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.