摘要
本研究以人类新型冠状病毒NL63 (human coronavirus NL63, HCoV-NL63)木瓜样蛋白酶(papain-like protease, PLP)为研究对象,首先构建了木瓜样蛋白酶1 (papain-like protease 1, PLP1)的突变体D1225A和木瓜样蛋白酶2 (papain-like protease 2, PLP2)的突变体D1849A表达载体。通过Western Blotting技术证实,PLP1及其突变体C1062A、H1212A、D1225A对泛素(ubiquitin, Ub)分子不具有去泛素化(deubiquitinase, DUB)活性,PLP2却明显不同,野生型PLP2及突变体D1849A具有较强的DUB活性,而突变体C1678A、H1836A则丧失了此活性,同时对泛素样分子——干扰素刺激基因15(interferon stimulate gene 15, ISG15)进行检测,得到了相同的结论,提示PLP2的第1678位半胱氨酸和第1836位组氨酸是其DUB活性所必需的位点,第1849位天冬氨酸属于非必需位点。
HCoV-NL63 PLP对宿主抗病毒天然免疫反应调控机制研究证实,PLP1及突变体C1062A、H1212A、D1225A均不能抑制干扰素-β(interferon-β, IFN-β)表达;PLP2和突变体D1849A却能强烈抑制IFN-β表达,但突变体C1678A、H1836却丧失了此功能。这表明,PLP2的干扰素拮抗活性与PLP2的DUB活性具有一定相关性。同时,对整个通路中关键蛋白调控的研究发现,PLP2与维甲酸诱导基因I (retinoic acid inducible gene I, RIG-I)、ERIS(endoplasmic reticulum IFN stimulator)相互作用,并利用自身的DUB活性特异性切除RIG-I、ERIS分子的多聚泛素化修饰,阻断其天然免疫反应,逃逸宿主防御系统。
This project researches on papain-like protease of human coronavirus NL63. We constructed the mutants PLP1 D1225A and PLP2 D1849A. Western Blotting assay showed that PLP2 and PLP2 D1849A not PLP1 and its mutants have deubiquitinating(DUB) activity. Meanwhile, ubiquitin-like molecule the interferon sitimulate gene (ISG) was detected, we proved that PLP2 has deISGylation activity. It was suggested that the DUB activity of PLP2 is not dependent on its protease activity perfectly C1678A and H1836A are necessary for its DUB activity, but not D1849A.
The research on PLP regulating innate immune response revealed that PLP2 not PLP1 supresses interferon (IFN) production, and this activity doesn’t dependent on PLP2 C1678 and PLP2 H1836. The results indicated that the IFN antagonist activity of PLP2 is related with its DUB activity. Further research manifested that PLP2 supresses IFN expression pathway by deubiquitinating of the retinoic acid inducible gene I (RIG-I) and the endoplasmic reticulum IFN stimulator (ERIS).
引文
1 Cavanagh D. Coronaviruses in poultry and other birds[J]. Avian Pathol, 2005, Vol.349(6): 439-448
2 Baker S C. Coronaviruses: from common colds to severe acute respiratory syndrome. Pediatr[J]. Infect Dis, 2004, Vol.23(11): 1049-1050
3 Lai M M, Cavanagh D. The molecular biology of coronaviruses[J]. Adv Virus Res, 1997, Vol.48: 91-100
4 Poon L L, Chu D K, Chan K H, et al. Identification of a novel coronavirus in bats[J]. J Virol, 2005, Vol.79(4): 2001-2009
5 Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome[J]. N Engl J Med, 2003, Vol.348(20): 1967-1976
6 Ksiazek T G, Erdman D, Goldsmith C S, et al. A novel coronavirus associated with severe acute respiratory syndrome[J]. N EnglJ Med, 2003, Vol.348(2): 1953-1966
7 Van der Hoek L, Pyrc K, Jebbink M F,et al. Identification of a new human coronavirus [J]. Nat Med, 2004, Vol.10(4): 368-373
8 Forster J, Ihorst G, Rieger C H, et al. Prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the PRI DE study)[J].Eur J Pediatr, 2004, Vol.163(12): 709-716
9 Esper F, Shapiro E D, Weibel C, Ferguson D, Landry M L, Kahn J S. Association between a novel human coronavirus and Kawasaki Disease[J]. J Infect Dis, 2005, Vol.191(4): 499-502
10 Esper F, Weibel C, Ferguson D, Landry M L, Kahn JS. Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children[J]. J Infect Dis, 2005, Vol.191(2): 492-498
11 Pyrc K, M F Jebbink, B Berkhout, van der Hoek.Genome structure and transcriptional regulation of human coronavirus NL63[J]. 2004, Virol J. Vol.17(1) 7
12 Sperry, S M, kazi L, Graham, Single-amino-acid substitutions in open reading frame (ORF) 1b-nsp14 and ORF 2a proteins of the coronavirus mouse hepatitis vius are attenuating in mice[J]. J Virol,2005, Vol.79(6): 3391-3400
