Characterization of the Two Overlapping Papain-like Proteinase Domains Encoded in Gene 1 of the Coronavirus Infectious Bronchitis Virus and Determination of the C-Terminal Cleavage Site of an 87-kDa Protein

Abstract

In a previous report, we showed that proteolytic processing of an 87-kDa mature viral protein from the coronavirus infectious bronchitis virus (IBV) 1a and 1a/1b polyproteins was mediated by two putative overlapping papain-like proteinase domains (PLPDs) encoded within the region from nucleotides 4243 to 5553 of ORF 1a (Liu et al., 1995). In this study, we demonstrate that only the first domain, PLPD-1, is responsible for this cleavage, as deletion of the second domain did not affect the formation of the 87-kDa protein. Site-directed mutagenesis studies further showed that a previously predicted nucleophilic cysteine residue (Cys1274) and a histidine residue (His1437) were essential for the proteinase activity, indicating that they may be important components of the catalytic center of the proteinase. Meanwhile, expression of a series of deletion mutants revealed that the 87-kDa protein was encoded by the 5'-most 2.6 kb of ORF 1a. Deletion and amino acid substitution mutation studies demonstrated that the Gly673-Gly674 dipeptide bond was most likely the cleavage site responsible for releasing the C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins.

Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products

“…We previously demonstrated that PLPD-1 encoded between nucleotides 4243 and 5576 was responsible for the cleavage of the 87-kDa protein at the Gly 673 -Gly 674 dipeptide bond (17), and the catalytic dyad for this activity was the Cys 1274 and His 1437 residues. We then used deletion and mutational analyses to test if either of the two PLPDs is involved in this novel cleavage activity.…”

“…A time course experiment was then carried out to further investigate the processing and maturation of the three N-terminal cleavage products (87-, 195-, and 41-kDa proteins). Immunoprecipitation using antiserum V59 (17,21) showed that the 87-kDa protein was detected from IBV-infected cells harvested at 10 h p.i. (Fig.…”

“…The Cys 1274 and His 1437 residues were revealed to be the catalytic dyad of this proteinase (17). In this paper, we report yet another cleavage event performed by PLPD-1.…”

“…This fragment was digested with NcoI and SnaBI, which digest the IBV sequence at nucleotide positions 3831 and 7999, respectively, and ligated into NcoI-and SnaBI-digested pIBV1a7, giving rise to pIBV1a6(ext). Finally, a PCR fragment covering nucleotides 2548 to 5759 was generated using (17,21), giving rise to pIBV1a3. The region coding for the 87-kDa protein (nucleotides 529 to 2545) was then deleted as described for pORF1a3, giving rise to pORF1a3⌬2.…”

“…In our previous reports, IBV PLPD-1 was shown to be responsible for cleavage of the 1a and 1a/1b polyproteins at the Gly 673 -Gly 674 dipeptide bond to release an 87-kDa N-terminal cleavage product (17,21). The Cys 1274 and His 1437 residues were revealed to be the catalytic dyad of this proteinase (17).…”