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Insights into the structure and activity of prototype foamy virus RNase H
BACKGROUND: RNase H is an endonuclease that hydrolyzes the RNA strand in RNA/DNA hybrids. Retroviral reverse transcriptases harbor a C-terminal RNase H domain whose activity is essential for viral replication. The RNase H degrades the viral genomic RNA after the first DNA strand is synthesized. Here...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305377/ https://www.ncbi.nlm.nih.gov/pubmed/22325739 http://dx.doi.org/10.1186/1742-4690-9-14 |
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author | Leo, Berit Hartl, Maximilian J Schweimer, Kristian Mayr, Florian Wöhrl, Birgitta M |
author_facet | Leo, Berit Hartl, Maximilian J Schweimer, Kristian Mayr, Florian Wöhrl, Birgitta M |
author_sort | Leo, Berit |
collection | PubMed |
description | BACKGROUND: RNase H is an endonuclease that hydrolyzes the RNA strand in RNA/DNA hybrids. Retroviral reverse transcriptases harbor a C-terminal RNase H domain whose activity is essential for viral replication. The RNase H degrades the viral genomic RNA after the first DNA strand is synthesized. Here, we report the biophysical and enzymatic properties of the RNase H domain of prototype foamy virus (PFV) as an independently purified protein. Sequence comparisons with other retroviral RNases H indicated that PFV RNase H harbors a basic protrusion, including a basic loop and the so-called C-helix, which was suggested to be important for activity and substrate binding and is absent in the RNase H domain of human immunodeficiency virus. So far, no structure of a retroviral RNase H containing a C-helix is available. RESULTS: RNase H activity assays demonstrate that the PFV RNase H domain is active, although its activity is about 200-fold reduced as compared to the full length protease-reverse transcriptase enzyme. Fluorescence equilibrium titrations with an RNA/DNA substrate revealed a K(D )for the RNase H domain in the low micromolar range which is about 4000-fold higher than that of the full-length protease-reverse transcriptase enzyme. Analysis of the RNase H cleavage pattern using a [(32)P]-labeled substrate indicates that the independent RNase H domain cleaves the substrate non-specifically. The purified RNase H domain exhibits a well defined three-dimensional structure in solution which is stabilized in the presence of Mg(2+ )ions. CONCLUSIONS: Our data demonstrate that the independent PFV RNase H domain is structured and active. The presence of the C-helix in PFV RNase H could be confirmed by assigning the protein backbone and calculating the chemical shift index using NMR spectroscopy. |
format | Online Article Text |
id | pubmed-3305377 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-33053772012-03-19 Insights into the structure and activity of prototype foamy virus RNase H Leo, Berit Hartl, Maximilian J Schweimer, Kristian Mayr, Florian Wöhrl, Birgitta M Retrovirology Research BACKGROUND: RNase H is an endonuclease that hydrolyzes the RNA strand in RNA/DNA hybrids. Retroviral reverse transcriptases harbor a C-terminal RNase H domain whose activity is essential for viral replication. The RNase H degrades the viral genomic RNA after the first DNA strand is synthesized. Here, we report the biophysical and enzymatic properties of the RNase H domain of prototype foamy virus (PFV) as an independently purified protein. Sequence comparisons with other retroviral RNases H indicated that PFV RNase H harbors a basic protrusion, including a basic loop and the so-called C-helix, which was suggested to be important for activity and substrate binding and is absent in the RNase H domain of human immunodeficiency virus. So far, no structure of a retroviral RNase H containing a C-helix is available. RESULTS: RNase H activity assays demonstrate that the PFV RNase H domain is active, although its activity is about 200-fold reduced as compared to the full length protease-reverse transcriptase enzyme. Fluorescence equilibrium titrations with an RNA/DNA substrate revealed a K(D )for the RNase H domain in the low micromolar range which is about 4000-fold higher than that of the full-length protease-reverse transcriptase enzyme. Analysis of the RNase H cleavage pattern using a [(32)P]-labeled substrate indicates that the independent RNase H domain cleaves the substrate non-specifically. The purified RNase H domain exhibits a well defined three-dimensional structure in solution which is stabilized in the presence of Mg(2+ )ions. CONCLUSIONS: Our data demonstrate that the independent PFV RNase H domain is structured and active. The presence of the C-helix in PFV RNase H could be confirmed by assigning the protein backbone and calculating the chemical shift index using NMR spectroscopy. BioMed Central 2012-02-10 /pmc/articles/PMC3305377/ /pubmed/22325739 http://dx.doi.org/10.1186/1742-4690-9-14 Text en Copyright ©2012 Leo et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Leo, Berit Hartl, Maximilian J Schweimer, Kristian Mayr, Florian Wöhrl, Birgitta M Insights into the structure and activity of prototype foamy virus RNase H |
title | Insights into the structure and activity of prototype foamy virus RNase H |
title_full | Insights into the structure and activity of prototype foamy virus RNase H |
title_fullStr | Insights into the structure and activity of prototype foamy virus RNase H |
title_full_unstemmed | Insights into the structure and activity of prototype foamy virus RNase H |
title_short | Insights into the structure and activity of prototype foamy virus RNase H |
title_sort | insights into the structure and activity of prototype foamy virus rnase h |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305377/ https://www.ncbi.nlm.nih.gov/pubmed/22325739 http://dx.doi.org/10.1186/1742-4690-9-14 |
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