Rabbit hemorrhagic disease virus: Identification of a cleavage site in the viral polyprotein that is not processed by the known calicivirus protease

The positive-strand RNA genome of the calicivirus rabbit hemorrhagic disease virus (RHDV) contains one long open reading frame (ORE) covering almost 95% of the genomic RNA. Translation of this ORE leads to a polyprotein that is proteolytically processed at eight sites. Site nos. 4 and 5 from the amino-terminus are located within the protein p41 and obviously belong to alternative cleavage pathways leading to p23/2 and p18 or p29 and p13. Seven of the eight cleavage sites were identified before and the flanking sequences fulfill the requirements of the known RHDV protease, so that it is very likely that all theses sites are cleaved by this enzyme. The last unknown cleavage site was no. 4, one of the two alternative sites within p41 that separates the nonstructural proteins p23/2 and the VPg precursor p18. Mutagenesis studies identified aspartic acid at position 936 and arginine at position 937 as the residues that flank the cleavage site. The sequence at the processing site is unusual for the RHDV 3C-like protease since other sites display glutamic acid or glutamine at the P1 site and glycine, aspartic acid, or threonine at the P1' site. Expression of a polyprotein fragment lacking the viral protease revealed that the newly identified site is not cleaved by the RHDV protease but by an unknown proteolytic activity.