ref-type="bibr">[26]</xref>. Purified 3CL<sup>pro</sup> at 0.2 to 2 µM were mixed with 35 µM of the substrate protein in buffer A. Cleavage of the substrate protein leads to a decrease in fluorescence at 530 nm when the reaction mixture was excited at 430 nm. The fluorescence intensity, monitored by EnVision 2101 Multilabel Plate Reader, was fitted to single exponential decay to obtain the observed rate constant (k<sub>obs</sub>). The protease activity against variant substrates was normalized against the WT activity to yield the relative activity. The assay was repeated in triplicate.</p></sec><sec id="s4c"><title>Correlation analysis</title><p>Structural properties of substituting residues, including side chain volume <xref rid="pone.0027228-Lee1" ref-type="bibr">[28]</xref>, hydrophobicity <xref rid="pone.0027228-Kyte1" ref-type="bibr">[29]</xref>, and α-helix and β-sheet propensities <xref rid="pone.0027228-Chou1" ref-type="bibr">[30]</xref>, were correlated with relative activity to determine correlation coefficients (r) and p-values.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0027228.s001"><label>Figure S1</label><caption><p><bold>Molecular modeling showing P3-Arg may interact with Glu-166 of 3CL<sup>pro</sup>.</bold> The model was based on the crystal structure of 3CLpro (grey) in complex with a peptide substrate ‘TSAVLQ↓SGFRK’ (yellow). P3-Val was replaced by P3-Arg using the program PyMOL. As shown, the invariant Glu-166 is in close proximity to P3-Arg, and may form favorable charge-charge interaction to P3-Arg.</p><p>(TIF)</p></caption><media xlink:href="pone.0027228.s001.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0027228.s002"><label>Table S1</label><caption><p>Relative activities of HCoV-NL63, HCoV-OC43, SARS-CoV and IBV 3CL<sup>pro</sup>. ND stands for non-detectable cleavage. The average and the standard deviation of three measurements are shown.</p><p>(DOC)</p></caption><media xlink:href="pone.0027228.s002.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0027228.s003"><label>Table S2</label><caption><p>Correlation between activity of 3CL<sup>pro</sup> and structural properties of substituting residues. The correlation coefficients and p-values (bracketed) are reported.</p><p>(DOC)</p></caption><media xlink:href="pone.0027228.s003.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0027228.s004"><label>Table S3</label><caption><p>Autocleavage sequences of 3CL<sup>pro</sup>. PEDV, TGEV, MHV, PHEV stand for porcine epidemic diarrhoea coronavirus, transmissible gastroenteritis coronavirus, mouse hepatitis coronavirus and porcine hemagglutinating encephalomyelitis coronavirus respectively.</p><p>(DOC)</p></caption><media xlink:href="pone.0027228.s004.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><fn-group><fn fn-type="COI-statement"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p><bold>Funding: </bold>This work was supported by the Research Fund for the Control of Infectious Diseases, Hong Kong SAR Government (project number: 09080282) (<ext-link ext-link-type="uri" xlink:href="http://www.fhb.gov.hk/grants/english/funds/funds_rfcid/funds_rfcid_abt/funds_rfcid_abt.html">http://www.fhb.gov.hk/grants/english/funds/funds_rfcid/funds_rfcid_abt/funds_rfcid_abt.html</ext-link>). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group><ref-list><title>References</title><ref id="pone.0027228-vanderHoek1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van der Hoek</surname><given-names>L</given-names></name><name><surname>Pyrc</surname><given-names>K</given-names></name><name><surname>Jebbink</surname><given-names>MF</given-names></name><name><surname>Vermeulen-Oost</surname><given-names>W</given-names></name><name><surname>Berkhout</surname><given-names>RJ</given-names></name><etal></etal></person-group><year>2004</year><article-title>Identification of a new human coronavirus.</article-title><source>Nat Med</source><volume>10</volume><fpage>368</fpage><lpage>373</lpage><pub-id pub-id-type="pmid">15034574</pub-id></element-citation></ref><ref id="pone.0027228-Hamre1"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hamre</surname><given-names>D</given-names></name><name><surname>Procknow</surname><given-names>JJ</given-names></name></person-group><year>1966</year><article-title>A new virus isolated from the human respiratory tract.</article-title><source>Proc Soc Exp Biol Med</source><volume>121</volume><fpage>190</fpage><lpage>193</lpage><pub-id pub-id-type="pmid">4285768</pub-id></element-citation></ref><ref id="pone.0027228-Woo1"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Chu</surname><given-names>CM</given-names></name><name><surname>Chan</surname><given-names>KH</given-names></name><name><surname>Tsoi</surname><given-names>HW</given-names></name><etal></etal></person-group><year>2005</year><article-title>Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia.</article-title><source>J Virol</source><volume>79</volume><fpage>884</fpage><lpage>895</lpage><pub-id pub-id-type="pmid">15613317</pub-id></element-citation></ref><ref id="pone.0027228-Kuiken1"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuiken</surname><given-names>T</given-names></name><name><surname>Fouchier</surname><given-names>RA</given-names></name><name><surname>Schutten</surname><given-names>M</given-names></name><name><surname>Rimmelzwaan</surname><given-names>GF</given-names></name><name><surname>van Amerongen</surname><given-names>G</given-names></name><etal></etal></person-group><year>2003</year><article-title>Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.</article-title><source>Lancet</source><volume>362</volume><fpage>263</fpage><lpage>270</lpage><pub-id pub-id-type="pmid">12892955</pub-id></element-citation></ref><ref id="pone.0027228-Peiris1"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peiris</surname><given-names>JS</given-names></name><name><surname>Lai</surname><given-names>ST</given-names></name><name><surname>Poon</surname><given-names>LL</given-names></name><name><surname>Guan</surname><given-names>Y</given-names></name><name><surname>Yam</surname><given-names>LY</given-names></name><etal></etal></person-group><year>2003</year><article-title>Coronavirus as a possible cause of severe acute respiratory syndrome.</article-title><source>Lancet</source><volume>361</volume><fpage>1319</fpage><lpage>1325</lpage><pub-id pub-id-type="pmid">12711465</pub-id></element-citation></ref><ref id="pone.0027228-Cheever1"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheever</surname><given-names>FS</given-names></name><name><surname>Daniels</surname><given-names>JB</given-names></name><name><surname>Pappenheimer</surname><given-names>AM</given-names></name><name><surname>Bailey</surname><given-names>OT</given-names></name></person-group><year>1949</year><article-title>A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin.</article-title><source>J Exp Med</source><volume>90</volume><fpage>181</fpage><lpage>210</lpage><pub-id pub-id-type="pmid">18137294</pub-id></element-citation></ref><ref id="pone.0027228-Binn1"><label>7</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Binn</surname><given-names>LN</given-names></name><name><surname>Lazar</surname><given-names>EC</given-names></name><name><surname>Keenan</surname><given-names>KP</given-names></name><name><surname>Huxsoll</surname><given-names>DL</given-names></name><name><surname>Marchwicki</surname><given-names>RH</given-names></name><etal></etal></person-group><year>1974</year><article-title>Recovery and characterization of a coronavirus from military dogs with diarrhea.</article-title><publisher-name>Proc Annu Meet U S Anim Health Assoc</publisher-name><fpage>359</fpage><lpage>366</lpage></element-citation></ref><ref id="pone.0027228-Poon1"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Poon</surname><given-names>LL</given-names></name><name><surname>Chu</surname><given-names>DK</given-names></name><name><surname>Chan</surname><given-names>KH</given-names></name><name><surname>Wong</surname><given-names>OK</given-names></name><name><surname>Ellis</surname><given-names>TM</given-names></name><etal></etal></person-group><year>2005</year><article-title>Identification of a novel coronavirus in bats.</article-title><source>J Virol</source><volume>79</volume><fpage>2001</fpage><lpage>2009</lpage><pub-id pub-id-type="pmid">15681402</pub-id></element-citation></ref><ref id="pone.0027228-Beaudette1"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beaudette</surname><given-names>FR</given-names></name><name><surname>Hudson</surname><given-names>CB</given-names></name></person-group><year>1937</year><article-title>Cultivation of the virus of infectious bronchitis.</article-title><source>J Am Vet Med Assoc</source><volume>90</volume><fpage>51</fpage><lpage>58</lpage></element-citation></ref><ref id="pone.0027228-Doyle1"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Doyle</surname><given-names>LP</given-names></name><name><surname>Hutchings</surname><given-names>LM</given-names></name></person-group><year>1946</year><article-title>A transmissible gastroenteritis virus in pigs.</article-title><source>J Am Vet Assoc</source><volume>108</volume><fpage>257</fpage><lpage>259</lpage></element-citation></ref><ref id="pone.0027228-Tyrrell1"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tyrrell</surname><given-names>DA</given-names></name><name><surname>Bynoe</surname><given-names>ML</given-names></name></person-group><year>1966</year><article-title>Cultivation of viruses from a high proportion of patients with colds.</article-title><source>Lancet</source><volume>1</volume><fpage>76</fpage><lpage>77</lpage><pub-id pub-id-type="pmid">4158999</pub-id></element-citation></ref><ref id="pone.0027228-Woo2"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Yuen</surname><given-names>KY</given-names></name></person-group><year>2009</year><article-title>Coronavirus diversity, phylogeny and interspecies jumping.</article-title><source>Exp Biol Med (Maywood)</source><volume>234</volume><fpage>1117</fpage><lpage>1127</lpage><pub-id pub-id-type="pmid">19546349</pub-id></element-citation></ref><ref id="pone.0027228-Snijder1"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snijder</surname><given-names>EJ</given-names></name><name><surname>Bredenbeek</surname><given-names>PJ</given-names></name><name><surname>Dobbe</surname><given-names>JC</given-names></name><name><surname>Thiel</surname><given-names>V</given-names></name><name><surname>Ziebuhr</surname><given-names>J</given-names></name><etal></etal></person-group><year>2003</year><article-title>Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.</article-title><source>J Mol Biol</source><volume>331</volume><fpage>991</fpage><lpage>1004</lpage><pub-id pub-id-type="pmid">12927536</pub-id></element-citation></ref><ref id="pone.0027228-Gorbalenya1"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gorbalenya</surname><given-names>AE</given-names></name><name><surname>Snijder</surname><given-names>EJ</given-names></name><name><surname>Spaan</surname><given-names>WJ</given-names></name></person-group><year>2004</year><article-title>Severe acute respiratory syndrome coronavirus phylogeny: toward consensus.</article-title><source>J Virol</source><volume>78</volume><fpage>7863</fpage><lpage>7866</lpage><pub-id pub-id-type="pmid">15254158</pub-id></element-citation></ref><ref id="pone.0027228-Gaunt1"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gaunt</surname><given-names>ER</given-names></name><name><surname>Hardie</surname><given-names>A</given-names></name><name><surname>Claas</surname><given-names>EC</given-names></name><name><surname>Simmonds</surname><given-names>P</given-names></name><name><surname>Templeton</surname><given-names>KE</given-names></name></person-group><year>2010</year><article-title>Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method.</article-title><source>J Clin Microbiol</source><volume>48</volume><fpage>2940</fpage><lpage>2947</lpage><pub-id pub-id-type="pmid">20554810</pub-id></element-citation></ref><ref id="pone.0027228-Dominguez1"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dominguez</surname><given-names>SR</given-names></name><name><surname>Robinson</surname><given-names>CC</given-names></name><name><surname>Holmes</surname><given-names>KV</given-names></name></person-group><year>2009</year><article-title>Detection of four human coronaviruses in respiratory infections in children: a one-year study in Colorado.</article-title><source>J Med Virol</source><volume>81</volume><fpage>1597</fpage><lpage>1604</lpage><pub-id pub-id-type="pmid">19626607</pub-id></element-citation></ref><ref id="pone.0027228-Cavanagh1"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cavanagh</surname><given-names>D</given-names></name></person-group><year>2007</year><article-title>Coronavirus avian infectious bronchitis virus.