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. 2006 Aug;80(16):7816–7831. doi: 10.1128/JVI.00532-06

FIG. 3.

FIG. 3.

Analysis of the cleavage site between NTPase and p18. (A) Alignment of the amino acid sequence of the putative junction of the MNV NTPase and p18 proteins with those of CV, MDV, NV, and SHV. Numbers correspond to the ORF1 polyprotein sequence of each virus, and asterisks indicate conserved amino acids. The cleavage site in this region, identified previously for human norovirus strains, is indicated with an arrow. (B) Schematic representation of the pF2R2Pro plasmid that was engineered to contain the MNV-1 NTPase-p18 junction and an active 3CL Pro. The chimeric protein was fused to the vector sequence (gray box) at its N terminus, and the entire VPg and p18 C-terminal sequences were deleted. (C) SDS-PAGE analysis of proteins encoded in pF2R2Pro when expressed in vitro and in E. coli. Lane 1, in vitro TNT translation products derived from pF2R2Pro (autoradiography); lane 2, IMAC-purified proteins isolated from the insoluble fraction of the induced bacterial cells carrying pF2R2Pro stained with Coomassie blue (Cblue); lanes 3 and 4, Western blot analysis of the same protein sample using PentaHis antibody (QIAGEN) and anti-Pro serum, respectively. The two major pF2R2Pro proteins were expressed in both in vitro translation reactions and bacteria. The N-terminal sequence of the 24-kDa protein (NKVYDFDAG) is shown.