Functional and structural analysis of the RNase H domain of the Moloney murine leukemia virus reverse transcriptase

The RNase H domain of the Moloney murine leukemia virus reverse transcriptase (Mo-MLV RT) and E. coli RNase H both possess a positively-charged α-helix (C-helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus RT (HIV RT) or avian sarcoma virus RT. Although a mutant of the Mo-MLV-RT lacking the C-helix (ΔC RT) retains DNA polymerase activity and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C-helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594,1597, R601 and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5′- and 3 ′-viral DNA ends. The mutant RTs were tested in vitro for polymerase activity, (−) strand strong stop synthesis, non-specific RNase H activity and proper cleavage at the polypurine tract-U3 junction. Mutant R601A was the most defective mutant. Mutants H594A, I597A and G602A had significant reductions in the rate of viral replication. Many of these mutants formed improper viral DNA ends and were defective in polypurine tract (PPT) recognition. The data suggest that the C-helix is an RNase H cleavage-specificity determinant for PPT recognition and for proper formation of the plus strand DNA 5′ end.; A crystallographic study resulted in the 1.6Å resolution structure of Mo-MLV RNase H ΔC using MAD (multi-wavelength anomalous diffraction) phasing. The overall polypeptide fold resembles other RNase H structures, a mixed five-stranded β-sheet surrounded by four or five α-helices. A superimposed model of Mo-MLV RNase H bound to RNA:DNA substrate composed of sequences from the HIV PPT was used to identify individual residues as potential substrate binding sites. The model also revealed that the positively-charged Mo-MLV C-helix and following loop may contact the substrate along the major groove, as well as the DNA and RNA backbone phosphates. The HIV RT p66 connection domain possesses a loop that resides in the same space as the Mo-MLV C-helix. HIV RT R358, H364 and T365 closely align with Mo-MLV RT R601, H594 and 1597, respectively. The alignment suggests that HIV RT has recruited other domains to perform the substrate interaction functions of its missing RNase H C-helix.