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Volume 83, Issue 3

Mutational analysis of the active centre of coronavirus 3C-like proteases Free

Abstract

Formation of the coronavirus replication–transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL, was determined. Comparative sequence analyses revealed that FIPV 3CL and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL domain and 25 mutants were expressed in and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL catalytic system employs His and Cys as the principal catalytic residues. Second, the amino acids Tyr and His, which are part of the conserved sequence signature Tyr–Met–His and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly and Asn, which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL mutants in which the equivalent Asn residue (HCoV 3CL Asn) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

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© Microbiology Society
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2002-03-01
2024-05-06
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References

  1. Allaire M., Chernaia M. M., Malcolm B. A., James M. N. 1994; Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases. Nature 369:72–76
     [Google Scholar]
  2. Almazán F., González J. M., Pénzes Z., Izeta A., Calvo E., Plana-Durán J., Enjuanes L. 2000; Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences, USA 97:5516–5521
     [Google Scholar]
  3. Baker S. C., Shieh C. K., Soe L. H., Chang M. F., Vannier D. M., Lai M. M. 1989; Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. Journal of Virology 63:3693–3699
     [Google Scholar]
  4. Bazan J. F., Fletterick R. J. 1988; Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proceedings of the National Academy of Sciences, USA 85:7872–7876
     [Google Scholar]
  5. Bergmann E. M., Mosimann S. C., Chernaia M. M., Malcolm B. A., James M. N. 1997; The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition. Journal of Virology 71:2436–2448
     [Google Scholar]
  6. Bonilla P. J., Hughes S. A., Weiss S. R. 1997; Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59. Journal of Virology 71:900–909
     [Google Scholar]
  7. Boniotti B., Wirblich C., Sibilia M., Meyers G., Thiel H. J., Rossi C. 1994; Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus. Journal of Virology 68:6487–6495
     [Google Scholar]
  8. Boursnell M. E. G., Brown T. D. K., Foulds I. J., Green P. F., Tomley F. M., Binns M. M. 1987; Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. Journal of General Virology 68:57–77
     [Google Scholar]
  9. Brierley I., Boursnell M. E., Binns M. M., Bilimoria B., Blok V. C., Brown T. D., Inglis S. C. 1987; An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV. EMBO Journal 6:3779–3785
     [Google Scholar]
  10. Cavanagh D. 1997; Nidovirales : a new order comprising Coronaviridae and Arteriviridae . Archives of Virology 142:629–633
     [Google Scholar]
  11. Chouljenko V. N., Lin X. Q., Storz J., Kousoulas K. G., Gorbalenya A. E. 2001; Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia. Journal of General Virology 82:2927–2933
     [Google Scholar]
  12. Cowley J. A., Dimmock C. M., Spann K. M., Walker P. J. 2000; Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses. Journal of General Virology 81:1473–1484
     [Google Scholar]
  13. de Groot R. J., Horzinek M. C. 1995; Feline infectious peritonitis. In The Coronaviridae pp 293–315 Edited by Siddell S. G. New York: Plenum Press;
     [Google Scholar]
  14. den Boon J. A., Snijder E. J., Chirnside E. D., de Vries A. A., Horzinek M. C., Spaan W. J. 1991; Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. Journal of Virology 65:2910–2920
     [Google Scholar]
  15. Denison M. R., Spaan W. J., van der Meer Y., Gibson C. A., Sims A. C., Prentice E., Lu X. T. 1999; The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis. Journal of Virology 73:6862–6871
     [Google Scholar]
  16. Dessens J. T., Lomonossoff G. P. 1991; Mutational analysis of the putative catalytic triad of the cowpea mosaic virus 24K protease. Virology 184:738–746
     [Google Scholar]
  17. Eleouet J. F., Rasschaert D., Lambert P., Levy L., Vende P., Laude H. 1995; Complete sequence (20 kilobases). of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology 206:817–822
     [Google Scholar]
  18. Gorbalenya A. E., Koonin E. V. 1993; Comparative analysis of the amino acid sequences of the key enzymes of the replication and expression of positive-strand RNA viruses. Validity of the approach and functional and evolutionary implications. Soviet Scientific Reviews Section D Physicochemical Biology Reviews 11:1–84
     [Google Scholar]
  19. Gorbalenya A. E., Snijder E. J. 1996; Viral cysteine proteinases. Perspectives in Drug Discovery and Design 6:64–86
     [Google Scholar]
  20. Gorbalenya A. E., Donchenko A. P., Blinov V. M., Koonin E. V. 1989a; Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Letters 243:103–114
     [Google Scholar]
  21. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. 1989b; Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Research 17:4847–4861
     [Google Scholar]
  22. Gorbalenya A. E., Koonin E. V., Lai M. M. 1991; Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and apthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Letters 288:201–205
     [Google Scholar]
  23. Grötzinger C., Heusipp G., Ziebuhr J., Harms U., Süss J., Siddell S. G. 1996; Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E. Virology 222:227–235
     [Google Scholar]
  24. Grubman M. J., Zellner M., Bablanian G., Mason P. W., Piccone M. E. 1995; Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus. Virology 213:581–589
     [Google Scholar]
  25. Hellen C. U., Facke M., Kräusslich H. G., Lee C. K., Wimmer E. 1991; Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220. Journal of Virology 65:4226–4231
     [Google Scholar]
  26. Henikoff S., Henikoff J. G. 1994; Position-based sequence weights. Journal of Molecular Biology 243:574–578
     [Google Scholar]
  27. Herold J., Raabe T., Schelle-Prinz B., Siddell S. G. 1993; Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology 195:680–691
     [Google Scholar]
  28. Herold J., Siddell S., Ziebuhr J. 1996; Characterization of coronavirus RNA polymerase gene products. Methods in Enzymology 275:68–89
     [Google Scholar]
  29. Herold J., Gorbalenya A. E., Thiel V., Schelle B., Siddell S. G. 1998; Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate. Journal of Virology 72:910–918
     [Google Scholar]
  30. Herold J., Siddell S. G., Gorbalenya A. E. 1999; A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold. Journal of Biological Chemistry 274:14918–14925
     [Google Scholar]
  31. Herrewegh A. A., Vennema H., Horzinek M. C., Rottier P. J., de Groot R. J. 1995; The molecular genetics of feline coronaviruses: comparative sequence analysis of the ORF7a/7b transcription unit of different biotypes. Virology 212:622–631
     [Google Scholar]
  32. Herrewegh A. A., Smeenk I., Horzinek M. C., Rottier P. J., de Groot R. J. 1998; Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus. Journal of Virology 72:4508–4514
     [Google Scholar]
  33. Heusipp G., Grötzinger C., Herold J., Siddell S. G., Ziebuhr J. 1997a; Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells. Journal of General Virology 78:2789–2794
     [Google Scholar]
  34. Heusipp G., Harms U., Siddell S. G., Ziebuhr J. 1997b; Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E. Journal of Virology 71:5631–5634
     [Google Scholar]
  35. Horzinek M. C., Lutz H., Pedersen N. C. 1982; Antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses. Infection and Immunity 37:1148–1155
     [Google Scholar]
  36. Jacobse-Geels H. E. L., Horzinek M. C. 1983; Expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrophage-like cells. Journal of General Virology 64:1859–1866
     [Google Scholar]
  37. Kanjanahaluethai A., Baker S. C. 2000; Identification of mouse hepatitis virus papain-like proteinase 2 activity. Journal of Virology 74:7911–7921
     [Google Scholar]
