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An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

Darcis G, Kula A, Bouchat S, Fujinaga K, Corazza F, Ait-Ammar A, Delacourt N, Melard A, Kabeya K, Vanhulle C, Van Driessche B, Gatot JS, Cherrier T, Pianowski LF, Gama L, Schwartz C, Vila J, Burny A, Clumeck N, Moutschen M, De Wit S, Peterlin BM, Rouzioux C, Rohr O, Van Lint C - PLoS Pathog. (2015)

Bottom Line: Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments.The potent effects of these two combination treatments were already detected 24 hours post-stimulation.These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Service of Molecular Virology, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), Gosselies, Belgium; Service des Maladies Infectieuses, Université de Liège, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium.

ABSTRACT
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

No MeSH data available.


Related in: MedlinePlus

Synergistic inducibility of HIV-1 LTR promoter by PKC agonist+BETi/HMBA combinatory treatments depends on NF-κB.Panel A. Jurkat cells were transiently transfected with the episomal plasmid containing the luciferase reporter gene driven either by the wild-type HIV LTR promoter (LTRwt-luc) or by the LTR promoter mutated in the two NF-κB binding sites (LTR-NFκBmut-luc). Twenty-four hours later, cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated conditions (transfection of the reporter plasmid without drug treatment) and arbitrarily set at values of 1. An experiment performed in triplicates representative of two independent experiments is shown. Panel B. Nuclear extracts were prepared from Jurkat cells which were mock-treated, treated with bryostatin-1 (10nM), JQ1 (0.5μM) or with bryostatin-1+JQ1 for different time periods. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe.
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ppat.1005063.g007: Synergistic inducibility of HIV-1 LTR promoter by PKC agonist+BETi/HMBA combinatory treatments depends on NF-κB.Panel A. Jurkat cells were transiently transfected with the episomal plasmid containing the luciferase reporter gene driven either by the wild-type HIV LTR promoter (LTRwt-luc) or by the LTR promoter mutated in the two NF-κB binding sites (LTR-NFκBmut-luc). Twenty-four hours later, cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated conditions (transfection of the reporter plasmid without drug treatment) and arbitrarily set at values of 1. An experiment performed in triplicates representative of two independent experiments is shown. Panel B. Nuclear extracts were prepared from Jurkat cells which were mock-treated, treated with bryostatin-1 (10nM), JQ1 (0.5μM) or with bryostatin-1+JQ1 for different time periods. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe.

Mentions: We assessed the molecular mechanisms underlying the synergistic induction of HIV-1 observed at the viral RNA and protein levels following individual or combined treatments with PKC agonists and BETi/HMBA (Figs 1–6). Firstly, we evaluated the involvement of two NF-κB binding sites located in the enhancer region of the HIV-1 promoter. We transiently transfected Jurkat cells either with episomal vector driving the luciferase expression from wild-type LTR (LTRwt-luc) or with similar reporter vector mutated in two NF-κB binding sites located in HIV LTR (LTR-NFκBmut-luc). Cells were stimulated with JQ1, I-BET, I-BET151, HMBA, prostratin, and bryostatin-1 alone or in combination. Combinations of PKC agonist+BETi/HMBA led to synergistic increases in luciferase activity arising from LTRwt-luc transfection (Fig 7A). We observed that lack of the two NF-κB binding sites located in HIV-1 LTR impaired the synergistic increase in luciferase activity following combined treatments. These data indicated the involvement of NF-κB protein binding to the viral promoter in the PKC agonist+BETi/HMBA synergies. To assess the effect of the drugs on the NF-κB binding activity, we next performed electrophoretic mobility shift assays (EMSAs) using an HIV-1 NF-κB probe and nuclear extracts from Jurkat T cells stimulated for various periods of time with JQ1, bryostatin-1 or bryostatin-1+JQ1 (Fig 7B). We detected an induction of NF-κB DNA-binding activity in response to a 60 min treatment with bryostatin-1 (Fig 7B, lane 7). JQ1 alone caused no induction of NF-κB binding activity even after a 240 min treatment (Fig 7B, lanes 2, 6, 10 and 14). When bryostatin-1 was combined with JQ1, an induction of NF-κB binding activity stronger than that obtained with bryostatin-1 alone was observed at 60 and 120 min time points (Fig 7B, compare lane 7 with lane 8 and lane 11 with lane 12). This effect faded away at 240 min time point, confirming transient NF-κB DNA- binding activity. Our results demonstrated that JQ1 increased bryostatin-1-induced NF-κB DNA-binding activity. This could explain, at least in part, the synergistic increases in HIV transcription following combined treatments observed in Fig 2.


An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

Darcis G, Kula A, Bouchat S, Fujinaga K, Corazza F, Ait-Ammar A, Delacourt N, Melard A, Kabeya K, Vanhulle C, Van Driessche B, Gatot JS, Cherrier T, Pianowski LF, Gama L, Schwartz C, Vila J, Burny A, Clumeck N, Moutschen M, De Wit S, Peterlin BM, Rouzioux C, Rohr O, Van Lint C - PLoS Pathog. (2015)

Synergistic inducibility of HIV-1 LTR promoter by PKC agonist+BETi/HMBA combinatory treatments depends on NF-κB.Panel A. Jurkat cells were transiently transfected with the episomal plasmid containing the luciferase reporter gene driven either by the wild-type HIV LTR promoter (LTRwt-luc) or by the LTR promoter mutated in the two NF-κB binding sites (LTR-NFκBmut-luc). Twenty-four hours later, cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated conditions (transfection of the reporter plasmid without drug treatment) and arbitrarily set at values of 1. An experiment performed in triplicates representative of two independent experiments is shown. Panel B. Nuclear extracts were prepared from Jurkat cells which were mock-treated, treated with bryostatin-1 (10nM), JQ1 (0.5μM) or with bryostatin-1+JQ1 for different time periods. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520688&req=5

ppat.1005063.g007: Synergistic inducibility of HIV-1 LTR promoter by PKC agonist+BETi/HMBA combinatory treatments depends on NF-κB.Panel A. Jurkat cells were transiently transfected with the episomal plasmid containing the luciferase reporter gene driven either by the wild-type HIV LTR promoter (LTRwt-luc) or by the LTR promoter mutated in the two NF-κB binding sites (LTR-NFκBmut-luc). Twenty-four hours later, cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated conditions (transfection of the reporter plasmid without drug treatment) and arbitrarily set at values of 1. An experiment performed in triplicates representative of two independent experiments is shown. Panel B. Nuclear extracts were prepared from Jurkat cells which were mock-treated, treated with bryostatin-1 (10nM), JQ1 (0.5μM) or with bryostatin-1+JQ1 for different time periods. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe.
Mentions: We assessed the molecular mechanisms underlying the synergistic induction of HIV-1 observed at the viral RNA and protein levels following individual or combined treatments with PKC agonists and BETi/HMBA (Figs 1–6). Firstly, we evaluated the involvement of two NF-κB binding sites located in the enhancer region of the HIV-1 promoter. We transiently transfected Jurkat cells either with episomal vector driving the luciferase expression from wild-type LTR (LTRwt-luc) or with similar reporter vector mutated in two NF-κB binding sites located in HIV LTR (LTR-NFκBmut-luc). Cells were stimulated with JQ1, I-BET, I-BET151, HMBA, prostratin, and bryostatin-1 alone or in combination. Combinations of PKC agonist+BETi/HMBA led to synergistic increases in luciferase activity arising from LTRwt-luc transfection (Fig 7A). We observed that lack of the two NF-κB binding sites located in HIV-1 LTR impaired the synergistic increase in luciferase activity following combined treatments. These data indicated the involvement of NF-κB protein binding to the viral promoter in the PKC agonist+BETi/HMBA synergies. To assess the effect of the drugs on the NF-κB binding activity, we next performed electrophoretic mobility shift assays (EMSAs) using an HIV-1 NF-κB probe and nuclear extracts from Jurkat T cells stimulated for various periods of time with JQ1, bryostatin-1 or bryostatin-1+JQ1 (Fig 7B). We detected an induction of NF-κB DNA-binding activity in response to a 60 min treatment with bryostatin-1 (Fig 7B, lane 7). JQ1 alone caused no induction of NF-κB binding activity even after a 240 min treatment (Fig 7B, lanes 2, 6, 10 and 14). When bryostatin-1 was combined with JQ1, an induction of NF-κB binding activity stronger than that obtained with bryostatin-1 alone was observed at 60 and 120 min time points (Fig 7B, compare lane 7 with lane 8 and lane 11 with lane 12). This effect faded away at 240 min time point, confirming transient NF-κB DNA- binding activity. Our results demonstrated that JQ1 increased bryostatin-1-induced NF-κB DNA-binding activity. This could explain, at least in part, the synergistic increases in HIV transcription following combined treatments observed in Fig 2.

Bottom Line: Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments.The potent effects of these two combination treatments were already detected 24 hours post-stimulation.These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Service of Molecular Virology, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), Gosselies, Belgium; Service des Maladies Infectieuses, Université de Liège, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium.

ABSTRACT
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

No MeSH data available.


Related in: MedlinePlus