Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2002 May 25;263(2):471–484. doi: 10.1006/viro.1999.9954

Further Requirements for Cleavage by the Murine Coronavirus 3C-like Proteinase: Identification of a Cleavage Site within ORF1b

Josefina D Piñón 1, Henry Teng 1, Susan R Weiss 1,1
PMCID: PMC7131300  PMID: 10544119

Abstract

The coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q939↓S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL↓Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL↓Zn site, as well as the previously identified HD1↓3C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD1↓3C and POL↓Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD1↓3C site was processed more efficiently than a polypeptide substrate carrying the POL↓Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase.

References

REFERENCES

  • 1.Baker S.C., Shieh C.K., Chang M.F., Vannier D.M., Lai M.M.C. Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. J. Virol. 1989;63:3693–3699. doi: 10.1128/jvi.63.9.3693-3699.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bonilla P.J., Gorbalenya A.E., Weiss S.R. Mouse hepatitis virus strain A59 RNA polymerase gene ORF 1a: heterogeneity among MHV strains. Virology. 1994;198:736–740. doi: 10.1006/viro.1994.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bonilla P.J., Hughes S.A., Piñon J.D., Weiss S.R. Characterization of the leader papain-like proteinase of MHV-A59: Identification of a new in vitro cleavage site. Virology. 1995;209:489–497. doi: 10.1006/viro.1995.1281. [DOI] [PubMed] [Google Scholar]
  • 4.Boursnell M.E., Brown T.D., Foulds I.J., Green P.F., Tomley F.M., Binns M.M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J. Gen. Virol. 1987;68:57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  • 5.Bredenbeek P.J., Pachuk C.J., Noten A.F., Charite J., Luytjes W., Weiss S.R., Spaan W.J. The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. Nucleic Acids Res. 1990;18:1825–1832. doi: 10.1093/nar/18.7.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Brierley I., Boursnell M.E.G., Binns M.M., Billimoria B., Blok V.C., Brown T.D.K., Inglis S.C. An efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus IBV. EMBO J. 1987;6:3779–3785. doi: 10.1002/j.1460-2075.1987.tb02713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cavanagh D. A new order comprising Coronaviridae and Arteriviridae. Arch. Virol. 1997;142:629–633. [PubMed] [Google Scholar]
  • 8.Denison M.R., Hughes S.A., Weiss S.R. Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine coronavirus MHV-A59. Virology. 1995;20:316–320. doi: 10.1006/viro.1995.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Denison M.R., Spaan W.J.M., van der Meer Y., Gibson C.A., Sims A.C., Prentice E., Lu X.T. 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. J. Virol. 1999;73:6862–6871. doi: 10.1128/jvi.73.8.6862-6871.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Denison M.R., Zoltick P.W., Hughes S.A., Giangreco B., Olson A.L., Perlman S., Leibowitz J.L., Weiss S.R. Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events. Virology. 1992;189:274–284. doi: 10.1016/0042-6822(92)90703-R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Denison M.R., Zoltick P.W., Leibowitz J.L., Pachuk C.J., Weiss S.R. Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59. J. Virol. 1991;65:3076–3082. doi: 10.1128/jvi.65.6.3076-3082.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.de Vries A.A.F., Horzinek F.M.C., Rottier P.J.M., de Groot R.J. The genome organization of the Nidovirales: Similarities and differences between arteri-, toro-, and coronaviruses. Semin. Virol. 1997;8:33–47. doi: 10.1006/smvy.1997.0104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Dougherty W.G., Semler B.L. Expression of virus-encoded proteinases: Functional and structural similarities with cellular enzymes. Microbiol. Rev. 1993;57:781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Eleouet J.F., Rasschaert D., Lambert P., Levy L., Vende P., Laude H. Complete sequence (20 kb) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology. 1995;206:817–822. doi: 10.1006/viro.1995.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Gorbalenya A.E., Koonin E.V., Donchenko A.P., Blinov V.M. Coronavirus genome: Prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Res. 1989;17:4847–4861. doi: 10.1093/nar/17.12.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Grötzinger C., Heusipp G., Ziebhur J., Harms U., Suss J., Siddell S.G. Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E. Virology. 1996;222:227–235. doi: 10.1006/viro.1996.0413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Herold J., Rabbe T., Schelle-Prinz B., Siddell S.G. Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology. 1993;195:680–691. doi: 10.1006/viro.1993.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Herold J., Siddell S.G. An “elaborated” pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA. Nucleic Acids Res. 1993;21:5838–5842. doi: 10.1093/nar/21.25.5838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Herold J., Siddell S.G., Ziebuhr J. Characterization of coronavirus RNA polymerase gene products. Methods Enzymol. 1996;275:68–89. doi: 10.1016/S0076-6879(96)75007-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Heusipp G., Grötzinger C., Herold J., Siddell S.G., Ziebuhr J. Identification and subcellular localization of a 41 kDa polyprotein 1ab processing product in human coronavirus 229E-infected cells. J. Gen. Virol. 1997;78:2789–2794. doi: 10.1099/0022-1317-78-11-2789. [DOI] [PubMed] [Google Scholar]
