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

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 I...

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Autores principales: Ng, Lisa F.P., Liu, D.X.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Academic Press. 2000
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131205/
https://www.ncbi.nlm.nih.gov/pubmed/10873746
http://dx.doi.org/10.1006/viro.2000.0330
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author Ng, Lisa F.P.
Liu, D.X.
author_facet Ng, Lisa F.P.
Liu, D.X.
author_sort Ng, Lisa F.P.
collection PubMed
description 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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spelling pubmed-71312052020-04-08 Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site Ng, Lisa F.P. Liu, D.X. Virology Article 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. Academic Press. 2000-06-20 2002-05-25 /pmc/articles/PMC7131205/ /pubmed/10873746 http://dx.doi.org/10.1006/viro.2000.0330 Text en Copyright © 2000 Academic Press. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
spellingShingle Article
Ng, Lisa F.P.
Liu, D.X.
Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title_full Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title_fullStr Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title_full_unstemmed Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title_short Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site
title_sort further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131205/
https://www.ncbi.nlm.nih.gov/pubmed/10873746
http://dx.doi.org/10.1006/viro.2000.0330
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