Mechanism of Inhibition of HIV-1 Reverse Transcriptase by Nonnucleoside Inhibitors

Spence, Rebecca A.; Kati, Warren M.; Anderson, Karen S.; Johnson, Kenneth A.

doi:10.1126/science.7532321

Cited by 494 publications

(448 citation statements)

References 42 publications

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“…The values of NERT inhibition for virus budded in the presence of EFV were not different before and after the wash procedure, suggesting that this NNRTI remains bound to the RT molecules inside the viral particles. These results agree with reports that named NNRTIs as rapid-equilibrium and tightly binding inhibitors (16,28). NVP is a rapid-equilibrium inhibitor and requires an excess of the drug over the enzyme concentration (27).…”

Section: Discussionsupporting

confidence: 83%

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

Aguiar

Costa

Pereira

et al. 2007

Antimicrob Agents Chemother

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Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC 50 s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.

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Section: Discussionsupporting

confidence: 83%

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

Aguiar

Costa

Pereira

et al. 2007

Antimicrob Agents Chemother

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“…NNRTIs are clinically approved anti-HIV drugs 36 that bind to a hydrophobic pocket 4 near the polymerase active site of RT to inhibit DNA synthesis allosterically 37 . We examined one such NNRTI, nevirapine, for its effects on the orientational dynamics of RT.…”

Section: Small-molecule Ligands Regulate Rt Binding Orientationmentioning

confidence: 99%

Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

173

216

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The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

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“…Inhibitors of RT fall into two groups: dideoxynucleoside analogues, which (as their metabolically activated triphosphates) cause premature termination of the extending DNA strand, and non-nucleoside inhibitors (NNIs; DeClercq, 1996). Kinetic (Spence et al, 1995) and structural studies have shown that NNIs, which are almost exclusively HIV-1 specific, function by an allosteric mechanism whereby binding of an NNI in an internal pocket of the RT causes distortion of the polymerase active site. The effectiveness of anti-AIDS therapies is compromised by the selection for drugresistant viral populations (Larder & Kemp, 1989;Schinazi et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

et al. 1998

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A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a highresolution limit of 3.7 ,~. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2,~. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

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Mechanism of Inhibition of HIV-1 Reverse Transcriptase by Nonnucleoside Inhibitors

Cited by 494 publications

References 42 publications

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

Dynamic binding orientations direct activity of HIV reverse transcriptase

Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

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