13 Zhu H, Hu S, Jona G, Zhu X, et al.Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray[J]. Proc Natl Acad Sci USA, 2006, Vol.103(11):4011-6
14 Diebuhr J. The coronavirus replicase[J]. Immunol, 2005, Vol.287: 57-94
15 Delmas B, Gelfi J, Haridon R, Vogel L K, Sjostrom H, Noren Laude H. AminopeptidaseN is a major receptor for the entero-pathogenic coronavirus TGEV[J]. Nature, 1992, Vol.357(6377): 417-420
16 Yeager C L, Ashmun R A, WilliamsR K, Cardellichio C B, Shapiro L H, Look A T , Holmes K V.Human aminopeptidase N is a receptor for human coronavirus 229E[J]. Nature, 1992, Vol.357(2): 420-422
17 Tresnan B, Levis R, Holmes K V. Feline aminopeptidase N serves as a receptor for feline canine porcine and human coronaviruses in serogroup[J]. J Virol, 1996, Vol.70(12) 8669-8674
18 Fouchier R A, Hartwig N G, Bestebroer T M, Niemeyer B, de Jong J C, Simon J H, Osterhaus A D. A previously undescribed coronavirus associated with respiratory disease in humans[J]. Proc Natl Acad Sci USA, 2004, Vol.101(16): 6212-6216
19 Hofmann H, K Pyrc, van der Hoek, M Geier, B Berkhout, and S Pohlmann. Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellμLar entry[J]. Proc Natl Acad Sci USA, 2005, Vol.102(22): 7988-7993
20 Hofmann H, G Simmons, A J Rennekamp, Chaipan C, Gramberg. Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors[J]. J Virol, 2006, Vol.80(17): 8639-8652
21 Huang I C, Bosch B J, Li F, et al. SARS coronavirus but not human coronavirus NL63 utilizes cathepsin L to infect ACE2- expressing cells[J]. J Biol Chem, 2005, Vol.281(6): 3198-3203
22 Thiel V, Ivanov K A, Putics, Hertzig T, Schelle B, Mechanisms and enzymes involved in SARS coronavirus genome expression[J]. J Gen Virol, 2003, Vol.84(9): 2305-2315
23 Goldsmith C S, Tatti K M, Ksiazek T G, Rollin P E, et al. Ultrastructural characterization of SARS coronavirus[J]. Emerg.Infect. Dis, 2004, Vol. 10(2):320-326
24 Gosert R, Kanjanahaluethai A , Egger D, Bienz K, Baker S C. RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol, 2002,Vol.76(8): 3697-3708
25 Hau, S K, Woo P C, Li K S, Huang Y, et al. Severe acute respiratory syndrome coronavirus -like virus in Chinese horseshoe bats[J]. Proc Nat Acad Sci USA, 2005, Vol.2(3): 14040-14045
26 Shi Z, Yu M, Ren W, et al. Bats are natural reservoirs of SARS-like coronaviruses[J]. Science, 2005, Vol.310(5748): 676-679
27 Liu, D X, and Brown T D. Characterisation and mutational analysis of an ORF
1a-encoding proteinase domain responsible for proteolytic processing of theinfectious bronchitis virus 1a/1b polyprotein[J]. Virology, 1995, Vol.209(2): 420-427
28 Lim K P , Liu D X .Characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination ofthe C-terminal cleavage site of an 87-kDa protein[J]. Virology, 1998, Vol.245(4): 303-312
29 Liu W, Tang F, Fontanet A, et al. Long-term SARS coronavirus excretion from patientcohort China[J]. Emerg Infect Dis, 2004, Vol.10(10): 1841-1843
30 lmeida, J D, and Tyrrell DA. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture[J]. J Gen Virol, 1967, Vol. 1(2): 175-178
31 CIntosh K, Dees J H, Becker W B, Kapikian A Z, Chanock R M. Recovery in tracheal organ cμLtures of novel viruses from patients with respiratory disease[J]. Proc Natl Acad Sci USA, 1967, Vol.57(4): 933-940
32 Holmes, Lippincott Williams K V , Wilkins, Philadelphia, et al. Identification of a novelcoronavirus in patients with severe acute respiratory syndrome[J]. N. Engl.J. Med, 1967, Vol.348(20): 1967-1976
33 Blanchard J E, Elowe N H, Huitema C, Fortin P D, et al. High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase[J]. Chem Biol, 2004, 11(10): 1445-1453.
