Further Characterisation of the Coronavirus IBV ORF 1a Products Encoded by the 3C-Like Proteinase Domain and the Flanking Regions

  • Lisa F. P. Ng
  • D. X. Liu
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 440)


Coronavirus IBV encodes a piconarvirus 3C-like proteinase. In a previous report, this proteinase was shown to undergo rapid degradation in vitro in reticulocyte lysate due to a posttranslational event involving ubiquitination of the protein. Several lines of evidence presented here indicate that the proteinase itself is stable. Translation of the IBV sequence from nucleotide 8864 to 9787 resulted in the synthesis of a 33 kDa protein, representing the full-length 3C-like proteinase. Pulse-chase and time-course experiments showed that this protein was stable in reticulocyte lysate for up to 2 hours. However, a 45 kDa protein encoded by the IBV sequence from nucleotide 8693 to 9911 underwent rapid degradation in reticulocyte lysate, but was stable in wheat germ extract, suggesting that an ATP-dependent protein degradation pathway may be involved in the turnover of the 45 kDa protein. To identify the IBV sequence responsible for the instability of this 45 kDa protein species, the region from nucleotide 8693 to 9787 was translated both in vitro and in vivo, leading to the synthesis of a stable 43 kDa protein. These results suggest that a destabilising signal may be located in the IBV sequences between the nucleotides 9787 and 9911. Meanwhile, protein aggregation was observed when the product encoded by the IBV sequence from nucleotide 9911 to 10510 was boiled for 5 minutes before being analysed in SDS-PAGE; when the same product was treated at 37°C for 15 minutes, however, protein aggregation was not detected. Deletion studies indicate that the presence of a hydrophobic domain downstream of the 3C-like proteinase-encoding region may be the cause for the aggregation of the product encoded by this region of ORF 1a.


Infectious Bronchitis Virus Rabbit Reticulocyte Lysate Wheat Germ Extract Destabilise Signal Kilodalton Polypeptide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Boursnell, M. E. G., T. D. K. Brown, I. J. Foulds, P. F. Green, F. M. Tomley, and M. M. Binns., 1987, Completion of the sequence of the genome of the Coronavirus avian infectious bronchitis virus, J. Gen. Virol. 68: 57–77.PubMedCrossRefGoogle Scholar
  2. Brierley, I., M. E. G. Boursnell, M. M. Binns, B. Billimoria, V. C. Blok, T. D. K. Brown, and S. C. Inglis., 1987, An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the Coronavirus IBV, EMBO. J. 6:3779–3785.PubMedGoogle Scholar
  3. Contreras, R., H. Cheroutre, W. Degrave, and W. Fiers., 1982, Simple efficient in vitro synthesis of capped RNA useful for direct expression of cloned DNA, Nucleic Acids Res. 10:6353–6362.PubMedCrossRefGoogle Scholar
  4. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss., 1986, Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase, Proc. Natl. Acad. Sci. USA 83:8122–8127.PubMedCrossRefGoogle Scholar
  5. Gorbalenya, A. E., E. Y. Koonin, A. P. Donchenko, and V. M. Blinov., 1989, Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis, Nucleic Acids Res. 17:4847–4860.PubMedCrossRefGoogle Scholar
  6. Laemmli, U. K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London) 227:680–685.Google Scholar
  7. Liu D. X., I, Brierley, K. W. Tibbies, and T. D. K. Brown, 1994, 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 68:5772–5780.PubMedGoogle Scholar
  8. Liu D. X., K. W. Tibbies, D. Cavanagh, T. D. K. Brown, and I. Brierley., 1995, Identification, expression, and processing of an 87 kDa polypeptide encoded by ORF 1a of the Coronavirus infectious bronchitis virus, Virology 208:48–57.PubMedCrossRefGoogle Scholar
  9. Liu D. X., and T. D. K. Brown., 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.PubMedCrossRefGoogle Scholar
  10. Liu D. X., H. Y. Xu, and T. D. K. Brown., 1997, Proteolytic processing of the Coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kilodalton polypeptide and determination of its cleavage sites, J. Virol. 71:48–57.Google Scholar
  11. Tibbies, K. W., I. Brierley, D. Cavanagh, and T. D. K. Brown., 1995, A region of the infectious bronchitis virus 1a polyprotein encoding the 3C-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate, J. Gen. Virol. 76:3059–3070.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Lisa F. P. Ng
    • 1
  • D. X. Liu
    • 1
  1. 1.Institute of Molecular AgrobiologySingapore

Personalised recommendations