Insights into the structure and activity of prototype foamy virus RNase H
| Autor: | Berit Leo, Florian Mayr, Maximilian J. Hartl, Birgitta M. Wöhrl, Kristian Schweimer |
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| Rok vydání: | 2012 |
| Předmět: |
lcsh:Immunologic diseases. Allergy
RNA Stability Magnetic Resonance Spectroscopy Cations Divalent Protein Conformation RNase P Molecular Sequence Data Ribonuclease H Coenzymes Biology C-helix RNase PH chemistry.chemical_compound PFV Virology Humans Magnesium Amino Acid Sequence RNase H basic loop Sequence Homology Amino Acid Research RNA Molecular biology NMR Reverse transcriptase Kinetics retroviral RNase H RNase MRP Infectious Diseases chemistry biology.protein Spumavirus lcsh:RC581-607 DNA |
| Zdroj: | Retrovirology, Vol 9, Iss 1, p 14 (2012) Retrovirology |
| ISSN: | 1742-4690 |
| Popis: | 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 KD 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 [32P]-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 Mg2+ 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. |
| Databáze: | OpenAIRE |
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