Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities
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- 作者：Cathryn A. Shaw-Reid ; Bradley Feuston ; Vandna Munshi ; Krista Getty ; Julie Krueger ; Daria J. Hazuda ; Michael A. Parniak ; Michael D. Miller ; and Dale Lewis
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：February 8, 2005
- 年：2005
- 卷：44
- 期：5
- 页码：1595 - 1606
- 全文大小：769K
- 年卷期：v.44,no.5(February 8, 2005)
- ISSN：1520-4995
文摘
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 polymerasedomain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze theimpact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. Thenucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas thepyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependentmanner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage productsdiffered substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 differentRT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNaseH domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggestingthat NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase Hactive 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 Hcleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, althoughthe NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKAcombinations were synergistic in inhibiting reverse transcription overall, indicating that regimensincorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.