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. 2009 May 8;388(3):462-74.
doi: 10.1016/j.jmb.2009.03.025. Epub 2009 Mar 13.

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

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Free PMC article

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

Greg L Beilhartz et al. J Mol Biol. .
Free PMC article

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.

Figures

Figure 1
Figure 1. Structures of Small Molecules used in this Study and Schematic of the Polymerase-Dependent Binding Mode of HIV RT
(A) Structures of inhibitors used in this study: β-thujaplicinol and PFA. (B) Schematic representation of pre-and post-translocated complexes of HIV-1 RT. The polymerase active center is represented by the green cylinder, which is either occupied by the 3’ end of the primer (pre-translocation) or available for nucleotide binding (post-translocation). RT is shown here shifted relative to its nucleic acid substrate by a single nucleotide. This difference (Δ 1nt) is also reflected at the RNase H active site (green arrow).
Figure 2
Figure 2. Inhibition of RNase H Activity by β-thujaplicinol and PFA
A) Inhibition of RNase H activity under steady-state conditions. Time-course reaction (0-12 mins) in the absence and presence of β-thujaplicinol (50 μM) and PFA (200 μM) under steady-state conditions. RNase H cleavages at positions -18 and -19 are marked post- and pre-translocation, respectively. The figure focuses on this part of the gel. (B) Time-course experiment (0.05 – 20 sec) in the presence and absence of β-thujaplicinol (50 μM) and PFA (200 μM). The protein trap heparin was added to all reactions at 4 mg/mL. (C) and (D) Results from (A) and (B) represent graphically, respectively. Error bars represent the standard deviation between three independent experimental replicates.
Figure 3
Figure 3. Effects of Ternary Complex Formation on RNase H Inhibition
(A) Left: Increasing dose-response of PFA (0 – 10 μM) on the polymerase-dependent substrate with a dTMP-terminated primer. Right: Increasing concentrations of the next template nucleotide (dGTP) were added from 0 to 10 μM to reactions containing a polymerase-dependent substrate terminated with ddTMP. (B) The data from (A) presented graphically showing both translocational trends and RNase H activity. (C) Steady-state RNase H activity measured in the linear phase of the reaction in the absence of ligands, and with PFA or dGTP, in the absence and presence of β-thujaplicinol.
Figure 4
Figure 4. Effects of Order-of-Addition on RNase H Inhibition
Time-course assay under pre-steady-state conditions. % Activity refers to the percentage of the total substrate converted to the -18 and -19 major reaction products. (Top) Reactions were pre-incubated with RT, Mg2+ and the RNA/DNA substrate in the orders shown in the absence of inhibitor. (Bottom) Same as top but in the presence of 50 μM β–thujaplicinol.
Figure 5
Figure 5. Effects of β-thujaplicinol on Polymerase-Independent RNase H activity
(A) Sequence of the polymerase-independent substrate. A primary RNase H cut is expected at the RNA/DNA junction +1, while ensuing secondary cuts are expected to occur upstream of the primary cut. (B) Time-course of RNase H activity (0 – 40’) on the chimeric substrate PBS-14r8d. B-thujaplicinol was added at a concentration of 50 μM, while PFA was added at a concentration of 200 μM. Primary and secondary cuts are indicated.
Figure 6
Figure 6. Altering Y501 of the RNase H primer grip affects β-thujaplicinol sensitivity
Structures of natural (F and W) and unnatural amino acid insertions (AzF and BpF) for Y501 are illustrated in addition to the IC50 for the mutant enzyme. IC50 values are the average of triplicate analyses.
Figure 7
Figure 7. Model of β-thujaplicinol binding site
An RT-substrate complex generated by superposition of HIV RT-RNA/DNA and human RNase H-RNA/DNA co-crystal structures ; is depicted in all panels. (A) Surface representation of β-thujaplicinol (pink, with red oxygen atoms) and the RNA nucleotide 17 bp from the primer 3’ terminus (blue). Steric interference between the inhibitor and the RNA is evident at the junction between the two surfaces, as well as in the stick representations near the ribose C4’ atom. RNA (dark blue ball and stick), active site Mg ions (red spheres), and active site residues D443, E478, D498, D549 (light blue ball-and-stick) are also highlighted. (B) Y501 rotation from the position observed in published crystal structures (grey) to stack with β-thujaplicinol (rotated Y501, white; β-thujaplicinol, pink). For illustrative purposes, RNA and DNA strands are shown as blue and red tubes, respectively; however, binding of substrate and β-thujaplicinol at the RNase H active site is thought to be mutually exclusive. (C) Same as B, except the complex is rotated to highlight the positioning of Y501 relative to L479, K476, and Q475 (yellow ball-and-stick), and β-thujaplicinol is not shown. (D) Same as C, with benzophenone substituted for the Y501 side chain. Note that while the extended side chain permits potential hydrophobic interactions with L479 and K476 that cannot occur with Y501, it is not likely to directly affect substrate binding.

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