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. 1999 Apr;73(4):2658-66.
doi: 10.1128/JVI.73.4.2658-2666.1999.

Expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides

H Teng  1 , J D Piñón, S R Weiss
Affiliations

Expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides

H Teng et al. J Virol. 1999 Apr.

Abstract

Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitro trans cleavage is more efficient at 22 degrees C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage in cis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.

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Figures

FIG. 1
FIG. 1
Functional domains of MHV-A59 gene 1 and plasmids encoding ORF1a polypeptides. (A) The two ORFs of gene 1, ORF1a and ORF1b, and their predicted functional domains: PLP-1 and PLP-2, X, picornavirus 3C-like proteinase (3CLpro), and hydrophobic (HD1 and HD2). NTPase, nucleoside triphosphatase. (B) Diagrams of plasmids used for transcription and translation of ORF1a polypeptides, along with relevant functional domains and restriction sites. The catalytic residues of PLP-1, Cys1121 and His1272, and the proposed catalytic residues of PLP-2, Cys1716 and His1873, are indicated. The T7 bacteriophage RNA polymerase promoter is designated T7. The cleavages sites for p28 (Gly247/Val248) and p65 (Ala823/Gly833) and the region of ORF1a polypeptide used to raise antiserum UP102 (11) are also indicated. (C) Plasmids used for expression of the PLP domains in E. coli. T7 and ptac are procaryotic promoters for RNA synthesis, and malE encodes MBP.
FIG. 2
FIG. 2
Overexpression and purification of recombinant PLP-1. Proteins were purified as described in Materials and Methods, analyzed by SDS–10% (A) or 4 to 15% (B) polyacrylamide gel electrophoresis, and stained with Coomassie blue. (A) Expression of PLP-1 from pET-PLP-1. Lane 1, supernatant after sonication and centrifugation; lane 2, pellet after sonication and centrifugation; lane 3, refolded PLP-1. PLP-1 is indicated by the arrow. (B) Purified MBP–PLP-1 H1272P and MBP–PLP-1 with (−) and without (+) factor Xa cleavage. MBP–PLP-1 (or MBP–PLP-1 H1272P), MBP, PLP-1, and factor Xa are indicated by arrows. The molecular masses of marker proteins are indicated to the left of each panel.
FIG. 3
FIG. 3
trans cleavage of viral substrates by PLP-1. (A) Cleavage of viral substrate translated in vitro from pSPN1S1. Lane 1, with MBP–PLP-1 enzyme; lane 2, with MBP–PLP-1 H1272P enzyme; lane 3, with refolded PLP-1. (B) Cleavage of viral substrate translated in vitro from pSPΔMscN1S1. Lane 1, with MBP–PLP-1 enzyme; lane 2, with MBP–PLP-1 H1272P enzyme; lane 3, with refolded PLP-1. (C) Immunoprecipitation with antiserum UP102 after cleavage of viral substrates translated in vitro from pSPN1S1 (lane 1), pSPΔMscN1S1 (lane 2), and pSPΔMAGN1S1 (lane 3) by MBP–PLP-1 enzyme. Samples were analyzed on SDS–10% polyacrylamide gels. Cleavage products are indicated by arrows. In panel B, p50 represents the carboxyl-terminal cleavage product resulting from cleavage at the p65 site. The molecular masses of marker proteins are indicated to the left of panel A. Much less p28 was produced with substrate translated from pSPΔMscN1S1 than with substrates translated from pSPN1S1 and pSPΔMAGN1S1 (B, lanes 1 and 3; C, lane 2). The p28 protein bands were visible upon prolonged exposure.
FIG. 4
FIG. 4
In vitro trans cleavage by recombinant PLP-1 is more efficient at room temperature. Radiolabeled viral polypeptides were synthesized in a coupled transcription-translation system using as templates pSPN1S1 (A) and pSPΔMscN1S1 (B). Viral substrates were incubated with recombinant MBP–PLP-1 fusion enzyme overnight at either 22°C (lane 1) or 30°C (lane 2), followed by electrophoresis on SDS–10% polyacrylamide gels. Cleavage products are indicated by the arrows. In panel B, cleavage at the p65 site generates the carboxyl-terminal p50 polypeptide. The molecular masses of marker proteins are indicated to the right of panel B.
FIG. 5
FIG. 5
In vitro trans cleavage efficiency of PLP-1 increases with increase substrate length. Plasmid pSPNK H1272P was digested with the enzymes indicated at the top followed by transcription and translation in the coupled system in the presence of [35S]methionine. The radiolabeled viral substrates were incubated with unlabeled mutant PLP-1 H1272P (A) or wild-type PLP-1 (B), also synthesized in the coupled transcription-translation using pET-PLP-1 H1272P or pET-PLP-1, respectively, as the templates. Cleavage products were immunoprecipitated with UP102 and then analyzed by SDS–10% polyacrylamide gel electrophoresis; p28 is indicated by the arrow. The molecular masses of marker proteins are indicated between the panels.
FIG. 6
FIG. 6
PLP-1 cleavage efficiency at the p28 and p65 sites increases with increased length of proteinase polypeptide. Plasmid pCITE P1P2a was digested with the enzymes listed at the top and used as templates in coupled transcription-translation reactions in the presence of unlabeled methionine. The translated polypeptides, serving as a source of PLP-1, were incubated in trans cleavage assays with viral substrates synthesized in coupled transcription-translation reactions in the presence of [35S]methionine (as described in Materials and Methods), using pSPN1S1 (A) and pSPΔMscN1S1 (B) as templates. Cleavage reactions were analyzed by SDS–10% polyacrylamide gel electrophoresis; cleavage products p28, p43, p50, and p70 are indicated by the arrows. p50 is the carboxyl-terminal product derived by cleavage at the p65 site. The molecular masses of marker proteins are indicated to the left of panel A.
FIG. 7
FIG. 7
Proteolytic activity could not be detected for PLP-2. (A) trans cleavage of [35S]methionine-labeled viral substrates encoded by pSPΔMscN1S1 with enzymes synthesized by coupled transcription-translation using as the template pCITE P1P2a (lane 1) or pCITE P1P2a H1272P (lane 2). (B) cis cleavage with [35S]methionine-labeled viral polypeptides synthesized by transcription-translation using as templates pSPNK (lane 1), pSPN1S2ΔD1049K1657 (lane 2), and pSPN1S2ΔF1085A1670 (lane 3), followed by immunoprecipitation with UP102. (C) trans cleavage carried out with MBP fusion enzymes using [35S]methionine-labeled viral polypeptides as substrates. Lane 1, MBP–PLP-1 (enzyme) and pSPN1S1-encoded polypeptide (substrate); lane 2, MBP–PLP-2 (enzyme) and pSPN1S1-encoded polypeptide (substrate); lane 3, MBP–PLP-1 (enzyme) and pSPΔMscN1S1-encoded polypeptide (substrate); lane 4, MBP–PLP-2 (enzyme) and pSPΔMscN1S1-encoded polypeptide (substrate). All cleavage reactions were analyzed by SDS–10% polyacrylamide gel electrophoresis; cleavage products p28, p43, p50, and p70 are indicated by the arrows. p50 is the carboxyl-terminal product derived by cleavage at the p65 site. The molecular masses of marker proteins are indicated to the left of panel A. The p28 protein bands were visible upon prolonged exposure.
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References

    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates and Wiley-Interscience; 1987. pp. 10.1.1–10.1.3.
    1. Baker S C, Shieh C K, Chang M F, Vannier D M, Lai M M C. Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. J Virol. 1989;63:3693–3699. - PMC - PubMed
    1. Baker S C, Yokomori K, Dong S, Carlisle R, Gorbalenya A E, Koonin E V, Lai M M C. Identification of the catalytic sites of a papain-like proteinase of murine coronavirus. J Virol. 1993;67:6056–6063. - PMC - PubMed
    1. Bonilla P J, Gorbalenya A E, Weiss S R. Mouse hepatitis virus strain A59 RNA polymerase gene ORF1a: heterogeneity among MHV strains. Virology. 1994;198:736–740. - PMC - PubMed
    1. Bonilla P J, Hughes S A, Piñón J D, Weiss S R. Characterization of the leader papain-like proteinase of MHV-A59: identification of a new in vitro cleavage site. Virology. 1995;209:489–497. - PubMed

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