Synergistic Activation of HIV-1 Expression by Deacetylase Inhibitors and Prostratin: Implications for Treatment of Latent Infection

Abstract

The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- κB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5′ Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-κB and degradation of cytoplasmic NF-κB inhibitor, IκBα . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8+-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4+ T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.

Histone deacetylase inhibitor Scriptaid reactivates latent HIV-1 promoter by inducing histone modification in in vitro latency cell lines

“…Our results showed that under the co-treatment of Scriptaid/prostratin or Scriptaid/TNF-·, HIV-1 expression was induced with higher proportions 6.4 or 37.4% of A7 cells than being treated by the Scriptaid alone, indicating that Scriptaid synergistically reactivates HIV-1 production with prostratin or TNF-· in the latency cell line model. These results are similar to other studies (26) in which the proportion of J-Lat cells displaying GFP epifluorescence was increased by prostratin + HDACI cotreatments, compared to treatments with the compounds alone. However, we did not find a synergistic activation in the induction with Scriptaid/5-Aza.…”

“…ChIP analyses were performed according to Milipore Company online protocol and a procedure described previously (26)(27)(28). Briefly, A7 cells (1x10 7 cells/100-mm dish) were treated with or without Scriptaid (200 nM) or TSA (200 nM) for 4 h, then crosslinked with formaldehyde to a final concentration of 1% for 10 min at 37˚C.…”

“…HDAC-2 and -3 can also associate with the HIV long terminal repeat (LTR), and play a role in the repression of LTR expression (14,17). It has been shown that the disruption of HDAC-1 recruitment to LTR, resulting from the inhibition of HDAC activity by global HDAC inhibitors, leads to LTR activation and the escape of viral expression in both cell line models and primary cells obtained from patients (10,(18)(19)(20)(21)(22)(23)(24)(25)(26). Furthermore, HDAC inhibitor valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA; vorinostat) can induce viral outgrowth from resting CD4 T cells of aviremic patients undergoing HAART (27,28).…”

Inhibitors of Histone Deacetylases

“…HeLa and 293T cells were maintained in DMEM medium supplemented with 10% FBS, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. CD8(ϩ)-depleted peripheral blood mononuclear cells were isolated from fresh whole blood (100 ml) of HIV-negative individuals, as described previously (38). To determine HDACI cytotoxicity, 1 ϫ 10 4 cells were plated in 96-well plates with varying concentrations of drug.…”

HIV-1 Reservoirs and Latency: Critical Barriers and Clues for HIV / AIDS Cure

“…However, combination of the different activators may play a synergistic role in the reactivation of latent viral reservoirs as demonstrated by some studies. For instance, Reuse and colleagues have demonstrated that combining valproic acid and prostratin or suberoylanilide hydroxamic acid (SAHA)-an HDAC inhibitor-and prostratin more efficiently reactivated HIV-1 production in cell lines and cells isolated from patients receiving HAART than each compound alone (Reuse et al, 2009;.…”