  38. Kean K. M., Teterina N. L., Marc D., Girard M. 1991; Analysis of putative active site residues of the poliovirus 3C protease. Virology 181:609–619
     [Google Scholar]
  39. Kimura M. 1983 The Neutral Theory of Molecular Evolution p– 367 Cambridge, NY: Cambridge University Press;
     [Google Scholar]
  40. Koonin E. V., Dolja V. V. 1993; Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28:375–430
     [Google Scholar]
  41. Lawson M. A., Semler B. L. 1991; Poliovirus thiol proteinase 3C can utilize a serine nucleophile within the putative catalytic triad. Proceedings of the National Academy of Sciences, USA 88:9919–9923
     [Google Scholar]
  42. Lee H. J., Shieh C. K., Gorbalenya A. E., Koonin E. V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M. M. 1991; The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180:567–582
     [Google Scholar]
  43. Lim K. P., Ng L. F., Liu D. X. 2000; Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products. Journal of Virology 74:1674–1685
     [Google Scholar]
  44. Liu D. X., Brown T. D. 1995; Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein. Virology 209:420–427
     [Google Scholar]
  45. Liu D. X., Brierley I., Tibbles K. W., Brown T. D. 1994; A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. Journal of Virology 68:5772–5780
     [Google Scholar]
  46. Liu D. X., Shen S., Xu H. Y., Wang S. F. 1998; Proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3C-like proteinase and identification of two novel cleavage products. Virology 246:288–297
     [Google Scholar]
  47. Lu Y., Denison M. R. 1997; Determinants of mouse hepatitis virus 3C-like proteinase activity. Virology 230:335–342
     [Google Scholar]
  48. Lu Y., Lu X., Denison M. R. 1995; Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59. Journal of Virology 69:3554–3559
     [Google Scholar]
  49. Lu X. T., Sims A. C., Denison M. R. 1998; Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro. Journal of Virology 72:2265–2271
     [Google Scholar]
  50. Matthews D. A., Smith W. W., Ferre R. A., Condon B., Budahazi G., Sisson W., Villafranca J. E., Janson C. A., McElroy H. E., Gribskov C. L. and others 1994; Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell 77:761–771
     [Google Scholar]
  51. Merrifield R. B. 1965; Automated synthesis of peptides. Science 150:178–185
     [Google Scholar]
  52. Mosimann S. C., Cherney M. M., Sia S., Plotch S., James M. N. 1997; Refined X-ray crystallographic structure of the poliovirus 3C gene product. Journal of Molecular Biology 273:1032–1047
     [Google Scholar]
  53. Ng L. F., Liu D. X. 2000; Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site. Virology 272:27–39
     [Google Scholar]
  54. Olsen C. W. 1993; A review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination. Veterinary Microbiology 36:1–37
     [Google Scholar]
  55. Page R. D. 1996; treeview: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12:357–358
     [Google Scholar]
  56. Rost B. 1996; PhD: predicting one-dimensional protein structure by profile-based neural networks. Methods in Enzymology 266:525–539
     [Google Scholar]
  57. Rost B., Casadio R., Fariselli P., Sander C. 1995; Transmembrane helices predicted at 95% accuracy. Protein Science 4:521–533
     [Google Scholar]
  58. Saitou N., Nei M. 1987; The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406–425
     [Google Scholar]
  59. Sawicki S. G., Sawicki D. L. 1998; A new model for coronavirus transcription. Advances in Experimental Medicine and Biology 440:215–219
     [Google Scholar]
  60. Sawicki D. L., Wang T., Sawicki S. G. 2001; The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus. Journal of General Virology 82:385–396
     [Google Scholar]
  61. Seybert A., Ziebuhr J., Siddell S. G. 1997; Expression and characterization of a recombinant murine coronavirus 3C-like proteinase. Journal of General Virology 78:71–75
     [Google Scholar]
  62. Seybert A., Hegyi A., Siddell S. G., Ziebuhr J. 2000a; The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5′-to-3′ polarity. RNA 6:1056–1068
     [Google Scholar]