  • 21.Heusipp G., Harms U., Siddell S.G., Ziebuhr J. Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E. J. Virol. 1997;71:5631–5634. doi: 10.1128/jvi.71.7.5631-5634.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Holmes K.V., Lai M.M.C. In: Fields Virology. Fields B.N., Knipe D.M., Howley P.M., editors. Lippincott–Raven; Philadelphia: 1996. Coronaviridae: The viruses and their replication; pp. 1075–1093. [Google Scholar]
  • 23.Houtman J.J., Fleming J.O. Pathogenesis of mouse hepatitis virus-induced demyelination. J. Neurovirol. 1996;6:361–376. doi: 10.3109/13550289609146902. [DOI] [PubMed] [Google Scholar]
  • 24.Hughes S.A., Bonilla P.J., Weiss S.R. Identification of the murine coronavirus p28 cleavage site. J. Virol. 1995;69:809–813. doi: 10.1128/jvi.69.2.809-813.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kim J.C., Spence R.A., Currier P.F., Lu X., Denison M.R. Coronavirus protein processing and RNA synthesis is inhibited by the cysteine proteinase inhibitor E64d. Virology. 1995;208:1–8. doi: 10.1006/viro.1995.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lai M.M.C. Coronavirus: Organization, replication and expression of genome. Annu. Rev. Microbiol. 1990;44:303–333. doi: 10.1146/annurev.mi.44.100190.001511. [DOI] [PubMed] [Google Scholar]
  • 27.Lee H.J., Shieh C.K., Gorbalenya A.E., Koonin E.V., LaMonica N., Tuler J., Bagdzhadzhyan A., Lai M.M.C. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991;180:567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Liu D.X., Brierley I., Tibbles K.W., Brown T.D.K. A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of coronavirus infectious bronchitis virus is processed by ORF1a products. J. Virol. 1994;68:5772–5780. doi: 10.1128/jvi.68.9.5772-5780.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liu D.X., Brown T.D. Characterization and mutational analysis of an ORF1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein. Virology. 1995;209:420–427. doi: 10.1006/viro.1995.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Liu D.X., Shen S., Xu H.Y., Wang S.F. 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. 1998;246:288–297. doi: 10.1006/viro.1998.9199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Liu D.X., Xu H.Y., Brown T.D.K. Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: Identification of a 10-kilodalton polypeptide and determination of its cleavage sites. J. Virol. 1997;71:1814–1820. doi: 10.1128/jvi.71.3.1814-1820.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lu Y., Lu X., Denison M.R. Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59. J. Virol. 1995;69:3554–3559. doi: 10.1128/jvi.69.6.3554-3559.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Lu X.T., Sims A.C., Denison M.R. 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. J. Virol. 1998;72:2265–2271. doi: 10.1128/jvi.72.3.2265-2271.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ng L.F.P., Liu D.X. Identification of a 24-kDa polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3C-like proteinase and determination of its cleavage sites. Virology. 1998;243:388–395. doi: 10.1006/viro.1998.9058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Piñón J.D., Mayreddy R.R., Turner J.D., Khan F.S., Bonilla P.J., Weiss S.R. Efficient autoproteolytic processing of the MHV-A59 3C-like proteinase from the flanking hydrophobic domains requires membranes. Virology. 1997;230:309–322. doi: 10.1006/viro.1997.8503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Seybert A., Ziebuhr J., Siddell S.G. Expression and characterization of a recombinant murine coronavirus 3C-like proteinase. J. Gen. Virol. 1997;78:71–75. doi: 10.1099/0022-1317-78-1-71. [DOI] [PubMed] [Google Scholar]
  • 37.Sims A.C., Lu X.T., Denison M.R. Expression, purification, and activity of recombinant MHV-A59 3CLpro. Adv. Exp. Med. Biol. 1998;440:129–134. doi: 10.1007/978-1-4615-5331-1_17. [DOI] [PubMed] [Google Scholar]
  • 38.Teng H., Piñón J.D., Weiss S.R. Expression of murine coronavirus recombinant papain-like proteinase: Efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides. J. Virol. 1999;73:2658–2666. doi: 10.1128/jvi.73.4.2658-2666.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Tibbles K.W., Brierley I., Cavanagh D., Brown T.D.K. Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus. J. Virol. 1996;70:1923–1930. doi: 10.1128/jvi.70.3.1923-1930.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.van Dinten L.C., Sietske R., Gorbalenya A.E., Snijder E.J. Proteolytic processing of the open reading frame 1b-encoded part of the arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication. J. Virol. 1999;73:2027–2037. doi: 10.1128/jvi.73.3.2027-2037.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ziebuhr J., Herold J., Siddell S.G. Characterization of a human coronavirus (strain 229E) 3C-like proteinase assay. J. Virol. 1995;69:4331–4338. doi: 10.1128/jvi.69.7.4331-4338.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ziebuhr J., Siddell S.G. 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. J. Virol. 1999;73:177–185. doi: 10.1128/jvi.73.1.177-185.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES