Identification of the cleavage sites of sapovirus open reading frame 1 polyprotein

Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI-GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH(2)-p11-p28-p35 (NTPase)-p32-p14 (VPg)-p70 (Pro-Pol)-p60 (VP1)-COOH. The cleavage site between p14 (VPg) and p70 (Pro-Pol) was identified as E(1055)/A(1056) by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription-translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1' position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.