SARS冠状病毒木瓜样蛋白酶生物学功能研究
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摘要
本研究利用分子克隆技术构建严重急性呼吸系统综合症冠状病毒(SARS-CoV)木瓜样蛋白酶(PLpro)的截断体和其蛋白酶活性缺失突变体,研究PLpro去泛素化、去ISG化和去SUMO化的分子机制。实验结果表明,PLpro蛋白的三个蛋白酶活性位点C1651、H1812、D1826对其去泛素活性均有影响,但C1651起着主要作用,而H1812和D1826起到辅助作用;C1651和H1812对PLpro蛋白去ISG15活性是必需的,而D1826的突变对其去ISG15活性无影响;游离态PLpro无去SUMO化活性,而带有跨膜域(transmemberane domain, TM)的PLpro-TM则有一定程度的去SUMO化活性;截去PLpro蛋白N末端泛素样结构域(Ubl)后,对PLpro的去泛素和去ISG15活性均无影响;从PLpro-TM中截去NAB-G2M结构域的实验证明,TM的核周内质网膜定位功能是PLpro-TM发挥去SUMO作用的原因,NAB-G2M结构域并不参与其去SUMO反应。
In this study, we constructed cut-off mutagenesis and site-direct mutagenesis through cloning technology to investigate the molecular mechanism of SARS-CoV papain-like protease (PLpro) on the deubiquitinating, deISGylating and deSUMOylating process. Our result shew that the proteolytic tried-residuce active sites all could affect the PLpro`s deubiquiting activity. Among them, the C1651 is the majority functional residuce, while the H1812 and D1826 play supporting role; residuces C1651 and H1812 are necessary for the PLpro`s deISGylating activity. However, mutation of the D1826 to alanine no affect on PLpro`s deISGylating activity; solutio-PLpro no deSUMOylating activity, truncating the ubiquitin-like domain from PLpro did not effect its deubiquiting and deISGylating activity; solution-PLpro no deSUMOylating activity, the PLpro involve transmembrane domain (TM), however, have certain degree of deSUMOlating activity; truncating the NAB-G2M domain from PLpro-TM did not effect its deSUMOlating activity , indicating that the nuclear-around-mamberane locating function of transmembrane domain playes an important role in the deSUMOlating activity of PLpro-TM , in which NAB-G2M domain is not involved.
In this study, we constructed cut-off mutagenesis and site-direct mutagenesis through cloning technology to investigate the molecular mechanism of SARS-CoV papain-like protease (PLpro) on the deubiquitinating, deISGylating and deSUMOylating process. Our result shew that the proteolytic tried-residuce active sites all could affect the PLpro`s deubiquiting activity. Among them, the C1651 is the majority functional residuce, while the H1812 and D1826 play supporting role; residuces C1651 and H1812 are necessary for the PLpro`s deISGylating activity. However, mutation of the D1826 to alanine no affect on PLpro`s deISGylating activity; solutio-PLpro no deSUMOylating activity, truncating the ubiquitin-like domain from PLpro did not effect its deubiquiting and deISGylating activity; solution-PLpro no deSUMOylating activity, the PLpro involve transmembrane domain (TM), however, have certain degree of deSUMOlating activity; truncating the NAB-G2M domain from PLpro-TM did not effect its deSUMOlating activity , indicating that the nuclear-around-mamberane locating function of transmembrane domain playes an important role in the deSUMOlating activity of PLpro-TM , in which NAB-G2M domain is not involved.
引文
1. Susan R. Weiss and Sonia Navas-Martin. Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus[J]. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 2005, Vol. 69(4): 635～664.
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3. Chi Wai Yip, Chung Chau Hon, Mang Shi et al. Phylogenetic perspectives on the epidemiology and origins of SARS and SARS-like coronaviruses[J]. Infection, Genetics and Evolution , 2009, 9: 1185～1196
4.李金萍.冠状病毒概述[J],生命科学仪器, 2007 , 5:43～45
5. Lissenberg A, Vrolijk MM, van Vliet AL, et al. Luxury at a cost:Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro[J]. J Virol 2005, 79: 15054～15063.
6.王承忠,戚正武. SARS病毒及相关冠状病毒的生物学特征[J].生物化学与生物物理学报.2003 , 35 (6) : 495～502
7. Cui, J., Han, N., Streicker, D.,et al. Evolutionary relationships between bat coronaviruses and their hosts[J]. Emerg. Infect. Dis. 2007, 13 (10), 1526～1532.
8. Lau, S.K., Woo, P.C., Li, K.S., et al. Severe acute respiratory syndrome coronaviruslikevirus in Chinese horseshoe bats[J]. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (39), 14040～14045.
