Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1371/journal.pone.0144638 http://hdl.handle.net/11449/177744 |
Resumo: | The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies. |
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spelling |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patientThe hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.National Institutes of HealthSão Paulo State University - UNESP Department of BiologyUniversity of Wisconsin- Milwaukee Department of Chemistry and BiochemistrySão José do Rio Preto Medical School Laboratory of VirologySão Paulo State University - UNESP Department of BiologyNational Institutes of Health: R01 AI088001Universidade Estadual Paulista (Unesp)University of Wisconsin- MilwaukeeLaboratory of VirologyProvazzi, Paola J. S. [UNESP]Mukherjee, SouravHanson, Alicia M.Nogueira, Mauricio L.Carneiro, Bruno M. [UNESP]Frick, David N.Rahal, Paula [UNESP]2018-12-11T17:26:54Z2018-12-11T17:26:54Z2015-12-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.1371/journal.pone.0144638PLoS ONE, v. 10, n. 12, 2015.1932-6203http://hdl.handle.net/11449/17774410.1371/journal.pone.01446382-s2.0-849555956042-s2.0-84955595604.pdf79910823626712120000-0001-5693-6148Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengPLoS ONE1,164info:eu-repo/semantics/openAccess2023-12-02T06:12:50Zoai:repositorio.unesp.br:11449/177744Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-12-02T06:12:50Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
title |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
spellingShingle |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient Provazzi, Paola J. S. [UNESP] |
title_short |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
title_full |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
title_fullStr |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
title_full_unstemmed |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
title_sort |
Analysis of the enzymatic activity of an NS3 helicase genotype 3a variant sequence obtained from a relapse patient |
author |
Provazzi, Paola J. S. [UNESP] |
author_facet |
Provazzi, Paola J. S. [UNESP] Mukherjee, Sourav Hanson, Alicia M. Nogueira, Mauricio L. Carneiro, Bruno M. [UNESP] Frick, David N. Rahal, Paula [UNESP] |
author_role |
author |
author2 |
Mukherjee, Sourav Hanson, Alicia M. Nogueira, Mauricio L. Carneiro, Bruno M. [UNESP] Frick, David N. Rahal, Paula [UNESP] |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) University of Wisconsin- Milwaukee Laboratory of Virology |
dc.contributor.author.fl_str_mv |
Provazzi, Paola J. S. [UNESP] Mukherjee, Sourav Hanson, Alicia M. Nogueira, Mauricio L. Carneiro, Bruno M. [UNESP] Frick, David N. Rahal, Paula [UNESP] |
description |
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-12-01 2018-12-11T17:26:54Z 2018-12-11T17:26:54Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1371/journal.pone.0144638 PLoS ONE, v. 10, n. 12, 2015. 1932-6203 http://hdl.handle.net/11449/177744 10.1371/journal.pone.0144638 2-s2.0-84955595604 2-s2.0-84955595604.pdf 7991082362671212 0000-0001-5693-6148 |
url |
http://dx.doi.org/10.1371/journal.pone.0144638 http://hdl.handle.net/11449/177744 |
identifier_str_mv |
PLoS ONE, v. 10, n. 12, 2015. 1932-6203 10.1371/journal.pone.0144638 2-s2.0-84955595604 2-s2.0-84955595604.pdf 7991082362671212 0000-0001-5693-6148 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
PLoS ONE 1,164 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
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