A REGION OF THE CORONAVIRUS INFECTIOUS-BRONCHITIS VIRUS 1A POLYPROTEIN ENCODING THE 3C-LIKE PROTEASE DOMAIN IS SUBJECT TO RAPID TURNOVER WHEN EXPRESSED IN RABBIT RETICULOCYTE LYSATE

In order to investigate the mechanisms involved in the processing of i nfectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studi es it was observed that the translation product encoded by one of thes e clones (pKT205) was poorly expressed. Biochemical and genetic analys es revealed that the basis for the poor expression was a post-translat ional event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which confe rred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5' end of the clone, while t he other, more C-terminal region, was effective in conferring instabil ity upon a heterologous protein to which it had been transferred. Thes e regions may influence the stability of the authentic viral protein(s ) in vivo and hence allow for the control of their expression and/or f unction at the level of proteolysis by cellular protease(s).