Insights into the structure and activity of prototype foamy virus RNase H

• Published in: Retrovirology 2012-02, Vol.9 (1), p.14-14, Article 14
• Main Authors: Leo, Berit, Hartl, Maximilian J, Schweimer, Kristian, Mayr, Florian, Wöhrl, Birgitta M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anisotropy; basic loop; C-helix; Cations, Divalent - metabolism; Coenzymes - metabolism; Crystal structure; Deoxyribonucleic acid; DNA; E coli; Enzymes; Experiments; HIV; Human immunodeficiency virus; Humans; Immunoassay; Kinetics; Leukemia; Magnesium - metabolism; Magnetic Resonance Spectroscopy; Molecular Sequence Data; NMR; Nuclear magnetic resonance spectroscopy; PFV; Physiological aspects; Protein Conformation; Proteins; retroviral RNase H; Reverse transcriptase; Ribonuclease H - chemistry; Ribonuclease H - isolation & purification; Ribonuclease H - metabolism; RNA Stability; Sequence Homology, Amino Acid; Spectrum analysis; Spumavirus - enzymology
• ISSN: 1742-4690; 1742-4690
• DOI: 10.1186/1742-4690-9-14

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. 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. 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.