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. 2005 Oct;79(20):12721–12731. doi: 10.1128/JVI.79.20.12721-12731.2005

FIG. 2.

FIG. 2.

Bacterially expressed HCoV-229E X domain converts Appr-1"-p to Appr. (A) The HCoV-229E X domain (pp1a/pp1ab residues 1265 to 1436) was expressed and purified as an MBP fusion protein (see Materials and Methods) and subsequently cleaved with factor Xa. This protein and a mutant derivative carrying two substitutions of conserved Asn residues were incubated for 3 h at 30°C with Appr-1"-p that was generated from Appr>p using A. thaliana CPDase (see Materials and Methods). Lanes: WT, incubation with HCoV-229E X (wild-type sequence); N1302A+N1305A, incubation with a mutant form of HCoV-229E X in which the pp1a/pp1ab Asn residues 1302 and 1305 were each substituted with Ala; CIP, incubation with alkaline phosphatase isolated from calf intestine. Reaction products were separated on cellulose TLC plates using solvent I and visualized under UV light. Marker nucleotides: AMP, ADP, ATP, Appr, Appr-1"-p, and Appr>p. (B and C) The reactions described in panel A were analyzed on polyethyleneimine-cellulose TLC plates using solvent II (B) and III (C), respectively.