Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2002 May 25;230(2):309–322. doi: 10.1006/viro.1997.8503

Efficient Autoproteolytic Processing of the MHV-A59 3C-like Proteinase from the Flanking Hydrophobic Domains Requires Membranes

Josefina D Piñón 1, Ravi R Mayreddy 1, Julie D Turner 1,1, Farah S Khan 1, Pedro J Bonilla 1,2, Susan R Weiss 1,3
PMCID: PMC7130731  PMID: 9143287

Abstract

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates.In vitroexpression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence ofN-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance thetransprocessing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed fromEscherichia coli.Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pellets. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.

References

REFERENCES

  • 1.Baker S.C., La Monica N., Shieh C.K., Lai M.M. Murine coronavirus gene 1 polyprotein contains an autoproteolytic activity. Adv. Exp. Med. Biol. 1990;276:283–289. doi: 10.1007/978-1-4684-5823-7_39. [DOI] [PubMed] [Google Scholar]
  • 2.Baker S.C., Yokomori K., Dong S., Carlisle R., Gorbalenya A.E., Koonin E.V., Lai M.M. Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus. J. Virol. 1993;67:6056–6063. doi: 10.1128/jvi.67.10.6056-6063.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bi W., Bonilla P.J., Holmes K.V., Weiss S.R., Leibowitz J.L. Intracellular localization of polypeptides encoded in mouse hepatitis virus open reading frame 1a. Adv. Exp. Med. Biol. 1995;380:251–258. doi: 10.1007/978-1-4615-1899-0_40. [DOI] [PubMed] [Google Scholar]
  • 4.Bienz K., Egger D., Pfister T. Characteristics of the poliovirus replication complex. Arch. Virol.—Supplementum. 1994;9:147–157. doi: 10.1007/978-3-7091-9326-6_15. [DOI] [PubMed] [Google Scholar]
  • 5.Bienz K., Egger D., Pfister T., Troxler M. Structural and functional characterization of the poliovirus replication complex. J. Virol. 1992;66:2740–2747. doi: 10.1128/jvi.66.5.2740-2747.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.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]
  • 7.Bonilla P.J., Hughes S.A., Piñón 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]
  • 8.Bonilla P.J., Hughes S.A., Weiss S.R. Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus MHV-A59. J. Virol. 1997;71:900–909. doi: 10.1128/jvi.71.2.900-909.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.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]
  • 10.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]
  • 11.Chambers T.J., Hahn C.S., Galler R., Rice C.M. Flavivirus genome organization, expression, and replication. [Review] Annu. Rev. Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. [DOI] [PubMed] [Google Scholar]
  • 12.den Boon J.A., Snijder E.J., Chirnside E.D., de V.A.A., Horzinek M.C., Spaan W.J. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. J. Virol. 1991;65:2910–2920. doi: 10.1128/jvi.65.6.2910-2920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.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]
  • 14.Dougherty W.G., Semler B.L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. [Review] Microbiol. Rev. 1993;57:781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Echeverri A.C., Dasgupta A. Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology. 1995;208:540–553. doi: 10.1006/viro.1995.1185. [DOI] [PubMed] [Google Scholar]
  • 16.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]
  • 17.Froshauer S., Kartenbeck J., Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J. Cell. Biol. 1988;107:2075–2086. doi: 10.1083/jcb.107.6.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.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]
  • 19.Grotzinger C., Heusipp G., Ziebuhr 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]
  • 20.Herold J., Raabe 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]
  • 21.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]
  • 22.Kolodziej P.A., Young R.A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  • 23.Lee H.J., Shieh C.K., Gorbalenya A.E., Koonin E.V., La M.N., Tuler J., Bagdzhadzhyan A., Lai M.M. 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]
  • 24.Lu X., Lu Y., Denison M.R. Intracellular and in vitro-translated 27-kDa proteins contain the 3C-like proteinase activity of the coronavirus MHV-A59. Virology. 1996;222:375–382. doi: 10.1006/viro.1996.0434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.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]
  • 26.Mostov K.E., DeFoor P., Fleischer S., Blobel G. Co-translational membrane integration of calcium pump protein without signal sequence cleavage. Nature. 1981;292:87–88. doi: 10.1038/292087a0. [DOI] [PubMed] [Google Scholar]
  • 27.Pachuk C.J., Bredenbeek P.J., Zoltick P.W., Spaan W.J., Weiss S.R. Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59. Virology. 1989;171:141–148. doi: 10.1016/0042-6822(89)90520-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Snijder E.J., Wassenaar A.L., Spaan W.J. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J. Virol. 1994;68:5755–5764. doi: 10.1128/jvi.68.9.5755-5764.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Snijder E.J., Wassenaar A.L., van D.L.C., Spaan W.J., Gorbalenya A.E. The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. J. Biol. Chem. 1996;271:4864–4871. doi: 10.1074/jbc.271.9.4864. [DOI] [PubMed] [Google Scholar]
  • 30.Tibbles K.W., Brierley I., Cavanagh D., Brown T.D. 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]
  • 31.Van Dinten L.C., Wassenaar A.L., Gorbalenya A.E., Spaan W.J., Snijder E.J. Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains. J. Virol. 1996;70:6625–6633. doi: 10.1128/jvi.70.10.6625-6633.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ziebuhr J., Herold J., Siddell S.G. Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. J. Virol. 1995;69:4331–4338. doi: 10.1128/jvi.69.7.4331-4338.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES