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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 agonist+BETi/HMBA combined treatments increase HIV-1 expression in a higher proportion of cells than the drug alone and synergistically enhance viral transcription.The J-Lat 9.2 cells (panel A) or CHME-5/HIV microglial cells (panel B) harbor latent HIV-1 provirus containing gfp gene. The 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 as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained from individual treatments. Panels C-F. Measurement of initiated and elongated HIV-1 transcripts following drug treatment. Total RNA was extracted from J-Lat 9.2 (panels C and D), U1 (panel E), CHME-5/HIV (panel F) cells which were mock-treated or treated with BETi, HMBA, bryostatin-1 and prostratin for 24 hours at concentrations described in Fig 2A and 2B. Initiated (primers TAR) or elongated (primers tat or env) transcripts were quantified by quantitative real-time RT-PCR. Values were normalized using β-actin gene primers and were presented as fold inductions relative to the values measured in mock-treated cells, which were arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.
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ppat.1005063.g002: PKC agonist+BETi/HMBA combined treatments increase HIV-1 expression in a higher proportion of cells than the drug alone and synergistically enhance viral transcription.The J-Lat 9.2 cells (panel A) or CHME-5/HIV microglial cells (panel B) harbor latent HIV-1 provirus containing gfp gene. The 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 as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained from individual treatments. Panels C-F. Measurement of initiated and elongated HIV-1 transcripts following drug treatment. Total RNA was extracted from J-Lat 9.2 (panels C and D), U1 (panel E), CHME-5/HIV (panel F) cells which were mock-treated or treated with BETi, HMBA, bryostatin-1 and prostratin for 24 hours at concentrations described in Fig 2A and 2B. Initiated (primers TAR) or elongated (primers tat or env) transcripts were quantified by quantitative real-time RT-PCR. Values were normalized using β-actin gene primers and were presented as fold inductions relative to the values measured in mock-treated cells, which were arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.

Mentions: We next investigated whether synergistic effects in viral p24 antigen production (Fig 1) following PKC agonist+BETi/HMBA co-treatments were (i) due to an enhanced HIV-1 expression from those cells whose transcription was already reactivated by the individual drugs or (ii) due to an increase in the number of cells expressing virus. We used the J-Lat 9.2 cell line in which transcriptional activation of the latent provirus can be detected in individual cells by flow cytometry since these cells harbor full-length latent HIV-1 provirus containing gfp gene in place of nef. In the absence of stimulation, the J-Lat 9.2 cells expressed no GFP, indicating the blockade of viral transcription (Fig 2A). Treatments with each compound releasing P-TEFb used individually did not increase the number of GFP-positive cells (Fig 2A). Bryostatin-1 was weaker than prostratin in inducing GFP expression (1.3% compared to 6.3%). However, when we examined the effects of either bryostatin-1 or prostratin combined with BETi/HMBA, we observed similar synergies. The Jurkat CD4+ T-cell-based J-Lat clones are the most studied cellular models of HIV-1 post-integration latency. However, it is critical to address whether similar effects could be observed in other latency models of other cellular origins. For instance, microglial cells are of special importance since they represent the primary host cells for HIV in the brain. Therefore, we took advantage of CHME-5/HIV latently infected microglial cells developed in Jonathan Karn’s laboratory [41]. The CHME-5/HIV cell line containing reporter GFP was treated with prostratin, bryostatin-1, BETi and HMBA alone or in combination. JQ1 was the strongest LRA among the compounds releasing P-TEFb and activated 4.65% of cells (Fig 2B). Conversely, both bryostatin-1 and prostratin activated 4% of cells. Importantly, the proportion of GFP-positive CHME-5/HIV cells was synergistically increased by combined treatments except for the PKC agonist+HMBA and prostratin+I-BET combinations. Combinations of PKC agonist+JQ1 led to the highest synergistic increases in the percentage of GFP-positive cells (12% for bryostatin-1+JQ1 and 15% for prostratin+JQ1).


