Abstract
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.
Chapter PDF
Similar content being viewed by others
Keywords
- 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.
References
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.
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.
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.
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.
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.
Laemmli, U. K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London) 227:680–685.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer Science+Business Media New York
About this chapter
Cite this chapter
Ng, L.F.P., Liu, D.X. (1998). Further Characterisation of the Coronavirus IBV ORF 1a Products Encoded by the 3C-Like Proteinase Domain and the Flanking Regions. In: Enjuanes, L., Siddell, S.G., Spaan, W. (eds) Coronaviruses and Arteriviruses. Advances in Experimental Medicine and Biology, vol 440. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-5331-1_21
Download citation
DOI: https://doi.org/10.1007/978-1-4615-5331-1_21
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-7432-9
Online ISBN: 978-1-4615-5331-1
eBook Packages: Springer Book Archive