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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

PKC agonists synergize with BETi in releasing active P-TEFb.Jurkat cells were mock-treated, treated with JQ1 (0.25μM), bryostatin-1 (5nM) alone or in combination for 1 hour (Panel A) or 24 hours (Panel B). Nuclear extracts were prepared from treated cells and subjected to immunoprecipitations (IP) with an anti-CDK9 antibody or the control IgG. The complexes were immunodetected for the presence of CycT1 and HEXIM-1 by Western blotting (right panels). Input controls for CDK9, CycT1 and HEXIM1 are presented (left panels). Levels of β-actin were measured to control protein loading. Panels A and B. Histograms represent quantification of band intensities normalized to CDK9 levels in the IP and then normalized to mock-treated condition. Panel C. HeLa cells expressing YC.P-TEFb and VN.CTD were left untreated or were treated as indicated for 1 hour. Venus-positive cells were detected by fluorescence microscopy (upper panels). Bright-field images were also taken (lower panels). Panel D. HeLa cells expressing YC.P-TEFb and VN.CTD were treated as outlined in C and Venus-positive cells were counted and averaged from three different areas. Error bars represent differences between counts of Venus-positive cells from the randomly chosen fields under the microscope. Panel E. Hela cells were transfected with the Hex1(-104)Luc reporter plasmid. At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated condition (transfection of the reporter plasmid without drug treatment) and arbitrarily set at a value of 1. An experiment performed in duplicate representative of two independent experiments is shown.
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ppat.1005063.g008: PKC agonists synergize with BETi in releasing active P-TEFb.Jurkat cells were mock-treated, treated with JQ1 (0.25μM), bryostatin-1 (5nM) alone or in combination for 1 hour (Panel A) or 24 hours (Panel B). Nuclear extracts were prepared from treated cells and subjected to immunoprecipitations (IP) with an anti-CDK9 antibody or the control IgG. The complexes were immunodetected for the presence of CycT1 and HEXIM-1 by Western blotting (right panels). Input controls for CDK9, CycT1 and HEXIM1 are presented (left panels). Levels of β-actin were measured to control protein loading. Panels A and B. Histograms represent quantification of band intensities normalized to CDK9 levels in the IP and then normalized to mock-treated condition. Panel C. HeLa cells expressing YC.P-TEFb and VN.CTD were left untreated or were treated as indicated for 1 hour. Venus-positive cells were detected by fluorescence microscopy (upper panels). Bright-field images were also taken (lower panels). Panel D. HeLa cells expressing YC.P-TEFb and VN.CTD were treated as outlined in C and Venus-positive cells were counted and averaged from three different areas. Error bars represent differences between counts of Venus-positive cells from the randomly chosen fields under the microscope. Panel E. Hela cells were transfected with the Hex1(-104)Luc reporter plasmid. At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated condition (transfection of the reporter plasmid without drug treatment) and arbitrarily set at a value of 1. An experiment performed in duplicate representative of two independent experiments is shown.

Mentions: To address the molecular mechanisms mediating the synergistic activation of HIV-1 transcription and production following the combined bryostatin-1+JQ1 treatment, we investigated the effect on P-TEB release of these LRAs alone or in combination. To this end, Jurkat cells were treated with JQ1, bryostatin-1 or a combination of both compounds for 1 hour and 24 hours. Nuclear extracts were prepared from treated cells and were used to perform immunoprecipitation experiments targeting CDK9 and Western blotting addressing the interaction of CDK9 with either HEXIM1 or CycT1. As shown in Fig 8A, for the 1 hour treatment, our results demonstrated a transient release of HEXIM1 from the CDK9/CycT1 (P-TEFb) complex after JQ1 treatment. These data are in agreement with a previous report from Peterlin and colleagues [31]. Bryostatin-1 treatment caused a weaker release of HEXIM1 from the P-TEFb complex than the JQ1 treatment (Fig 8A, compare lane 8 with lane 9). Interestingly, the combined treatment bryostatin-1+JQ1 led to a much stronger and synergistic HEXIM1 release than the individual treatments. These results indicated that that combined treatment increased the global availability of the active form of P-TEFb. After 24 hours of treatment (Fig 8B), we observed no P-TEFb release but a potentiated interaction between HEXIM1 and P-TEFb. This 24 hour effect contrasted with the 1 hour short-term effect and indicated reassembly of P-TEFb in the inactive 7SK snRNP complex. These results are consistent with the study of Liu et al. reporting that transient release of P-TEFb results in upregulation of its immediate target gene, HEXIM1, and reincorporation of P-TEFb into the 7SK snRNP [48]. Consistently, we also observed an increased expression of HEXIM1 after the 24 hours treatment compared to the 1 hour treatment (Fig 8B, input panel), thereby favoring a negative feedback mechanism of P-TEFb activation.


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)

PKC agonists synergize with BETi in releasing active P-TEFb.Jurkat cells were mock-treated, treated with JQ1 (0.25μM), bryostatin-1 (5nM) alone or in combination for 1 hour (Panel A) or 24 hours (Panel B). Nuclear extracts were prepared from treated cells and subjected to immunoprecipitations (IP) with an anti-CDK9 antibody or the control IgG. The complexes were immunodetected for the presence of CycT1 and HEXIM-1 by Western blotting (right panels). Input controls for CDK9, CycT1 and HEXIM1 are presented (left panels). Levels of β-actin were measured to control protein loading. Panels A and B. Histograms represent quantification of band intensities normalized to CDK9 levels in the IP and then normalized to mock-treated condition. Panel C. HeLa cells expressing YC.P-TEFb and VN.CTD were left untreated or were treated as indicated for 1 hour. Venus-positive cells were detected by fluorescence microscopy (upper panels). Bright-field images were also taken (lower panels). Panel D. HeLa cells expressing YC.P-TEFb and VN.CTD were treated as outlined in C and Venus-positive cells were counted and averaged from three different areas. Error bars represent differences between counts of Venus-positive cells from the randomly chosen fields under the microscope. Panel E. Hela cells were transfected with the Hex1(-104)Luc reporter plasmid. At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated condition (transfection of the reporter plasmid without drug treatment) and arbitrarily set at a value of 1. An experiment performed in duplicate representative of two independent experiments is shown.
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Related In: Results  -  Collection

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

ppat.1005063.g008: PKC agonists synergize with BETi in releasing active P-TEFb.Jurkat cells were mock-treated, treated with JQ1 (0.25μM), bryostatin-1 (5nM) alone or in combination for 1 hour (Panel A) or 24 hours (Panel B). Nuclear extracts were prepared from treated cells and subjected to immunoprecipitations (IP) with an anti-CDK9 antibody or the control IgG. The complexes were immunodetected for the presence of CycT1 and HEXIM-1 by Western blotting (right panels). Input controls for CDK9, CycT1 and HEXIM1 are presented (left panels). Levels of β-actin were measured to control protein loading. Panels A and B. Histograms represent quantification of band intensities normalized to CDK9 levels in the IP and then normalized to mock-treated condition. Panel C. HeLa cells expressing YC.P-TEFb and VN.CTD were left untreated or were treated as indicated for 1 hour. Venus-positive cells were detected by fluorescence microscopy (upper panels). Bright-field images were also taken (lower panels). Panel D. HeLa cells expressing YC.P-TEFb and VN.CTD were treated as outlined in C and Venus-positive cells were counted and averaged from three different areas. Error bars represent differences between counts of Venus-positive cells from the randomly chosen fields under the microscope. Panel E. Hela cells were transfected with the Hex1(-104)Luc reporter plasmid. At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated condition (transfection of the reporter plasmid without drug treatment) and arbitrarily set at a value of 1. An experiment performed in duplicate representative of two independent experiments is shown.
Mentions: To address the molecular mechanisms mediating the synergistic activation of HIV-1 transcription and production following the combined bryostatin-1+JQ1 treatment, we investigated the effect on P-TEB release of these LRAs alone or in combination. To this end, Jurkat cells were treated with JQ1, bryostatin-1 or a combination of both compounds for 1 hour and 24 hours. Nuclear extracts were prepared from treated cells and were used to perform immunoprecipitation experiments targeting CDK9 and Western blotting addressing the interaction of CDK9 with either HEXIM1 or CycT1. As shown in Fig 8A, for the 1 hour treatment, our results demonstrated a transient release of HEXIM1 from the CDK9/CycT1 (P-TEFb) complex after JQ1 treatment. These data are in agreement with a previous report from Peterlin and colleagues [31]. Bryostatin-1 treatment caused a weaker release of HEXIM1 from the P-TEFb complex than the JQ1 treatment (Fig 8A, compare lane 8 with lane 9). Interestingly, the combined treatment bryostatin-1+JQ1 led to a much stronger and synergistic HEXIM1 release than the individual treatments. These results indicated that that combined treatment increased the global availability of the active form of P-TEFb. After 24 hours of treatment (Fig 8B), we observed no P-TEFb release but a potentiated interaction between HEXIM1 and P-TEFb. This 24 hour effect contrasted with the 1 hour short-term effect and indicated reassembly of P-TEFb in the inactive 7SK snRNP complex. These results are consistent with the study of Liu et al. reporting that transient release of P-TEFb results in upregulation of its immediate target gene, HEXIM1, and reincorporation of P-TEFb into the 7SK snRNP [48]. Consistently, we also observed an increased expression of HEXIM1 after the 24 hours treatment compared to the 1 hour treatment (Fig 8B, input panel), thereby favoring a negative feedback mechanism of P-TEFb activation.

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