The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding

Retrovirology. 2012 Sep 10:9:73. doi: 10.1186/1742-4690-9-73.


Background: The 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.

Results: The 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.

Conclusions: The 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA, Viral / genetics
  • DNA, Viral / metabolism
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Ribonuclease H / chemistry*
  • Ribonuclease H / genetics
  • Ribonuclease H / metabolism*
  • Sequence Alignment
  • Spumavirus / chemistry
  • Spumavirus / enzymology*
  • Spumavirus / genetics
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*


  • DNA, Viral
  • RNA, Viral
  • Viral Proteins
  • Ribonuclease H

Associated data

  • PDB/2LSN