Further characterisation of the coronavirus IBV ORF 1a products encoded by the 3C-like proteinase domain and the flanking regions.
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 10,510 was boiled for 5 minutes before being analysed in SDS-PAGE; when the same product was treated at 37 degrees 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.
Ng
L F
LF
Institute of Molecular Agrobiology, Singapore.
Liu
D X
DX
D000818:Animals; D002522:Chlorocebus aethiops; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D001351:Infectious bronchitis virus / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; D003546:Cysteine Endopeptidases; C000706208:3C-like proteinase, Avian infectious bronchitis virus; D000086782:Coronavirus 3C Proteases