</article-title><source>Vet Res</source><volume>38</volume><fpage>281</fpage><lpage>297</lpage><pub-id pub-id-type="pmid">17296157</pub-id></element-citation></ref><ref id="pone.0027228-Ignjatovic1"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ignjatovic</surname><given-names>J</given-names></name><name><surname>Sapats</surname><given-names>S</given-names></name></person-group><year>2000</year><article-title>Avian infectious bronchitis virus.</article-title><source>Rev Sci Tech</source><volume>19</volume><fpage>493</fpage><lpage>508</lpage><pub-id pub-id-type="pmid">10935276</pub-id></element-citation></ref><ref id="pone.0027228-Lin1"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>CW</given-names></name><name><surname>Tsai</surname><given-names>CH</given-names></name><name><surname>Tsai</surname><given-names>FJ</given-names></name><name><surname>Chen</surname><given-names>PJ</given-names></name><name><surname>Lai</surname><given-names>CC</given-names></name><etal></etal></person-group><year>2004</year><article-title>Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.</article-title><source>FEBS Lett</source><volume>574</volume><fpage>131</fpage><lpage>137</lpage><pub-id pub-id-type="pmid">15358553</pub-id></element-citation></ref><ref id="pone.0027228-Yang1"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Xie</surname><given-names>W</given-names></name><name><surname>Xue</surname><given-names>X</given-names></name><name><surname>Yang</surname><given-names>K</given-names></name><name><surname>Ma</surname><given-names>J</given-names></name><etal></etal></person-group><year>2005</year><article-title>Design of wide-spectrum inhibitors targeting coronavirus main proteases.</article-title><source>PLoS Biol</source><volume>3</volume><fpage>e324</fpage><pub-id pub-id-type="pmid">16128623</pub-id></element-citation></ref><ref id="pone.0027228-Li1"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>SY</given-names></name><name><surname>Chen</surname><given-names>C</given-names></name><name><surname>Zhang</surname><given-names>HQ</given-names></name><name><surname>Guo</surname><given-names>HY</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><etal></etal></person-group><year>2005</year><article-title>Identification of natural compounds with antiviral activities against SARS-associated coronavirus.</article-title><source>Antiviral Res</source><volume>67</volume><fpage>18</fpage><lpage>23</lpage><pub-id pub-id-type="pmid">15885816</pub-id></element-citation></ref><ref id="pone.0027228-Wu1"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname><given-names>CY</given-names></name><name><surname>Jan</surname><given-names>JT</given-names></name><name><surname>Ma</surname><given-names>SH</given-names></name><name><surname>Kuo</surname><given-names>CJ</given-names></name><name><surname>Juan</surname><given-names>HF</given-names></name><etal></etal></person-group><year>2004</year><article-title>Small molecules targeting severe acute respiratory syndrome human coronavirus.</article-title><source>Proc Natl Acad Sci U S A</source><volume>101</volume><fpage>10012</fpage><lpage>10017</lpage><pub-id pub-id-type="pmid">15226499</pub-id></element-citation></ref><ref id="pone.0027228-Chen1"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Gui</surname><given-names>C</given-names></name><name><surname>Luo</surname><given-names>X</given-names></name><name><surname>Yang</surname><given-names>Q</given-names></name><name><surname>Gunther</surname><given-names>S</given-names></name><etal></etal></person-group><year>2005</year><article-title>Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro.</article-title><source>J Virol</source><volume>79</volume><fpage>7095</fpage><lpage>7103</lpage><pub-id pub-id-type="pmid">15890949</pub-id></element-citation></ref><ref id="pone.0027228-Fan1"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fan</surname><given-names>K</given-names></name><name><surname>Ma</surname><given-names>L</given-names></name><name><surname>Han</surname><given-names>X</given-names></name><name><surname>Liang</surname><given-names>H</given-names></name><name><surname>Wei</surname><given-names>P</given-names></name><etal></etal></person-group><year>2005</year><article-title>The substrate specificity of SARS coronavirus 3C-like proteinase.