The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency

Abstract

Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4(+) T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4(+) T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs.

Importance: Antiretroviral therapy (ART) reduces HIV-1 replication to very low levels, but the virus persists in latently infected memory CD4(+) T cells, representing a long-lasting source of resurgent virus upon ART interruption. Based on the mode of action of didehydro-cortistatin A (dCA), a Tat-dependent transcription inhibitor, our work highlights an alternative approach to current HIV-1 eradication strategies to decrease the latent reservoir. In our model, dCA blocks the Tat feedback loop initiated after low-level basal reactivation, blocking transcriptional elongation and hence viral production from latently infected cells. Therefore, dCA combined with ART would be aimed at delaying or halting ongoing viral replication, reactivation, and replenishment of the latent viral reservoir. Thus, the latent pool of cells in an infected individual would be stabilized, and death of the long-lived infected memory T cells would result in a continuous decay of this pool over time, possibly culminating in the long-awaited sterilizing cure.

Copyright © 2015 Mousseau et al.

Figures

FIG 1

dCA inhibits HIV reactivation after TCR stimulation from CD4+ T cells isolated from virally suppressed subjects. (A) CD4+ T cells were isolated from PBMCs from nine virally suppressed infected individuals carefully selected that did not display spontaneous viral production upon in vitro culture to better reflect latency. Activation of viral production from latency with anti-CD3/CD28 beads was performed in the presence of ARVs with or without 100 nM dCA. Viral genomic RNAs from viral particles released in the supernatants were extracted 6 days later and analyzed by ultrasensitive RT-qPCR. NS, nonstimulated; ND, not detected. (B) Summary of the nine subjects. The two-tailed paired t test was used for statistical comparisons.

FIG 2

dCA inhibits residual transcription in HIV-1 latently infected cell line models. (A) dCA inhibits NL4-3 virus expression to undetectable levels in HeLa-CD4 cells (left panel). Latently infected HeLa-CD4 cells were treated with DMSO control or dCA for 239 days (100 nM dCA used from 0 to 118 days and then at 10 nM afterward). dCA treatment was stopped at day 24 (TS1) and day 103 (TS2). TS, treatment stop. Capsid p24 in the supernatants was assayed in an ELISA (detection limit, 3.1 pg/ml). Data are representative of two independent experiments (right panel). For analysis of viral mRNA expression, cDNAs from total RNA extracted at day 239 were quantified by RT-qPCR using primers to the Vpr region. Results were normalized as number of viral mRNA copies per GAPDH mRNA. Viral mRNA generated in the DMSO control was set to 100%. Data are representative of two analyses (days 129 and 239). RT-qPCR data are reported as means ± SD. (B) dCA inhibits viral production in the OM-10.1 cell line to almost-undetectable levels (left panel). OM-10.1 cells were split and treated on average every 3 days in the presence of ARVs with or without dCA. Capsid production was quantified via a p24 ELISA. Data are representative of four independent experiments with treatment of cells ranging from 42 to 226 days (right panel). For analysis of viral mRNA expression in dCA-treated OM-10.1 cells, cDNAs from total RNA (day 197) were quantified, normalized, and are reported as described for panel A using primers to the Gag-Pol region. Data are representative of three analyses at days 102, 163, and 197. (C and D) Effects of dCA on J-Lat 6.3 and 10.6 clones (left panels). Cells were split and treated on average every 3 days with or without 10 nM dCA. Capsid production was quantified via a p24 ELISA, and results for the DMSO controls were set to 100%. Data are reported as means ± SD of two or three independent experiments, respectively. Significant effects of dCA (****, P < 0.0001) and time (****, P < 0.0001) were determined by a two-way repeated-measures ANOVA with Bonferroni correction post hoc (n = 3 per group) (right panels). dCA inhibited viral transcription in J-Lat clones. Viral mRNA levels (day 9) were quantified, normalized, and are reported as described for panel A, using Gag-Pol primers. Data are representative of two independent experiments.

FIG 3

dCA reduces RNAP II recruitment to the HIV promoter and inhibits viral transcription elongation from latently infected HeLa-CD4 cells. (A) Schematic representation of the HIV genome and primer localizations. (B) RNAP II ChIP results with latently infected HeLa-CD4 cells treated long term with DMSO or dCA, at day 172. As controls, DMSO-treated cells were activated with TNF-α for 8 h or inhibited with α-amanitin (α-ama) for 48 h. Data are presented as percentages of input, with the average IgG background subtracted. Data for RT-qPCR (using the indicated primers) are reported as means ± SD. Results are representative of two independent ChIP assays. (C) GAPDH was used as the reference gene, with amplicons to the promoter or ORF.

FIG 4

dCA inhibits viral reactivation from latently infected HeLa-CD4 cells. (A) PMA-iono treatment failed to reactivate virus from dCA-mediated latency in HeLa-CD4 cells. Cells treated with DMSO or dCA at 10 nM were activated or not for 24 h with a combination of PMA and iono. Data are presented as means ± SD of two independent experiments performed at days 131 and 152 post-dCA treatment (n = 2). (B) Exogenous Tat reactivates virus from latency in dCA-treated HeLa-CD4 cells. Cells were evaluated at day 125 post-dCA treatment in the presence or absence of dCA by transfecting an empty vector control or a Tat-Flag-expressing plasmid, and p24 production was determined via an ELISA. Results are representative of three independent transfection experiments.

