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

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.Retroviral reverse transcription describes the formation of a double-stranded DNA using the single-stranded viral RNA genome as a template. The process is catalyzed by the viral reverse transcriptase which harbors a polymerase and an RNase H domain.In most retroviruses reverse transcription takes place after the virus has entered the host cell. Spumaviruses, or foamy viruses (FVs), belong to a subfamily of the retroviridae and follow a distinct replication pattern unique among retroviruses: (a) reverse transcription occurs predominantly in the virus producing cell (b) the pol-gene coding for the viral enzymes is expressed from an independently spliced mRNA and (c) the viral protease is not cleaved off from the Pol precursor protein, leading to a mature protease-reverse transcriptase (PR-RT) [1-5]. Thus the mature PR-RT of FVs harbors a protease, polymerase and RNase H domain, each possessing a distinct enzymatic activity [4].Retroviral RNases H are domains of the RT enzymes and degrade the RNA strand of the RNA/DNA hy