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Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis

Two virus-encoded proteinases are responsible for proteolysis of potyvirus polyproteins. One of these, HC-Pro, is a multifunctional protein that autolytically cleaves at its carboxyl-terminus (J. C. Carrington et al., 1989, EMBO J. 8, 365–370). To identify the class of proteinase to which HC-Pro bel...

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Autores principales: Oh, Chan-Seok, Carrington, James C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Published by Elsevier Inc. 1989
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131030/
https://www.ncbi.nlm.nih.gov/pubmed/2688301
http://dx.doi.org/10.1016/0042-6822(89)90582-5
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author Oh, Chan-Seok
Carrington, James C.
author_facet Oh, Chan-Seok
Carrington, James C.
author_sort Oh, Chan-Seok
collection PubMed
description Two virus-encoded proteinases are responsible for proteolysis of potyvirus polyproteins. One of these, HC-Pro, is a multifunctional protein that autolytically cleaves at its carboxyl-terminus (J. C. Carrington et al., 1989, EMBO J. 8, 365–370). To identify the class of proteinase to which HC-Pro belongs, tobacco etch virus (TEV) HC-Pro mutants containing single amino acid substitutions at serine, cysteine, aspartic acid, and histidine positions were synthesized by in vitro transcription and translation and were tested for autoproteolytic activity. Combinations of these residues are constituents of the active sites of diverse groups of cellular and viral proteinases. Only those positions that were strictly conserved among four potyvirus HC-Pro proteolytic domains (for which sequences have been deduced) were mutagenized. Of the 19 mutant proteinases synthesized and tested, only those with alterations at Cys-649 and His-722 were defective for HC-Pro autolytic activity. Most of the other mutant proteinases exhibited no impairments in processing kinetics experiments. The spectrum of essential residues, as defined by this genetic analysis, supports the hypothesis that HC-Pro most closely resembles members of the cysteine-type family of proteinases.
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spelling pubmed-71310302020-04-08 Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis Oh, Chan-Seok Carrington, James C. Virology Regular Article Two virus-encoded proteinases are responsible for proteolysis of potyvirus polyproteins. One of these, HC-Pro, is a multifunctional protein that autolytically cleaves at its carboxyl-terminus (J. C. Carrington et al., 1989, EMBO J. 8, 365–370). To identify the class of proteinase to which HC-Pro belongs, tobacco etch virus (TEV) HC-Pro mutants containing single amino acid substitutions at serine, cysteine, aspartic acid, and histidine positions were synthesized by in vitro transcription and translation and were tested for autoproteolytic activity. Combinations of these residues are constituents of the active sites of diverse groups of cellular and viral proteinases. Only those positions that were strictly conserved among four potyvirus HC-Pro proteolytic domains (for which sequences have been deduced) were mutagenized. Of the 19 mutant proteinases synthesized and tested, only those with alterations at Cys-649 and His-722 were defective for HC-Pro autolytic activity. Most of the other mutant proteinases exhibited no impairments in processing kinetics experiments. The spectrum of essential residues, as defined by this genetic analysis, supports the hypothesis that HC-Pro most closely resembles members of the cysteine-type family of proteinases. Published by Elsevier Inc. 1989-12 2004-01-30 /pmc/articles/PMC7131030/ /pubmed/2688301 http://dx.doi.org/10.1016/0042-6822(89)90582-5 Text en Copyright © 1989 Published by Elsevier Inc. 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 Regular Article
Oh, Chan-Seok
Carrington, James C.
Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title_full Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title_fullStr Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title_full_unstemmed Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title_short Identification of essential residues in potyvirus proteinase HC-pro by site-directed mutagenesis
title_sort identification of essential residues in potyvirus proteinase hc-pro by site-directed mutagenesis
topic Regular Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131030/
https://www.ncbi.nlm.nih.gov/pubmed/2688301
http://dx.doi.org/10.1016/0042-6822(89)90582-5
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