FIG 5

dCA inhibits viral reactivation from several models of latency. (A) dCA-mediated viral latency in OM-10.1 cells is refractory to reactivation by SAHA, TNF-α, or prostratin. Cells (DMSO controls or treated with dCA at 10 nM) (shown in Fig. 2B) were activated in the presence of ARVs with SAHA or TNF-α for 24 h or prostratin (pros) for 9 h, plus 100 nM dCA for the dCA-treated cells. Supernatant was collected at the end of the activation period and analyzed in a p24 ELISA. Data are presented as means ± SD of two independent activation experiments performed at days 219 and 226 post-dCA treatment (n = 5). (B and C) dCA inhibits viral reactivation from J-Lat 10.6 and 6.3 clones. At day 0, these clones were activated with SAHA, TNF-α, or prostratin in the presence or absence of 100 nM dCA for 24 h. SAHA was not able to activate J-Lat 6.3 above the limit of detection of the assay. Viral production was analyzed and is presented as described for panel in A, as means of two independent activation experiments (n = 4). Percentages represent the percent inhibition. The two-tailed paired t test was used for statistical comparisons. ND, not detectable.

FIG 6

dCA does not affect HIV-1 transcription in the ACH-2 or U1 models of HIV latency. (A and B) ACH-2 and U1 cells were infected with a virus with a mutant TAR or Tat, respectively (left panels). To analyze the effect of dCA on viral production in ACH-2 and U1 cells, cells were split and treated every 3 days on average with or without 10 nM dCA. Capsid production was quantified in a p24 ELISA. Data were normalized for each point to results with the DMSO control (as 100%) and are presented as means ± SD of three independent experiments. For analysis of the effects of dCA and time, a two-way repeated-measures ANOVA was performed. For ACH-2 cells (A), significant effects of dCA treatment (**, P = 0.0037) and time (***, P < 0.0002) are shown. For U1 cells (B), significant effects of dCA treatment (**, P = 0.0065) are shown. After post hoc Bonferroni correction (n = 3 per group), the statistical significance of results is indicated as follows: NS, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (right panels). dCA does not inhibit viral transcription in ACH-2 or U1 cells. On the indicated day, viral mRNA was extracted and analyzed by RT-qPCR using Gag-Pol primers. RT-qPCR data are presented as means ± SD and are representative of at least two independent experiments. (C and D) dCA does not inhibit viral reactivation in ACH-2 or U1 cells. Cells were grown for 15 days with ARVs and in the presence or absence of dCA at 10 nM. At days 9 and 15, DMSO-treated (blue) or dCA-treated (red) cells and also treated with ARVs were activated with SAHA, TNF-α, or prostratin for 24 h in the presence or absence of 100 nM dCA. Supernatant was collected at the end of the activation period and analyzed in a p24 ELISA. Data are presented as means ± SD of two independent activation experiments performed at days 9 and 15 (n = 4) except for U1 cells (SAHA and prostratin treatments, for which n = 2, due to the use of a different activation protocol at day 9). Percentages indicate the percent inhibition. The two-tailed paired t test was used for statistical comparisons. ND, not detectable.

FIG 7

dCA maintains a state of latency and blocks reactivation in primary expanded CD4+ T cells. (A) Schematic of generation of expanded CD4+ T cells and reactivation. CD4+ T cells were sorted from patient PBMCs and initially expanded with 1 µg/ml PHA, 100 U/ml IL-2, and irradiated autologous feeder PBMCs. Cells were then grown for 22 days in the presence of IL-2 and either ARVs alone or ARVs plus 100 nM dCA. At day 22, cells were split into 6 groups and either stimulated with prostratin or nonstimulated, with treatment stopped (TS) or continued for an additional 6 days before measuring HIV RNA viral production. (B) Limited viral rebound upon dCA removal in expanded primary CD4+ T cells derived from patients A and B. At day 22, the ARVs or ARVs plus dCA cultured CD4+ T cells were washed, and all drugs were removed. Particle-associated HIV genomic RNAs were quantified by RT-qPCR at day 6 after TS (day 28). (C) Inhibition of viral reactivation upon prostratin stimulation in primary CD4+ T cells expanded in the presence of dCA. At day 22, the ARVs or ARVs plus dCA cultured CD4+ T cells were stimulated with prostratin or left unstimulated. Quantification was done as described for panel B (day 28). ND, nondetected. Data are presented as means ± standard errors of the means (n = 2).

FIG 8

Hypothetical approach to a functional HIV cure. (1) Upon HIV infection, there is a sharp increase of the viral load in circulating plasma of infected individuals. (2) The viral load sharply decreases to below the limit of detection (+ T cell reactivation (4). The addition of a Tat inhibitor such as dCA to an ART regimen could promote and maintain a state of latency, possibly allowing for ART interruption without viral rebound. dCA may also prevent reservoir replenishment. With time, patients may potentially observe a reduction in the size of the viral reservoir and relief from chronic inflammation caused by ongoing low-level virus production.