Identification of a new cleavage site of the 3C-like protease of rabbit haemorrhagic disease virus

The calicivirus rabbit haemorrhagic disease virus (RHDV) possesses a 3C-lik e protease which processes the RHDV polyprotein. In order to monitor the pr oteolytic activity of the RHDV 3C-like protease on its putative target sequ ences, i.e. the 10 EG dipeptide bonds distributed along the large polyprote in, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion prot ein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the co ding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequ ences of the fusion proteins were cloned downstream of the T7 promoter and the proteolytic activity was evaluated by measuring the luciferase activity in both in vitro and 'in vivo' systems. The EG dipeptide bonds at position s 718-719, 1108-1109 and 1767-1768 were confirmed as cleavage sites and a n ew cleavage site EG (143-144) was identified.