Effects of Efavirenz Binding on the Subunit Equilibria of HIV-1 Reverse Transcriptase

Venezia, Carl F.; Howard, Kathryn J.; Ignatov, M.E.; Holladay, Leslie A.; Barkley, Mary D.

doi:10.1021/bi051915z

Cited by 54 publications

(119 citation statements)

References 39 publications

(79 reference statements)

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“…S4C, S4D, and S4E, respectively. the p51 homodimer (K d ϭ 230 M) (25). For each of the RT bait-prey combinations strong signals were obtained, whereas background signals were consistently low (supplemental Fig.…”

Section: In Situ Monitoring Of Ppis and Their Modulation In Response mentioning

confidence: 96%

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Lievens

Gerlo

Lemmens

et al. 2014

Molecular & Cellular Proteomics

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Probably every cellular process is governed by proteinprotein interaction (PPIs), which are often highly dynamic in nature being modulated by in-or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent ␤-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken A protein's function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI) 1 maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra-and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow

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Section: In Situ Monitoring Of Ppis and Their Modulation In Response mentioning

confidence: 96%

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Lievens

Gerlo

Lemmens

et al. 2014

Molecular & Cellular Proteomics

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“…It has been demonstrated that some NNRTIs act as chemical enhancers of HIV-1 RT heterodimerization (Tachedjian et al, 2001;Venezia et al, 2006). To date, efavirenz was found to be the most potent enhancer of RT heterodimerization, whereas nevirapine has a weak effect and delavirdine has no effect at all (Tachedjian et al, 2001).…”

Section: Nnrtis Act As Potent Inhibitors Of Hiv-1 Rt Dimerizationmentioning

confidence: 99%

Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors

2008

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The nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a therapeutic class of compounds that are routinely used, in combination with other antiretroviral drugs, to treat HIV-1 infection. NNRTIs primarily block HIV-1 replication by preventing RT from completing reverse transcription of the viral single-stranded RNA genome into DNA. However, some NNRTIs, such as efavirenz, have been shown to inhibit the late stages of HIV-1 replication by interfering with HIV-1 Gag-Pol polyprotein processing, while others, such as the pyrimidinediones, have been shown to inhibit both HIV-1 RT-mediated reverse transcription and HIV-1/HIV-2 viral entry. Accordingly, in this review we describe the multiple mechanisms by which NNRTIs inhibit HIV-1 reverse transcription (and in some cases HIV-2 reverse transcription) and other key steps involved in HIV-1/ HIV-2 replication.

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“…First, RT truncation mutations involving the Trp repeat motif significantly impair PR-mediated Gag processing (30). Second, efavirenz (EFV)-a nonnucleoside reverse transcriptase inhibitor (NNRTI) that greatly enhances HIV-1 RT dimerization in vitro (46,47)-is capable of suppressing virion production by enhancing the efficiency of PR-mediated Gag and Gag-Pol cleavage (14,45). However, a W401A alanine substitution mu-tation that abrogates RT dimerization in vitro (49) almost completely negates the EFV inhibitory effect on virion production (9).…”

Section: Gag-polmentioning

confidence: 99%

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

Chiang

Wang

Pan

et al. 2010

J Virol

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HIV-1 protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. PR activation is thought to be triggered by appropriate Gag-Pol/Gag-Pol interaction; factors affecting this interaction either enhance or reduce PR-mediated cleavage efficiency, resulting in markedly reduced virion production or the release of inadequately processed virions. We previously showed that a Gag-Pol deletion mutation involving the reverse transcriptase tryptophan (Trp) repeat motif markedly impairs PR-mediated virus maturation and that an alanine substitution at W401 (W401A) or at both W401 and W402 (W401A/ W402A) partially or almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the Trp repeat motif may contribute to the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency, W402 alanine or leucine substitution significantly reduces virus production. However, W402 replacement with phenylalanine does not significantly affect virus particle assembly or processing, but it does markedly impair viral infectivity in a single-cycle infection assay. Our results demonstrate that a single amino acid substitution at HIV-1 RT can radically affect virus assembly by enhancing Gag cleavage efficiency, suggesting that in addition to contributing to RT biological function during the early stages of virus replication, the HIV-1 RT tryptophan repeat motif in a Gag-Pol context may play an important role in suppressing the premature activation of PR during late-stage virus replication.

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Effects of Efavirenz Binding on the Subunit Equilibria of HIV-1 Reverse Transcriptase

Cited by 54 publications

References 39 publications

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

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