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

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

Retrovirology 9: 14

ISSN/ISBN: 1742-4690

PMID: 22325739

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.

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