</article-title><source>Biochem Biophys Res Commun</source><volume>329</volume><fpage>934</fpage><lpage>940</lpage><pub-id pub-id-type="pmid">15752746</pub-id></element-citation></ref><ref id="pone.0027228-Goetz1"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goetz</surname><given-names>DH</given-names></name><name><surname>Choe</surname><given-names>Y</given-names></name><name><surname>Hansell</surname><given-names>E</given-names></name><name><surname>Chen</surname><given-names>YT</given-names></name><name><surname>McDowell</surname><given-names>M</given-names></name><etal></etal></person-group><year>2007</year><article-title>Substrate specificity profiling and identification of a new class of inhibitor for the major protease of the SARS coronavirus.</article-title><source>Biochemistry</source><volume>46</volume><fpage>8744</fpage><lpage>8752</lpage><pub-id pub-id-type="pmid">17605471</pub-id></element-citation></ref><ref id="pone.0027228-Chuck1"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chuck</surname><given-names>CP</given-names></name><name><surname>Chong</surname><given-names>LT</given-names></name><name><surname>Chen</surname><given-names>C</given-names></name><name><surname>Chow</surname><given-names>HF</given-names></name><name><surname>Wan</surname><given-names>DC</given-names></name><etal></etal></person-group><year>2010</year><article-title>Profiling of substrate specificity of SARS-CoV 3CL<sup>pro</sup>.</article-title><source>PLoS ONE</source><volume>5</volume><fpage>e13197</fpage><pub-id pub-id-type="pmid">20949131</pub-id></element-citation></ref><ref id="pone.0027228-Hegyi1"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hegyi</surname><given-names>A</given-names></name><name><surname>Ziebuhr</surname><given-names>J</given-names></name></person-group><year>2002</year><article-title>Conservation of substrate specificities among coronavirus main proteases.</article-title><source>J Gen Virol</source><volume>83</volume><fpage>595</fpage><lpage>599</lpage><pub-id pub-id-type="pmid">11842254</pub-id></element-citation></ref><ref id="pone.0027228-Lee1"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>S</given-names></name><name><surname>Tikhomirova</surname><given-names>A</given-names></name><name><surname>Shalvardjian</surname><given-names>N</given-names></name><name><surname>Chalikian</surname><given-names>TV</given-names></name></person-group><year>2008</year><article-title>Partial molar volumes and adiabatic compressibilities of unfolded protein states.</article-title><source>Biophys Chem</source><volume>134</volume><fpage>185</fpage><lpage>199</lpage><pub-id pub-id-type="pmid">18342425</pub-id></element-citation></ref><ref id="pone.0027228-Kyte1"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kyte</surname><given-names>J</given-names></name><name><surname>Doolittle</surname><given-names>RF</given-names></name></person-group><year>1982</year><article-title>A simple method for displaying the hydropathic character of a protein.</article-title><source>J Mol Biol</source><volume>157</volume><fpage>105</fpage><lpage>132</lpage><pub-id pub-id-type="pmid">7108955</pub-id></element-citation></ref><ref id="pone.0027228-Chou1"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chou</surname><given-names>PY</given-names></name><name><surname>Fasman</surname><given-names>GD</given-names></name></person-group><year>1978</year><article-title>Prediction of the secondary structure of proteins from their amino acid sequence.</article-title><source>Adv Enzymol Relat Areas Mol Biol</source><volume>47</volume><fpage>45</fpage><lpage>148</lpage><pub-id pub-id-type="pmid">364941</pub-id></element-citation></ref><ref id="pone.0027228-Xue1"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xue</surname><given-names>X</given-names></name><name><surname>Yu</surname><given-names>H</given-names></name><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Xue</surname><given-names>F</given-names></name><name><surname>Wu</surname><given-names>Z</given-names></name><etal></etal></person-group><year>2008</year><article-title>Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design.