34 Zhu Z, Dimitrov A S,Chakraborti, et al. Development of human monoclonal antibodies against diseases caused by emerging and biodefense-related viruses[J]. CCRNP, 2006, Vol.4(1): 573-66
35 Yam W C, Chan K H, Poon L L, Guan Y, et al. Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus[J]. J Clin Microbiol, 2003, Vol.41(10):4521-4
36 Arden K E, Nissen M D, Sloots T P, Mackay I M. New human coronavirus HCoV-NL63 associated with severe lower respiratory tract disease in Australia[J]. J Med Virol, 2005, Vol.75(3): 455-462
37 Ebihara T, Endo R, Ma X, Ishiguro N, Kikuta H. Detection of human coronavirus NL63 in young children with bronchiolitis[J]. J Med Virol, 2005, Vol.75: 463-465
38 Suzuki A, Okamoto M, Ohmi A, Watanabe O, Miyabayashi S Nishimura H. Detection of human coronavirus-NL63 in children in Japan[J]. Pediatr Infect Dis J, 2005, Vol.24(7): 645-646
39 Vabret A, Dina J, Gouarin S, Petitjean J, Corbet S, Freymuth F. Detection of thenew human coronavirus HKU1: a report of 6 cases[J]. Clin Infect Dis, 2006, Vol.42(5): 634-639
40 Chiu S S, Chan K H, Chu K W, Kwan S W, Guan Y, Poon L L, Peiris J S .Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong China. Clin Infect Dis, 2005, Vol.40(12): 1721-1729
41 Hendley J O, Fishburne H B, Gwaltney J M Jr. Coronavirus infections in working adults. Eight-year study with 229 E and OC43[J]. Am Rev Respir Dis, 1972, Vol.105(4): 805-811
42 Endo R, Ma X, Ju N, et al. Lack of association between New Haven coronavirus and Kawasaki disease[J]. Infect Dis, 2005, Vol.192(2): 351-352
43 Piao J H, Jin L H, et al. Epidemiological investigation of Kawasaki disease in Jilin province of China from 2000 to 2008[J]. J Infect Dis, 2010, Vol.26: 1-7
44 Shimizu C, Shike H, Baker S C, et al. Human coronavirus NL63 is not detectedin the respiratory tracts of children with acute Kawasaki disease[J]. J Infect Dis, 2005, Vol.192(10): 1767-1771
45 Kawasaki T, Kosaki F, Okawa S, A new infantile acute febrile mucocutaneous lymph node syndrome (MLNS) prevailing in Japan[J]. Pediatrics, 1974, Vol.54(3):271-276
46 lmeida J D, Tyrrell D A. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture[J]. J Gen Virol, 1967, Vol.1(2): 175-178
47 Rowe T, Gao G, Hogan, Crystal R J, Voss R G, et al. Macaque model for severe acute respiratory syndrome [J]. J Virol, 2004, Vol.78: 11401-11404
48 Jin M S, Lee J O. Structures of the toll-like receptor family and its ligand complexes [J]. Immunity, 2008, Vol.29(20): 182-191
49 Martinon F, Tschopp J. Inflammatory caspases and inflammasomes: master switches of inflammation [J]. Cell Death Differ, 2007, Vol.14(1): 10-22
50 Kakahasi K, Yoneyama M, Nishihori T, et al. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses[J]. Mol Cell, 2008, Vol.29(4): 428 - 440
51 Fneyama M, Fujita T. Structural mechanism of RNA recognition by the RIG-Ilike Receptors[J]. Immunity, 2008, Vol.29(2):178-181
52 Eamming D, Horvath C M. Regulation of signal transduction by enzymatically inactive antiviral RNA helicase proteins MDA5, RIG-I and LGP2 [J]. J Biol Chem, 2009, Vol.284(15): 9700-9712
