title: HIV-1 reverse transcriptase can simultaneously engage its DNA/RNA substrate at both DNA polymerase and RNase H active sites: implications for RNase H inhibition.
abstract: Reverse transcriptase of the human immunodeficiency virus possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study, we demonstrate that ligands capable of trapping the 3'-end of the primer at the polymerase active site affect the specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single-turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative of post-translocated and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3'-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor beta-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip, and have shown that the inhibitor is unable to bind to a preformed reverse transcriptase-DNA/RNA complex. In conclusion, the bound nucleic acid substrate and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site.
authors.affiliation: Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada.
authors.forename: Greg L
authors.initials: GL
authors.lastname: Beilhartz
authors.forename: Michaela
authors.initials: M
authors.lastname: Wendeler
authors.forename: Noel
authors.initials: N
authors.lastname: Baichoo
authors.forename: Jason
authors.initials: J
authors.lastname: Rausch
authors.forename: Stuart
authors.initials: S
authors.lastname: Le Grice
authors.forename: Matthias
authors.initials: M
authors.lastname: Götte
mesh_terms: D020134:Catalytic Domain; D004279:DNA, Viral / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase