title: Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.
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.
authors.affiliation: Department of Microbiology, School of Medicine, Box 357242, University of Washington, Seattle, WA 98195, USA.
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Schultz
authors.forename: Miaohua
authors.initials: M
authors.lastname: Zhang
authors.forename: James J
authors.initials: JJ
authors.lastname: Champoux
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009711:Nucleotides / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H