Abstract
BackgroundThe ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity.ResultsThe solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding.ConclusionsThe structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.
Highlights
The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses
NMR spectroscopy of prototype foamy virus (PFV) RNase H revealed the existence of the socalled “basic protrusion” which consists of an α-helix, referred to as the “C-helix” followed by a basic loop [5]
The higher tc of PFV RNase H as compared to the isolated RNase H domain of HIV-Reverse transcriptase (RT) (9.7 ns, [21]) reflects the larger size of the PFV RNase H (165 aa vs. 138 aa, 18 kDa vs. 15.3 kDa), and confirms the monomeric character of the domain already demonstrated by size exclusion chromatography [5]
Summary
The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. We report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. That the isolated RNase H domain of PFV exhibits activity, albeit to a much lower extent than the mature full length PR-RT [5]. This behavior is comparable to that of the separate RNase H domain of MoMLV [6,7,8]. XMRV is highly related to MoMLV and appears to have arisen through recombination events during passaging of human tumors in mice [14]
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