Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities

Abstract

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

“…The increase in resistance to EFV was more than 8-fold for T-3, 3.6-fold for T-4, and 2.5-fold for T-8; the combination of L100I and K103N in T-6 pol elevated EFV White bars represent the average IC 50 for the wild-type RT control; gray bars represent RTs containing pol domains originated from the patients' virus; black bars represent both pol and CN originated from the patients' virus. Numbers in front of black bars represent fold changes, and asterisks within the black bars indicate a statistically significant difference in IC 50 within each pair of RTs connected by a bracket (t test, P Ͻ 0.05). Numbers within bars represent the maximum drug concentration used for the testing that was not sufficient to inhibit viral replication.…”

“…Numbers within bars represent the maximum drug concentration used for the testing that was not sufficient to inhibit viral replication. (E and F) IC 50 determinations for the viruses containing specific CN mutation(s) from treatment-experienced patients' RTs to NVP (E) and EFV (F). Numbers in front of gray bars represent fold changes, and asterisks within the gray bars indicate a statistically significant difference in IC 50 versus the black bar for each group connected by a bracket (t test, P Ͻ 0.05).…”

“…4E), but not ETR. We assume that the K d for the ETR-RT complex is comparable to or lower than that for the EFV-RT complex, because the IC 50 s for NVP, DLV, and EFV correlate with their K d values, and the IC 50 for ETR (1.1 nM) is lower than that for EFV (1.9 nM) (Fig. 4E).…”

“…Biochemical and structural analysis of the inhibition of reverse transcription by NNRTIs reveals that their binding induces conformational changes in RT that distort the precise geometry of the DNA polymerase catalytic site; these conformational changes affect the alignment of the primer terminus and slow down phosphodiester bond formation, as well as restrict domain motions and DNA translocation (48,51,52,54). Some NNRTIs can modulate RNase H activity through long-range interactions and, depending upon the structure of the RNA-DNA hybrid substrate, can lead to the inhibition or stimulation of RNase H activity (21,25,37,45,50). These NNRTIs alter the RNase H cleavage site specificity and rates of the reaction (21), resulting in the accumulation of secondary cleavage products (37,45), but do not affect the activity of the isolated RNase H domain (25).…”

HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor Dihydroxy Benzoyl Naphthyl Hydrazone Bound at a Novel Site

“…3,4-Dihydroxybenzhydrazide and other acid hydrazides were obtained from TransWorld Chemical (Rockville, MD). [ 3 H]-TTP and the homopolymeric template-primer poly(rA)-oligo (dT) [12][13][14][15][16][17][18] were products of Amersham Bio-sciences. The oligonucleotides 5′-GAU CUG AGC CUG GGA GCU-fluorescein-3′ and 5′-dabcyl-AGC TCC CAG GCT CAG ATC-3′ were synthesized and provided as an annealed RNA-DNA duplex by TriLink Biotechnologies (San Diego, CA).…”