53 Cui S, Eisenacher K, Kirchhofer A, et al. The C-terminal regμLatory domain is the RNA 5-triphosphate sensor of RIG-I [J]. Mol Cell, 2008, Vol.29(2): 169–179
54 Lin R, Lacoste J, Nakhaei P, et al. Dissociation of a MAVS/ IPS-1 /VISA /Cardif -IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3-4A proteolytic cleavage[J]. J Virol,2006, Vol.80(12): 6072-6083
55 Zhong B, Yang Y, Shu H B, et al. The Adaptor Protein MITA Links Virus-Sensing Receptors to IRF-3 Transcription Factor Activation[J]. Immunity, 2008, Vol.29(4): 538-550
56 Sun W X, Li Y, Chen L B, et al. ERIS, an endoplasmic reticμLum IFN stimμLator, activates innate immune signaling through dimerization[J]. Proc Natl Acad Sci USA, 2009, Vol.106(21): 8653-8658
57 Gack M U, Shin Y C, Joo C H, et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity[J]. Nature, 2007, Vol.446(7138): 916-920
58 Lin R, Yang L, Sun Q, et al. Negative regulation of the retinoic acid-inducible gene I-induced antiviral state by the ubiquitin-editing protein A20[J]. J Biol Chem, 2006, Vol.281(4):2095-2103
59 Nobuhiko K, Qui P, Karen M, et al. DUBA: A Deubiquitinase That Regulates Type I Interferon Production[J]. Science, 2007, Vol.318(5856): 1628-1632
60 Arimoto K, Takahashi H, Hishiki T, et al. Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125[J]. Proc Natl Acad Sci USA , 2007, Vol.104(18): 7500-7505
61 Dahai Zheng, Gang Chen1,Beichu Guo, Genhong Cheng, Hong Tang. PLP2, a potent deubiquitinase from murine hepatitis virus,strongly inhibits cellular type I interferon production[J]. Cell Research, 2008, Vol.18(11):1105-1113
62 Gack, M U, Kirchhofer A, Shin Y C, et al. Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction[J]. Proc Nat Acad Sci USA, 2008, Vol.105(43): 16743-16748
63 Guo B, Cheng G. Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK[J]. J Biol Chem, 2007, Vol.282(16): 11817-11826
64 Joureiro J, Ploegh H L. Antigen presentation and the ubiquitin–proteasome system in host–pathogen interactions[J]. Adv Immunol, 2006, Vol.92: 225-305
65 Vinuesa C G. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity[J]. Nature, 2005, Vol.435(7041): 452-458
66 Venuprasad, K. et al. The E3 ubiquitin ligase Itch regμLates expression of transcription factor Foxp3 and airway inflammation by enhancing the function of transcription factor TIEG1[J]. Nature Immunol, 2008, Vol.9(3): 245-253
67 Jeon, M S. Essential role of the E3 ubiquitin ligase Cbl-b in T cell anergyinduction[J]. Immunity, 2004, Vol.21(2): 167-177
68 Bongiovanni A, Nastasi T. E3 ubiquitin ligases as regulators of membrane protein trafficking and degradation[J]. Traffic, 2005, Vol.6(6): 429-441
69 Vijay G Bhoj,Zhijian J, Chen. Ubiquitylation in innate and adaptive immunity[J]. Nature Review, 2009, Vol.458(7237): 430-437
70 Bennett, Harper E J. DNA damage: ubiquitin marks the spot[J]. Nature Struct Mol Biol, 2008, Vol.15(1): 20-22
71 Zhongbin Chen, Yanhua Wang, Kiira Ratia, et al. Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63[J]. J.VIROLOGY, 2007, Vol.6(1): 6007-6018
72 Wan Y Y, Chi, xie M, et al. The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development,survival and function [J].Nature Immunol, 2006, Vol. 7(8): 851-858
