De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are...

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Autores principales:	Pilar Clemente-Casares, Alberto J López-Jiménez, Itxaso Bellón-Echeverría, José Antonio Encinar, Elisa Martínez-Alfaro, Ricardo Pérez-Flores, Antonio Mas
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/19ddb3067d4245359f3c0dc954995d8e
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spelling	oai:doaj.org-article:19ddb3067d4245359f3c0dc954995d8e2021-11-18T06:56:04ZDe novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.1932-620310.1371/journal.pone.0018515https://doaj.org/article/19ddb3067d4245359f3c0dc954995d8e2011-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21490973/?tool=EBIhttps://doaj.org/toc/1932-6203Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max)/K(m) ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in V(max) values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.Pilar Clemente-CasaresAlberto J López-JiménezItxaso Bellón-EcheverríaJosé Antonio EncinarElisa Martínez-AlfaroRicardo Pérez-FloresAntonio MasPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 4, p e18515 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pilar Clemente-Casares Alberto J López-Jiménez Itxaso Bellón-Echeverría José Antonio Encinar Elisa Martínez-Alfaro Ricardo Pérez-Flores Antonio Mas De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
description	Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max)/K(m) ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in V(max) values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.
format	article
author	Pilar Clemente-Casares Alberto J López-Jiménez Itxaso Bellón-Echeverría José Antonio Encinar Elisa Martínez-Alfaro Ricardo Pérez-Flores Antonio Mas
author_facet	Pilar Clemente-Casares Alberto J López-Jiménez Itxaso Bellón-Echeverría José Antonio Encinar Elisa Martínez-Alfaro Ricardo Pérez-Flores Antonio Mas
author_sort	Pilar Clemente-Casares
title	De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
title_short	De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
title_full	De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
title_fullStr	De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
title_full_unstemmed	De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.
title_sort	de novo polymerase activity and oligomerization of hepatitis c virus rna-dependent rna-polymerases from genotypes 1 to 5.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/19ddb3067d4245359f3c0dc954995d8e
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De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

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