FIG. 5.
Cloning, expression, and trans cleavage of p1a-22 in vitro. (A) Schematic of the carboxy-terminal region of the ORF 1a polyprotein. The putative p1a-22 domain is shown as filled rectangles. The locations of the sequenced LQ_S cleavage site and the putative IQ_S and LQ_A cleavage sites are shown by filled arrows, and the location of the LQ_N motif is shown by the open arrowhead. The boxed amino acid residues show the amino- and carboxy-terminal residues expressed from the cloned constructs. Residues in parentheses (MQ or MG) were contributed by the primers used to clone the fragments by PCR. The fragment number (1 through 3) and the calculated masses of the individual fragments are shown at the bottom of the diagram, and the constructs are numbered according to the fragments comprising them. (B) The results of the expression of constructs shown in panel A, along with products of cleavage by r3CLpro, are shown. Lane 1 shows p1a-22 immunoprecipitated from MHV-infected DBT cells (DBT RIP), and the location of p1a-22 along with molecular mass standards (in kilodaltons) is indicated to the left. Proteins expressed from construct 2, 1-2, 2-3, or 1-3 were incubated in the presence (+) or absence of r3CLpro and proteinase inhibitor E64 (PI). The masses of expressed proteins and cleavage products are shown to the right of the gel. (C) The products of expression and r3CLpro cleavage were immunoprecipitated with B4 antiserum. All lanes correspond to the proteins seen directly above them in panel B. The levels of contrast of lanes 11 and 12 were equally altered (Photoshop version 4.0) to show the minor 22-kDa cleavage product in lane 12 that was easily seen on the original fluorogram.