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 agonist+BETi/HMBA combined treatments increase HIV-1 expression in a higher proportion of cells than the drug alone and synergistically enhance viral transcription.The J-Lat 9.2 cells (panel A) or CHME-5/HIV microglial cells (panel B) harbor latent HIV-1 provirus containing gfp gene. The 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 as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained from individual treatments. Panels C-F. Measurement of initiated and elongated HIV-1 transcripts following drug treatment. Total RNA was extracted from J-Lat 9.2 (panels C and D), U1 (panel E), CHME-5/HIV (panel F) cells which were mock-treated or treated with BETi, HMBA, bryostatin-1 and prostratin for 24 hours at concentrations described in Fig 2A and 2B. Initiated (primers TAR) or elongated (primers tat or env) transcripts were quantified by quantitative real-time RT-PCR. Values were normalized using β-actin gene primers and were presented as fold inductions relative to the values measured in mock-treated cells, which were arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.
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Related In: Results  -  Collection

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

ppat.1005063.g002: PKC agonist+BETi/HMBA combined treatments increase HIV-1 expression in a higher proportion of cells than the drug alone and synergistically enhance viral transcription.The J-Lat 9.2 cells (panel A) or CHME-5/HIV microglial cells (panel B) harbor latent HIV-1 provirus containing gfp gene. The 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 as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained from individual treatments. Panels C-F. Measurement of initiated and elongated HIV-1 transcripts following drug treatment. Total RNA was extracted from J-Lat 9.2 (panels C and D), U1 (panel E), CHME-5/HIV (panel F) cells which were mock-treated or treated with BETi, HMBA, bryostatin-1 and prostratin for 24 hours at concentrations described in Fig 2A and 2B. Initiated (primers TAR) or elongated (primers tat or env) transcripts were quantified by quantitative real-time RT-PCR. Values were normalized using β-actin gene primers and were presented as fold inductions relative to the values measured in mock-treated cells, which were arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.
Mentions: We next investigated whether synergistic effects in viral p24 antigen production (Fig 1) following PKC agonist+BETi/HMBA co-treatments were (i) due to an enhanced HIV-1 expression from those cells whose transcription was already reactivated by the individual drugs or (ii) due to an increase in the number of cells expressing virus. We used the J-Lat 9.2 cell line in which transcriptional activation of the latent provirus can be detected in individual cells by flow cytometry since these cells harbor full-length latent HIV-1 provirus containing gfp gene in place of nef. In the absence of stimulation, the J-Lat 9.2 cells expressed no GFP, indicating the blockade of viral transcription (Fig 2A). Treatments with each compound releasing P-TEFb used individually did not increase the number of GFP-positive cells (Fig 2A). Bryostatin-1 was weaker than prostratin in inducing GFP expression (1.3% compared to 6.3%). However, when we examined the effects of either bryostatin-1 or prostratin combined with BETi/HMBA, we observed similar synergies. The Jurkat CD4+ T-cell-based J-Lat clones are the most studied cellular models of HIV-1 post-integration latency. However, it is critical to address whether similar effects could be observed in other latency models of other cellular origins. For instance, microglial cells are of special importance since they represent the primary host cells for HIV in the brain. Therefore, we took advantage of CHME-5/HIV latently infected microglial cells developed in Jonathan Karn’s laboratory [41]. The CHME-5/HIV cell line containing reporter GFP was treated with prostratin, bryostatin-1, BETi and HMBA alone or in combination. JQ1 was the strongest LRA among the compounds releasing P-TEFb and activated 4.65% of cells (Fig 2B). Conversely, both bryostatin-1 and prostratin activated 4% of cells. Importantly, the proportion of GFP-positive CHME-5/HIV cells was synergistically increased by combined treatments except for the PKC agonist+HMBA and prostratin+I-BET combinations. Combinations of PKC agonist+JQ1 led to the highest synergistic increases in the percentage of GFP-positive cells (12% for bryostatin-1+JQ1 and 15% for prostratin+JQ1).

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