  63. Seybert A., van Dinten L. C., Snijder E. J., Ziebuhr J. 2000b; Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases. Journal of Virology 74:9586–9593
     [Google Scholar]
  64. Siddell S. G. 1995; The Coronaviridae : an introduction. In The Coronaviridae pp 1–10 Edited by Siddell S. G. New York: Plenum Press;
     [Google Scholar]
  65. Snijder E. J., Wassenaar A. L., van Dinten L. C., Spaan W. J., Gorbalenya A. E. 1996; The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. Journal of Biological Chemistry 271:4864–4871
     [Google Scholar]
  66. Spaan W., Delius H., Skinner M., Armstrong J., Rottier P., Smeekens S., van der Zeijst B. A., Siddell S. G. 1983; Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO Journal 2:1839–1844
     [Google Scholar]
  67. Stephensen C. B., Casebolt D. B., Gangopadhyay N. N. 1999; Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay. Virus Research 60:181–189
     [Google Scholar]
  68. Swofford D. L. 2000 paup*: Phylogenetic Analysis using Parsimony, version 4 Sunderland: Sinauer Associates;
     [Google Scholar]
  69. Thiel V., Herold J., Schelle B., Siddell S. G. 2001a; Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. Journal of General Virology 82:1273–1281
     [Google Scholar]
  70. Thiel V., Herold J., Schelle B., Siddell S. G. 2001b; Viral replicase gene products suffice for coronavirus discontinuous transcription. Journal of Virology 75:6676–6681
     [Google Scholar]
  71. Thompson J. D., Higgins D. G., Gibson T. J. 1994; Improved sensitivity of profile searches through the use of sequence weights and gap excision. Computer Applications in the Biosciences 10:19–29
     [Google Scholar]
  72. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25:4876–4882
     [Google Scholar]
  73. van Marle G., Dobbe J. C., Gultyaev A. P., Luytjes W., Spaan W. J., Snijder E. J. 1999; Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proceedings of the National Academy of Sciences, USA 96:12056–12061
     [Google Scholar]
  74. Vennema H., Rossen J. W., Wesseling J., Horzinek M. C., Rottier P. J. 1992; Genomic organization and expression of the 3′ end of the canine and feline enteric coronaviruses. Virology 191:134–140
     [Google Scholar]
  75. Vennema H., Poland A., Foley J., Pedersen N. C. 1998; Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virology 243:150–157
     [Google Scholar]
  76. Yao Z., Jones D. H., Grose C. 1992; Site-directed mutagenesis of herpesvirus glycoprotein phosphorylation sites by recombination polymerase chain reaction. PCR Methods & Applications 1:205–207
     [Google Scholar]
  77. Yount B., Curtis K. M., Baric R. S. 2000; Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model. Journal of Virology 74:10600–10611
     [Google Scholar]
  78. Yu S. F., Lloyd R. E. 1991; Identification of essential amino acid residues in the functional activity of poliovirus 2A protease. Virology 182:615–625
     [Google Scholar]
  79. Ziebuhr J., Siddell S. G. 1999; Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab. Journal of Virology 73:177–185
     [Google Scholar]
  80. Ziebuhr J., Herold J., Siddell S. G. 1995; Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. Journal of Virology 69:4331–4338
     [Google Scholar]
  81. Ziebuhr J., Heusipp G., Siddell S. G. 1997; Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase. Journal of Virology 71:3992–3997
     [Google Scholar]
  82. Ziebuhr J., Snijder E. J., Gorbalenya A. E. 2000; Virus-encoded proteinases and proteolytic processing in the Nidovirales . Journal of General Virology 81:853–879
     [Google Scholar]
  83. Ziebuhr J., Thiel V., Gorbalenya A. E. 2001; 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. Journal of Biological Chemistry 276:33220–33232
     [Google Scholar]
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Mutational analysis of the active centre of coronavirus 3C-like proteases
J. Gen. Virol. 83, 581 (2002); https://doi.org/10.1099/0022-1317-83-3-581
/content/journal/jgv/10.1099/0022-1317-83-3-581
/content/journal/jgv/10.1099/0022-1317-83-3-581
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