Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus

Baker, Susan C.; Yokomori, Kyoko; Dong, Shangdong; Carlisle, Rachel E.; Gorbalenya, Alexander E.; Koonin, Eugene V.; Lai, Michael M. C.

doi:10.1128/jvi.67.10.6056-6063.1993

Cited by 97 publications

(87 citation statements)

References 36 publications

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“…Similar enzymatic activities were also reported for the first papain-like proteinase domain of human and murine coronaviruses. In mouse hepatitis virus (MHV), two Nterminal cleavage products (p28 and p65) have been reported (Hughes et al, 1995;Bonilla et al, 1997;Baker et al, 1989Baker et al, , 1993Dong and Baker, 1994;Gao et al, 1996;Schiller et al, 1998). The same domain of human coronavirus (HCV) processes the two polyproteins to release p9 (Herold et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site

Liu

2000

Virology

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Coronavirus infectious bronchitis virus (IBV) encodes a trypsin-like proteinase (3C-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene 1-encoded polyproteins. In our previous studies, the proteinase was identified as a 33-kDa protein in IBV-infected cells, and its catalytic center was shown to consist of H(2820) and C(2922) residues. It is released from the 1a and 1a/1b polyproteins by autoprocessing at two Q-S dipeptide bonds (Q(2779)-S(2780) and Q(3086)-S(3087)). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q(2779)-S(2780) site is tolerant to mutations at the P1 position. Deletion of the C-terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids, suggesting that the extreme C-terminal region may be dispensable for the proteolytic activity of the proteinase. Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q(2779)-S(2780) site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q(3086)-S(3087) site. The occurrence of both cleavage events in intact cells is potentially rapid and efficient, as no intermediate cleavage products covering the proteinase were detected in either IBV-infected or transfected cells. Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV-infected cells and in cells expressing the 3C-like proteinase alone, indicating that additional roles in viral replication might be played by this protein. Finally, a Q-A (Q(3379)-A(3380)) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase.

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Section: Introductionmentioning

confidence: 99%

Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site

Liu

2000

Virology

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“…The cleavage of p28 by PCP-1 was inhibited by leupeptin in both in vivo and in vitro reactions (Denison and Perlman, 1986;Baker et al, 1989). Furthermore, the catalytic cysteine and histidine residues of the PCP-1 domain have been identified (Baker et al, 1993). The cleavage site recognized by PCP-1 for the cleavage of p28 has been determined to be the Gly-247/Val-248 dipeptide bond (Dong and Baker, 1994;Hughes et al, 1995).…”

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confidence: 99%

“…It is likely that a number of distinct proteolytic events are required to generate the functional MHV RNA polymerase. To date, a number of proteins have been identified from MHV infected cells, including p28, p72, p65, p50, p240 and p290 (Denison and Perlman, 1987;Denison et al, 1992Denison et al, , 1995 this report), but only the proteolytic pathway for generating p28 has been elucidated (Baker et al, 1993;Dong and Baker, 1994;Hughes et al, 1995). There has been no report of the cleavage activity of the PCP-2 domain.…”

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confidence: 99%

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

1996

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The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

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“…The kinetics of nsp1 expression suggests that it might have an early regulatory role during the viral life cycle. Nsp1 is the first mature protein processed from the gene 1 polyprotein and is likely cleaved quickly following translation of PL1 pro within nsp3 (Baker et al, 1989;Baker et al, 1993;Denison and Perlman, 1987;Denison et al, 1992Denison et al, , 1995Denison and Perlman, 1986). MHV mutants that are incapable of liberating nsp1 from the nascent polyprotein exhibit delayed replication, diminished peak titers, small plaques, and reduced RNA synthesis compared to wild-type controls .…”

Section: Figmentioning

confidence: 99%

Atlas of coronavirus replicase structure

et al. 2014

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The international response to SARS-CoV has produced an outstanding number of protein structures in a very short time. This review summarizes the findings of functional and structural studies including those derived from cryoelectron microscopy, small angle X-ray scattering, NMR spectroscopy, and X-ray crystallography, and incorporates bioinformatics predictions where no structural data is available. Structures that shed light on the function and biological roles of the proteins in viral replication and pathogenesis are highlighted. The high percentage of novel protein folds identified among SARS-CoV proteins is discussed.

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Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus

Cited by 97 publications

References 36 publications

Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site

Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

Atlas of coronavirus replicase structure

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