Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H

J Mol Biol. 2010 Mar 19;397(1):161-78. doi: 10.1016/j.jmb.2010.01.059. Epub 2010 Feb 1.

Abstract

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution / genetics
  • Base Sequence
  • DNA / genetics
  • HIV-1 / enzymology*
  • Molecular Sequence Data
  • Moloney murine leukemia virus / enzymology*
  • Nucleotides / genetics*
  • Protein Structure, Tertiary
  • RNA-Directed DNA Polymerase / metabolism
  • Ribonuclease H / chemistry
  • Ribonuclease H / isolation & purification
  • Ribonuclease H / metabolism*

Substances

  • Nucleotides
  • DNA
  • RNA-Directed DNA Polymerase
  • Ribonuclease H