</article-title><source>J Virol</source><volume>82</volume><fpage>2515</fpage><lpage>2527</lpage><pub-id pub-id-type="pmid">18094151</pub-id></element-citation></ref><ref id="pone.0027228-Anand1"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Anand</surname><given-names>K</given-names></name><name><surname>Ziebuhr</surname><given-names>J</given-names></name><name><surname>Wadhwani</surname><given-names>P</given-names></name><name><surname>Mesters</surname><given-names>JR</given-names></name><name><surname>Hilgenfeld</surname><given-names>R</given-names></name></person-group><year>2003</year><article-title>Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.</article-title><source>Science</source><volume>300</volume><fpage>1763</fpage><lpage>1767</lpage><pub-id pub-id-type="pmid">12746549</pub-id></element-citation></ref><ref id="pone.0027228-Zhao1"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>Q</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Xue</surname><given-names>F</given-names></name><name><surname>Zou</surname><given-names>Y</given-names></name><name><surname>Chen</surname><given-names>C</given-names></name><etal></etal></person-group><year>2008</year><article-title>Structure of the main protease from a global infectious human coronavirus, HCoV-HKU1.</article-title><source>J Virol</source><volume>82</volume><fpage>8647</fpage><lpage>8655</lpage><pub-id pub-id-type="pmid">18562531</pub-id></element-citation></ref><ref id="pone.0027228-Jenkins1"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jenkins</surname><given-names>GM</given-names></name><name><surname>Rambaut</surname><given-names>A</given-names></name><name><surname>Pybus</surname><given-names>OG</given-names></name><name><surname>Holmes</surname><given-names>EC</given-names></name></person-group><year>2002</year><article-title>Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis.</article-title><source>J Mol Evol</source><volume>54</volume><fpage>156</fpage><lpage>165</lpage><pub-id pub-id-type="pmid">11821909</pub-id></element-citation></ref><ref id="pone.0027228-Lai1"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lai</surname><given-names>MM</given-names></name></person-group><year>1992</year><article-title>RNA recombination in animal and plant viruses.</article-title><source>Microbiol Rev</source><volume>56</volume><fpage>61</fpage><lpage>79</lpage><pub-id pub-id-type="pmid">1579113</pub-id></element-citation></ref><ref id="pone.0027228-Pasternak1"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pasternak</surname><given-names>AO</given-names></name><name><surname>Spaan</surname><given-names>WJ</given-names></name><name><surname>Snijder</surname><given-names>EJ</given-names></name></person-group><year>2006</year><article-title>Nidovirus transcription: how to make sense...?</article-title><source>J Gen Virol</source><volume>87</volume><fpage>1403</fpage><lpage>1421</lpage><pub-id pub-id-type="pmid">16690906</pub-id></element-citation></ref><ref id="pone.0027228-Duffy1"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Duffy</surname><given-names>S</given-names></name><name><surname>Shackelton</surname><given-names>LA</given-names></name><name><surname>Holmes</surname><given-names>EC</given-names></name></person-group><year>2008</year><article-title>Rates of evolutionary change in viruses: patterns and determinants.</article-title><source>Nat Rev Genet</source><volume>9</volume><fpage>267</fpage><lpage>276</lpage><pub-id pub-id-type="pmid">18319742</pub-id></element-citation></ref><ref id="pone.0027228-Zhu1"><label>38</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>L</given-names></name><name><surname>George</surname><given-names>S</given-names></name><name><surname>Schmidt</surname><given-names>MF</given-names></name><name><surname>Al-Gharabli</surname><given-names>SI</given-names></name><name><surname>Rademann</surname><given-names>J</given-names></name><etal></etal></person-group><year>2011</year><article-title>Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease.</article-title><publisher-name>Antiviral Res</publisher-name><comment>doi:<ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.antiviral.2011.08.001">10.1016/j.antiviral.2011.08.001</ext-link></comment></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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