Skip to main content
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2002 May 25;246(2):288–297. doi: 10.1006/viro.1998.9199

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

DX Liu 1,1, S Shen 1, HY Xu 1, SF Wang 1
PMCID: PMC7131314  PMID: 9657947

Abstract

We have previously reported that the 3C-like proteinase of the coronavirus infectious bronchitis virus (IBV) is responsible for processing of the 1a and 1a/1b polyproteins to three mature products of 24, 10, and 100 kDa (Liuet al.,1994, 1997; Ng and Liu, 1998). The C-terminal cleavage site of the 100-kDa protein was defined to be the Q891(1b)-S892(1b)dipeptide bond encoded by nucleotides 15,129 to 15,134 (Liu and Brown, 1995). In this report, other cleavage sites of the 3C-like proteinase in the polyprotein encoded by the ORF 1b region were mapped by coexpression, deletion, and site-directed mutagenesis studies. Using two ORF 1b-specific antisera, V58 and V17, three more Q-S(G) dipeptide bonds, encoded by nucleotides 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, were demonstrated to be the cleavage sites of the 3C-like proteinase. Cleavage at these four positions would result in the release of four mature products with molecular masses of approximately 68, 58, 39, and 35 kDa. Among them, the 39- and 35-kDa proteins were specifically identified in IBV-infected cells. Taken together with the 100-kDa protein previously identified, these results suggest that the ORF 1b region of IBV mRNA1 may be able to encode five mature products.

References

REFERENCES

  • 1.Alonso-Caplen F.V., Matsuoka Y., Wilcox G.E., Compans R.W. Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin. Virus Res. 1984;1:153–167. doi: 10.1016/0168-1702(84)90070-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Boursnell M.E.G., Brown T.D.K., 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]
  • 3.Brierley I., Boursnell M.E.G., Binns M.M., Bilimoria 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]
  • 4.Brierley I., Digard P., Inglis S.C. Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot. Cell. 1989;57:537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Denison M., Perlman S. Identification of putative polymerase gene product in cells infected with murine coronavirus A59. Virology. 1987;157:565–568. doi: 10.1016/0042-6822(87)90303-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.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;207:316–320. doi: 10.1006/viro.1995.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Fuerst T.R., Niles E.G., Studier F.W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA. 1986;83:8122–8127. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.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]
  • 9.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]
  • 10.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]
  • 11.Heusipp G., Grotzinger C., Herold J., Siddell S.G., Ziebuhr J. Identification and subcellular localization of a 41 kDa, polyprotein lab 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]
  • 12.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  • 13.Lee H-J., Shieh C-K., Gorbalenya A.E., Koonin E.V., Monica N.L., 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]
  • 14.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 of the C-terminal cleavage site of an 87 kDa protein. Virology. 1998;245:303–312. doi: 10.1006/viro.1998.9164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Liu D.X., Inglis S.C. Association of the infectious bronchitis virus 3c protein with the virion envelope. Virology. 1991;185:911–917. doi: 10.1016/0042-6822(91)90572-S. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Liu D.X., Brown T.D.K. Characterisation and mutational analysis of an ORF 1a-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]
  • 17.Liu D.X., Brierley I., Tibbles K.W., Brown T.D.K. A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. J. Virol. 1994;68:5772–5780. doi: 10.1128/jvi.68.9.5772-5780.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Liu D.X., Tibbles K.W., Cavanagh D., Brown T.D.K., Brierley I. Identification, expression, and processing of an 87-kDa polypeptide encoded by ORF 1a of the coronavirus infectious bronchitis virus. Virology. 1995;208:48–57. doi: 10.1006/viro.1995.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Liu D.X., Xu H.Y., Brown T.D.K. Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: Identification of a 10 kDa 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]
  • 20.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]
  • 21.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]
  • 22.Stern D.F., Sefton B.M. Coronavirus multiplication: Location of genes for virion proteins on the avian infectious bronchitis virus genome. J. Virol. 1984;50:22–29. doi: 10.1128/jvi.50.1.22-29.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.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]
  • 24.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