9. Li, W., Shi, Z., Yu, M., et al. Bats are natural reservoirs of SARS-like coronaviruses[J]. Science 2005b, 310 (5748), 676～679.
10. Guan, Y., Zheng, B.J., He,et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern[J]. China. Science. 2003, 302 (5643), 276～278.
11.周丽萍,,罗仕伟,贺东生. SARS冠状病毒的分子生物学研究进展[J].生物技术通讯. 2006, 17(1), 75～77
12. Shi, Z., Hu, Z.,. A review of studies on animal reservoirs of the SARS coronavirus[J].Virus Res. 2008, 133 (1), 74～87.
13. Kan, B.; Wang, M.; Jing, H.;et al. Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms[J]. J.Virol. 2005, 79(18), 11892～11900.
14. Li, W.; Moore, M.J.; Vasilieva, N.; et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus [J]. Nature, 2003, 426(6965), 450～454.
15. Song, H.-D.; Tu, C.-C.; Zhang, G.-W., et al. Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human[J]. Proc. Natl. Acad. Sci. USA, 2005, 102(7), 2430～2435.
16. Kap-Sun Yeung, Nicholas A. Meanwell. Recent Developments in the Virology and Antiviral Research of Severe Acute Respiratory Syndrome Coronavirus[J]. Infectious Disorders-Drug Targets. 2007, 7: 29～41
17. Zhang, J.X.; Zhang, S.Y.; Wang, P.; et al. Prevalence and genetic diversity of coronaviruses in bats from China[J]. J. Virol., 2006, 80(15), 7481～7490.
18. Rota, P.A.; Oberste, M.S.; Monroe, S.S.; et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome [J]. Science, 2003, 300(5624), 1394～1399.
19. Stanley Perlman, Jason Netland. Coronaviruses post-SARS: Update on replication and pathogenesis[J]. Nat Rev Microbiol. 2009, 7(6): 439～450
20. Benjamin W. Neuman,Jeremiah S. Joseph,et al. Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3[J]. JOURNAL OF VIROLOGY . 2008, 82(11): 5279～5294
21.杨宇东,孙莉,陈忠斌. SARS冠状病毒PLpro蛋白酶的结构与功能[J].中国生物化学与分子生物学报. 2010, 26(1), 15～21
22. Chatterjee A, JohnsonM A, Serrano P, et al. Nuclearmagnetic resonance structure shows that the severe acute resp iratory syndrome coronavirus-unique domain contains amacrodomain fold [J]. J Virol, 2009, 83 (4) : 1823～1836
23. Kiira Ratia, Kumar Singh Saikatendu, Bernard D.et al Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme[J]. PNAS, 2006, 103, 5717～5722
24. Herold, J., Siddell, S. G. & Gorbalenya, A. E. A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold[J]. J. Biol. Chem. 1999 , 274(21), 14918～14925.
25. Ziebuhr, J., Thiel, V. & Gorbalenya, A. E. The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond[J]. J. Biol. Chem. 2001, 276(35), 33220～33232.
26. Naina Barretto, Dalia Jukneliene, Kiira Ratia, et al. The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity. JOURNAL OF VIROLOGY[J], 2005, 79(24) , 15189～15198
27. Thiel, V., Ivanov, K. A., Putics, A., et al. Mechanisms and enzymes involved inSARS coronavirus genome expression[J]. J. Gen.Virol. 2003. 84(pt9), 2305～2315.
28. Harcourt, B. H., Jukneliene, D., Kanjanahaluethai, A., et al. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity[J]. J. Virol. 2004. 78(24), 13600～13612.
29. Harcourt B H, Jukneliene D, Kanjanahaluethai A, et al. Identification of severe acute resp iratory syndrome coronavirus replicase p roducts and characterization of papain-like p rotease activity [J]. J Virol, 2004, 78 (24) : 13600～13612
30. Perlman S, Holmes K V. Advances in Experimental Medicine and Biology [M]. Sp ringerUS Press, 2006, 581: 37～41
31. Lindner H A, Lytvyn V, Qi H, et al. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like p rotease [J]. Arch Biochem Biophys, 2007, 466 (1) : 8～14
32.阳凯,王琛.宿主细胞应答病毒感染的细胞信号转导研究新进展[J].生物化学与物理进展, 2006,33(12):1138～1145.
