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-like protease which processes the RHDV polyprotein. In order to monitor the proteolytic activity of the RHDV 3C-like protease on its putative target sequences, i.e. the 10 EG dipeptide bonds distributed along the large polyprotein, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion protein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the coding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequences 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 positions 718-719, 1108-1109 and 1767-1768 were confirmed as cleavage sites and a new cleavage site EG (143-144) was identified.
Joubert
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P
Laboratoire de Virologie et Barrière d'Espèce, Unité de Pathologie Aviaire et de Parasitologie, INRA de Tours, 37380 Nouzilly, France.
Pautigny
C
C
Madelaine
M F
MF
Rasschaert
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D005814:Genes, Viral; D017929:Hemorrhagic Disease Virus, Rabbit / Q000201:enzymology* / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D008156:Luciferases; C089391:3C-like protease, rabbit hemorrhagic disease virus; D003546:Cysteine Endopeptidases