RNase H activity: Structure, specificity, and function in reverse transcription

Abstract

This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3′ end-directed, and RNA 5′ enddirected. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5′ end-directed and DNA 3′ end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance. KeywordsRNase H; reverse transcriptase; human immunodeficiency virus; type 1 (HIV -1); Moloney murine leukemia virus (MoMLV); reverse transcription; polypurine tract (PPT)

Human Immunodeficiency Virus Reverse Transcriptase Displays Dramatically Higher Fidelity under Physiological Magnesium Conditions In Vitro

“…Both activities are divalent cation dependent, and the polymerase active site contains two divalent cation binding sites. Models for one or two cation binding sites have also been proposed for RNase H (3)(4)(5)(6)(7)(8)(9).…”

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

“…Conversely, polymerase-independent cuts collectively refer to RNase H cleavage events that occur, whereas the polymerase active site is not positioned at the 3Ј-primer terminus. Variations of this mechanism have been proposed (13). This activity is required for non-specifically degrading the transcribed viral RNA genome during (Ϫ)-strand DNA synthesis, as well as for creating and removing the (ϩ)-strand primer or polypurine tract, and for removing the (Ϫ)-strand or tRNA primer (14 -19).…”

Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

“…HIV-1 RT is a heterodimer composed of two peptide subunits, p66 and p51. The polymerase active site is located at the N-terminus of the p66 subunit, whereas its C-terminal end contains the RNase H active domain [5]. The RNase H domain of HIV-1 RT (HIV-RH) plays a role in many steps of reverse transcription, such as the generation of an RNA primer for synthesis of the (+)-strand DNA, the degradation of the viral genomic RNA in the intermediate RNA·DNA hybrid, and the removal of host tRNA and plus-strand primers [2,6,7].…”

“…HIV-RH forms a central five-stranded β-sheet surrounded by four α-helices. The core domain of the RNase H active site contains a highly-conserved DEDD motif that consists of four acidic residues, D443, E478, D498, and D549 [5,7]. The hydrolysis of the scissile phosphodiester bonds catalyzed by RNase H requires divalent metal ions, preferably Mg 2+ [6,8].…”