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, 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.
Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstr, 30, D-95447 Bayreuth, Germany.
Berit
B
Leo
Maximilian J
MJ
Hartl
Kristian
K
Schweimer
Florian
F
Mayr
Birgitta M
BM
Wöhrl
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020871:RNA Stability; D016914:Ribonuclease H / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016092:Spumavirus / Q000201:enzymology*
D002413:Cations, Divalent; D003067:Coenzymes; D016914:Ribonuclease H; D008274:Magnesium