33. Akira S, Takeda K. Toll - like receptor signaling[J]. Nat Rcv Immunol,2004, 4(7): 499- 511.
34. Qi Baochang, Huang Yue, Rowe D, et al. VISA- A pass to innate immunity[J]. Int J Biochem Cell Biol, 2007,39:287～291.
35. Garcia-Sastre A, Biron C A. Type 1 interferons and the virus- host relationship: a lesson in détente [J]. Science, 2006, 312 (5775) : 879～882
36. Devaraj S G, Wang N, Chen Z, et al. Regulation of IRF-3-dependent innate immunity by the papain2like p rotease domain of the severe acute resp iratory syndrome coronavirus [J]. J Biol Chem, 2007, 282 (44) : 32208～32221
37. Frieman M, Ratia K, Johnston R E, et al. Severe acute resp iratory syndrome coronavirus papain-like p rotease ubiquitin- like domain and catalytic domain regulate antagonism of IRF-3 and NF-κB signaling [J]. J Virol, 2009, 83 (13) : 6689～6705
38. O. Kerscher, R. Felberbaum, M. Hochstrasser, Modification of proteins by ubiquitin and ubiquitin-like proteins[J], Annu. Rev. Cell. Dev. Biol. 2006, 22: 159～180.
39.宋春雷,刘红林.组蛋白的泛素化与去泛素化修饰[J].细胞生物学杂志. 2007, 29: 51-55
40. Tom AM Groothuis, Nico P Dantuma, Jacques Neefjes et al. Ubiquitin crosstalk connecting cellular processes[J]. Cell Division 2006, 1:21
41.白勇,孙安盛,顾振纶等.泛素-蛋白酶体通路在细胞生存和肿瘤发生中的作用[J].中国药理学通报. 2008, 24 (10) : 1268～1271
42.赵天锁,任贺,郝继辉.泛素-蛋白酶体通路的研究进展[J].山东医药, 2009, 49: 113-114
43.曹建南,郭汉城.泛素-蛋白酶体系统在核因子κB途径中的作用[J].医学综述. 2009, 15(3), 398～400
44.肖兰博,李力力,曹亚.泛素在信号转导中的作用[J].生命的化学. 2007, 27(6): 527-531
45.邓彬蔚,张令强,贺福初.蛋白泛素途径及其非降解功能研究新进展[J].军事医学科学院院刊. 2006, 30(6): 565～569
46. GackM U, Shin Y C, Joo C H, et al. TRIM-5 RING-finger E3 ubiquitin ligase is essential for R IG-I-mediated antiviral activity [J]. Nature, 2007, 446 (7138) : 916～920
47. Huye L E, Ning S, KelliherM, et al. Interferon regulatory factor 7 is activated by a viral oncop rotein through RIP-dependent ubiquitination [J]. Mol Cell Biol, 2007, 27 (8): 2910～2918
48.胡日查,杨宇东,孙莉等.泛素样蛋白ISG15在抗病毒天然免疫中的作用[J].生物技术通讯. 2010, 21(1): 94～99
49. Ian F. Pitha-Rowe and Paula M. Pitha. Viral Defense, Carcinogenesis and ISG15: Novel Roles for an Old ISG. Cytokine Growth Factor[J]. Rev. 2007 ; 18(5-6): 409～417.
50.陈泉,施蕴渝.小范素相关修饰物SUMO研究进展.生命科学, 2004, 16 (1) : 1～6
51.韦玮,张浩,毛建平等.蛋白质SUMO化修饰研究进展[J].中国生物工程杂志. 2008, 28 (7) : 122～126
52. Melchior F. SUMO——nonclassical ubiquitin[J]. Annu Rev Cell Dev Biol. 2000 , 16 :591- 626
53. Xiang-Dong Li, Yang Shen,Ya-Feng Qiu,et al. SUMOylation: An Important Post-translational Modification in vivo[J]. Chinese Journal of Cell Biology. 2008, 30: 142～146
54. Min Hu, Pingwei Li1, Ling Song. Structure and mechanisms of the proteasomeassociated deubiquitinating enzyme USP14[J]. The EMBO Journal. 2005, 24, 3747～3756
2.芮伟,张其鹏,石磊等. SARS冠状病毒基因组、蛋白质与侵入宿主细胞过程的研究近况[J].中华医学杂志. 2003. 83 (11): 913～921.
3. Chi Wai Yip, Chung Chau Hon, Mang Shi et al. Phylogenetic perspectives on the epidemiology and origins of SARS and SARS-like coronaviruses[J]. Infection, Genetics and Evolution , 2009, 9: 1185～1196
4.李金萍.冠状病毒概述[J],生命科学仪器, 2007 , 5:43～45
5. Lissenberg A, Vrolijk MM, van Vliet AL, et al. Luxury at a cost:Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro[J]. J Virol 2005, 79: 15054～15063.
6.王承忠,戚正武. SARS病毒及相关冠状病毒的生物学特征[J].生物化学与生物物理学报.2003 , 35 (6) : 495～502
7. Cui, J., Han, N., Streicker, D.,et al. Evolutionary relationships between bat coronaviruses and their hosts[J]. Emerg. Infect. Dis. 2007, 13 (10), 1526～1532.
8. Lau, S.K., Woo, P.C., Li, K.S., et al. Severe acute respiratory syndrome coronaviruslikevirus in Chinese horseshoe bats[J]. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (39), 14040～14045.
9. Li, W., Shi, Z., Yu, M., et al. Bats are natural reservoirs of SARS-like coronaviruses[J]. Science 2005b, 310 (5748), 676～679.
10. Guan, Y., Zheng, B.J., He,et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern[J]. China. Science. 2003, 302 (5643), 276～278.
11.周丽萍,,罗仕伟,贺东生. SARS冠状病毒的分子生物学研究进展[J].生物技术通讯. 2006, 17(1), 75～77
12. Shi, Z., Hu, Z.,. A review of studies on animal reservoirs of the SARS coronavirus[J].Virus Res. 2008, 133 (1), 74～87.
13. Kan, B.; Wang, M.; Jing, H.;et al. Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms[J]. J.Virol. 2005, 79(18), 11892～11900.
14. Li, W.; Moore, M.J.; Vasilieva, N.; et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus [J]. Nature, 2003, 426(6965), 450～454.
15. Song, H.-D.; Tu, C.-C.; Zhang, G.-W., et al. Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human[J]. Proc. Natl. Acad. Sci. USA, 2005, 102(7), 2430～2435.
16. Kap-Sun Yeung, Nicholas A. Meanwell. Recent Developments in the Virology and Antiviral Research of Severe Acute Respiratory Syndrome Coronavirus[J]. Infectious Disorders-Drug Targets. 2007, 7: 29～41
17. Zhang, J.X.; Zhang, S.Y.; Wang, P.; et al. Prevalence and genetic diversity of coronaviruses in bats from China[J]. J. Virol., 2006, 80(15), 7481～7490.
18. Rota, P.A.; Oberste, M.S.; Monroe, S.S.; et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome [J]. Science, 2003, 300(5624), 1394～1399.
19. Stanley Perlman, Jason Netland. Coronaviruses post-SARS: Update on replication and pathogenesis[J]. Nat Rev Microbiol. 2009, 7(6): 439～450
20. Benjamin W. Neuman,Jeremiah S. Joseph,et al. Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3[J]. JOURNAL OF VIROLOGY . 2008, 82(11): 5279～5294
21.杨宇东,孙莉,陈忠斌. SARS冠状病毒PLpro蛋白酶的结构与功能[J].中国生物化学与分子生物学报. 2010, 26(1), 15～21
22. Chatterjee A, JohnsonM A, Serrano P, et al. Nuclearmagnetic resonance structure shows that the severe acute resp iratory syndrome coronavirus-unique domain contains amacrodomain fold [J]. J Virol, 2009, 83 (4) : 1823～1836
23. Kiira Ratia, Kumar Singh Saikatendu, Bernard D.et al Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme[J]. PNAS, 2006, 103, 5717～5722
24. Herold, J., Siddell, S. G. & Gorbalenya, A. E. A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold[J]. J. Biol. Chem. 1999 , 274(21), 14918～14925.
25. Ziebuhr, J., Thiel, V. & Gorbalenya, A. E. The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond[J]. J. Biol. Chem. 2001, 276(35), 33220～33232.
26. Naina Barretto, Dalia Jukneliene, Kiira Ratia, et al. The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity. JOURNAL OF VIROLOGY[J], 2005, 79(24) , 15189～15198
27. Thiel, V., Ivanov, K. A., Putics, A., et al. Mechanisms and enzymes involved inSARS coronavirus genome expression[J]. J. Gen.Virol. 2003. 84(pt9), 2305～2315.
28. Harcourt, B. H., Jukneliene, D., Kanjanahaluethai, A., et al. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity[J]. J. Virol. 2004. 78(24), 13600～13612.
29. Harcourt B H, Jukneliene D, Kanjanahaluethai A, et al. Identification of severe acute resp iratory syndrome coronavirus replicase p roducts and characterization of papain-like p rotease activity [J]. J Virol, 2004, 78 (24) : 13600～13612
30. Perlman S, Holmes K V. Advances in Experimental Medicine and Biology [M]. Sp ringerUS Press, 2006, 581: 37～41
31. Lindner H A, Lytvyn V, Qi H, et al. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like p rotease [J]. Arch Biochem Biophys, 2007, 466 (1) : 8～14
32.阳凯,王琛.宿主细胞应答病毒感染的细胞信号转导研究新进展[J].生物化学与物理进展, 2006,33(12):1138～1145.
33. Akira S, Takeda K. Toll - like receptor signaling[J]. Nat Rcv Immunol,2004, 4(7): 499- 511.
34. Qi Baochang, Huang Yue, Rowe D, et al. VISA- A pass to innate immunity[J]. Int J Biochem Cell Biol, 2007,39:287～291.
35. Garcia-Sastre A, Biron C A. Type 1 interferons and the virus- host relationship: a lesson in détente [J]. Science, 2006, 312 (5775) : 879～882
36. Devaraj S G, Wang N, Chen Z, et al. Regulation of IRF-3-dependent innate immunity by the papain2like p rotease domain of the severe acute resp iratory syndrome coronavirus [J]. J Biol Chem, 2007, 282 (44) : 32208～32221
37. Frieman M, Ratia K, Johnston R E, et al. Severe acute resp iratory syndrome coronavirus papain-like p rotease ubiquitin- like domain and catalytic domain regulate antagonism of IRF-3 and NF-κB signaling [J]. J Virol, 2009, 83 (13) : 6689～6705
38. O. Kerscher, R. Felberbaum, M. Hochstrasser, Modification of proteins by ubiquitin and ubiquitin-like proteins[J], Annu. Rev. Cell. Dev. Biol. 2006, 22: 159～180.
39.宋春雷,刘红林.组蛋白的泛素化与去泛素化修饰[J].细胞生物学杂志. 2007, 29: 51-55
40. Tom AM Groothuis, Nico P Dantuma, Jacques Neefjes et al. Ubiquitin crosstalk connecting cellular processes[J]. Cell Division 2006, 1:21
41.白勇,孙安盛,顾振纶等.泛素-蛋白酶体通路在细胞生存和肿瘤发生中的作用[J].中国药理学通报. 2008, 24 (10) : 1268～1271
42.赵天锁,任贺,郝继辉.泛素-蛋白酶体通路的研究进展[J].山东医药, 2009, 49: 113-114
43.曹建南,郭汉城.泛素-蛋白酶体系统在核因子κB途径中的作用[J].医学综述. 2009, 15(3), 398～400
44.肖兰博,李力力,曹亚.泛素在信号转导中的作用[J].生命的化学. 2007, 27(6): 527-531
45.邓彬蔚,张令强,贺福初.蛋白泛素途径及其非降解功能研究新进展[J].军事医学科学院院刊. 2006, 30(6): 565～569
46. GackM U, Shin Y C, Joo C H, et al. TRIM-5 RING-finger E3 ubiquitin ligase is essential for R IG-I-mediated antiviral activity [J]. Nature, 2007, 446 (7138) : 916～920
47. Huye L E, Ning S, KelliherM, et al. Interferon regulatory factor 7 is activated by a viral oncop rotein through RIP-dependent ubiquitination [J]. Mol Cell Biol, 2007, 27 (8): 2910～2918
48.胡日查,杨宇东,孙莉等.泛素样蛋白ISG15在抗病毒天然免疫中的作用[J].生物技术通讯. 2010, 21(1): 94～99
49. Ian F. Pitha-Rowe and Paula M. Pitha. Viral Defense, Carcinogenesis and ISG15: Novel Roles for an Old ISG. Cytokine Growth Factor[J]. Rev. 2007 ; 18(5-6): 409～417.
50.陈泉,施蕴渝.小范素相关修饰物SUMO研究进展.生命科学, 2004, 16 (1) : 1～6
51.韦玮,张浩,毛建平等.蛋白质SUMO化修饰研究进展[J].中国生物工程杂志. 2008, 28 (7) : 122～126
52. Melchior F. SUMO——nonclassical ubiquitin[J]. Annu Rev Cell Dev Biol. 2000 , 16 :591- 626
53. Xiang-Dong Li, Yang Shen,Ya-Feng Qiu,et al. SUMOylation: An Important Post-translational Modification in vivo[J]. Chinese Journal of Cell Biology. 2008, 30: 142～146
54. Min Hu, Pingwei Li1, Ling Song. Structure and mechanisms of the proteasomeassociated deubiquitinating enzyme USP14[J]. The EMBO Journal. 2005, 24, 3747～3756