73 Amerik A K,Hochstrasser M. Mechanism and function of deubiquitylating enzymes[J]. Biochim Biophys Acta, 2004, Vol.1695(5): 189-207
74 Sulea T, Lindner H A, Purisima E O, Menard R. Deubiquitination,acute resoiratory svndrome coronavirus papain-like protease[J]. J Virol, 2005, Vol.79(7): 4550-4551
75 Mukhopadhyay, Riezman D. Proteasome-independent functions of ubiquitin in endocytosis and signaling[J]. Science, 2007, Vol.315(5809): 201-205
76 Mashimo T, Hadjebi O, Amair-Pinedo F. Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase. [J]. PLos genet, 2009, Vol.5(6): 629-641
77 Ciechanover A, The ubiquitin proteolytic system: from a vague idea,through basic mechanisms and onto human diseases and drug targeting[J]. Neurology, 2006, Vol.66(2): S7-S19
78 Lenschow, D J, GiannakopouLos N V, Gunn L J, et al. Identification of interferon stimulated gene 15 as an antivirus molecμLe during Sindbis virus infection in vivo[J]. J Virol, 2005, Vol.79(3): 13974-13983
79 Wang S Y, Imaizumi T, et al. Negative feedback regulation of RIG-Imediated antiviral signaling by interferon-induced ISG15 conjugation [J]. J Virol, 2008, Vol.82(3): 1474 -1483
80 Murray C J L, Lopex A D, Marheres C D, et al. The global burden of disease 2000 project: aims methods and data sources World Health Organization: Global programme on evidence for health policy[R]. Geneva, 2001.
81梁万年,黄若刚,赵悦等. SARS病毒及其流行特征[J].中国全科医学, 2003, Vol.6(7):527-530
82 Australian Government Department of Health and Ageing.Pandemic (H1N1) Update BuLletin[R], Canberra, 2009.
83 Hscott J. Convergence of the NF-kappaB and IRF pathways in the regulation of the innate antiviral response[J]. Cytokine , Growth Factor Reviews, 2007, Vol.18(5-6): 483-490
84 Rowzard J B, Ranjan P, Sambhara S, et al. Antiviral defense: RIG-Ing the immune system to STING[J]. Cytokine,Growth Factor Reviews, 2009, Vol.20(1): 1-5
85 Kira S, Uematsu S, Takeuchi O, et al. Pathogen recognition and innate Immunity[J]. Cell, 2006, Vol.124(4): 783-801
86 Kematsu S, Akira S. Toll-like receptors and innate immunity[J]. JMol Med, 2006, Vol.84(6): 712-725
87 Perry A K, Chen G, Zheng D, et al. The host type I interferon response to viral and bacterial infections[J]. Cell Res, 2005, Vol.15(6): 407-422
88 Zhou H, Perlman S. Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by doublestranded RNA[J]. J Virol, 2007, Vol.81(1): 568-574
89 Friedman C S, Donnell M A, Legarda-Addison D, et al. The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response[J]. EMBO, 2008, Vol. 9(9): 930-936
90 Ingham K C, Brew S, et al. Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry and modular requirements[J]. J B C, 2004, Vol.279(41): 429458-53
91 Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K, et al. Cell type-specific involvement of RIG-I in antiviral response[J]. Immunity, 2005, Vol.23()(1): 19-26
92 Zhong B, Zhang L, Shu H B, et al. The Ubiquitin Ligase RNF5 Regulates Antiviral Responses by Mediating Degradation of the Adaptor Protein MITA[J]. Immunity, 2009, Vol.30(3): 397-407