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

• Published in: Biochemistry (Easton) Vol. 44; no. 5; pp. 1595 - 1606
• Main Authors: Shaw-Reid, Cathryn A, Feuston, Bradley, Munshi, Vandna, Getty, Krista, Krueger, Julie, Hazuda, Daria J, Parniak, Michael A, Miller, Michael D, Lewis, Dale
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-02-2005
• Subjects: Anti-HIV Agents - chemistry; Anti-HIV Agents - pharmacology; Binding Sites; Butyrates - chemistry; Butyrates - pharmacology; Catalysis - drug effects; Dideoxynucleotides; DNA-Directed DNA Polymerase - metabolism; Drug Combinations; Drug Synergism; Foscarnet - chemistry; Foscarnet - pharmacology; HIV Reverse Transcriptase - antagonists & inhibitors; HIV Reverse Transcriptase - metabolism; Human immunodeficiency virus 1; Hydrolysis; Kinetics; Nucleic Acid Synthesis Inhibitors; Oxazines - chemistry; Oxazines - pharmacology; Reverse Transcriptase Inhibitors - chemistry; Reverse Transcriptase Inhibitors - pharmacology; Ribonuclease H - antagonists & inhibitors; Ribonuclease H - metabolism; Thiophenes - chemistry; Thiophenes - pharmacology; Thymine Nucleotides - chemistry; Thymine Nucleotides - pharmacology; Zidovudine - analogs & derivatives; Zidovudine - chemistry; Zidovudine - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0486740

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.