Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E-coli

Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprote in. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3C(pro)) sequence together with Ranking sequences f rom a region of the viral genome 3'-distal to the putative helicase-encodin g region. Expression from these vectors in E. coli resulted in discrete pro tein products with smaller than expected molecular sizes. This confirmed th at an active viral protease was produced in E. coli and that the protease w as capable of cleaving the expressed protein at defined sites. Expressed cl eavage products surrounding the protease region of the viral polyprotein we re separated by SDS-PAGE, transferred to PVDF membranes and subjected to N- terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N t erminus of the putative VPg (E-961/GKNKG) and also at the protease/polymera se boundary (E-1280/GGDKG). The N terminus of the protease was released fro m the VPg C terminus at an EA dipeptide in the sequence E-1099/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.