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. 2014 Jan;69(1):28-33.
doi: 10.1093/jac/dkt338. Epub 2013 Sep 1.

Unique characteristics of histone deacetylase inhibitors in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells

Liang Shan  1 , Sifei Xing, Hung-Chih Yang, Hao Zhang, Joseph B Margolick, Robert F Siliciano
Affiliations

Unique characteristics of histone deacetylase inhibitors in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells

Liang Shan et al. J Antimicrob Chemother. 2014 Jan.

Abstract

Objectives: The latent reservoir for HIV-1 in resting memory CD4+ T cells is a major barrier to eradication. In vitro models involving transformed cell lines have been used to search for small molecules that reactivate latent HIV-1. Histone deacetylase (HDAC) inhibitors can reverse HIV-1 latent infection. Most studies on HDAC inhibitors have been performed in cell line models that differ in important aspects from the resting CD4+ T cells that harbour latent HIV-1 in vivo. Therefore, we evaluated the potency and kinetics of HDAC inhibitors in a primary cell model of HIV-1 latency that involves resting CD4+ T cells.

Methods: A green fluorescent protein (GFP)-expressing reporter virus NL4-3-Δ6-drGFP was used to generate latent infection in Bcl-2-transduced primary CD4+ T cells. Seventeen HDAC inhibitors were tested in this primary cell model. The effects of these HDAC inhibitors on the reactivation of latent HIV-1 were determined and compared with anti-CD3 and anti-CD28 co-stimulation.

Results: In Bcl-2-transduced primary CD4+ T cells, short-term treatment with HDAC inhibitors resulted in very limited reactivation of latent HIV-1, while prolonged treatment greatly enhanced drug efficacy. The effects of HDAC inhibitors in reactivating latent HIV-1 correlated with their inhibitory effects on class I HDACs. Importantly, HIV-1 reactivated by HDAC inhibitors can quickly re-establish latent infection upon drug removal.

Conclusions: We identified unique features of HDAC inhibitor-induced reactivation of latent HIV-1 in primary CD4+ T cells. Our findings may be useful for the design of eradication trials.

Keywords: cytotoxicity; latent reservoir; vorinostat.

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Figures

Figure 1.
Figure 1.
Comparison of the ability of different HDAC inhibitors to reactivate latent HIV-1. (a–d) Limited short-term effects of HDAC inhibitors on reactivation of latent HIV-1. Latently infected Bcl-2-transduced resting memory CD4+ T cells were treated with different HDAC inhibitors at 200 nM (a) or at the indicated concentrations (c) for 2 days. The fraction of GFP-positive cells was measured by flow cytometry. The effect of HDAC inhibitors was normalized to the effect of anti-CD3 plus anti-CD28 antibodies. *The concentration of VPA was 5 mM. Error bars represent SEM, n = 3. (b) Freshly isolated resting CD4+ T cells (grey bars) or Bcl-2-transduced resting CD4+ T cells (black bars) were cultured in the presence of HDAC inhibitors at 200 nM for 2 days. (d) Bcl-2-transduced resting CD4+ T cells were cultured in the presence of HDAC inhibitors at the indicated concentrations for 2 days. Cell death was determined by MTT assay (Promega) and the value was normalized to untreated controls. Error bars represent SEM, n = 3. (e and f) Correlation between reactivation of latent HIV-1 and inhibition of the indicated HDACs by HDAC inhibitors. Reactivation of latent HIV-1 by 1 μM HDAC inhibitors was measured as described above. Values of inhibition constants were obtained from a previous study. APHA, 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxypropenamide.
Figure 2.
Figure 2.
Unique characteristics of HDAC inhibitors in reactivating latent HIV-1. (a and b) Prolonged treatment with HDAC inhibitors leads to effective virus reactivation. Latently infected Bcl-2-transduced resting memory CD4+ T cells were treated with different HDAC inhibitors for 8 days. The fraction of GFP-positive cells was measured by flow cytometry at indicated timepoints (a) or at day 8 (b). The effect of HDAC inhibitors was normalized to the effect of anti-CD3 plus anti-CD28 antibodies at day 2. *Drugs caused significant cell death. (c) Bcl-2-transduced resting CD4+ T cells were cultured in the presence of HDAC inhibitors at the indicated concentrations for 8 days. Cell death was determined by MTT assay (Promega) and the value normalized to untreated controls. Error bars represent SEM, n = 3. (d and e) Induction of latent HIV-1 gene expression by HDAC inhibitors is reversible after drug removal. (d) HIV-1 latently infected Bcl-2-transduced resting memory CD4+ T cells were treated with 1 μM vorinostat or anti-CD3 plus anti-CD28 antibodies for 4 days. Vorinostat was removed or anti-CD3 plus anti-CD28 antibodies were removed at day 4 and cells were cultured for an additional 4 days without treatment. On day 8, 1 μM vorinostat was added back or anti-CD3 plus anti-CD28 antibodies were added back to the culture. (e) Cells were treated with indicated HDAC inhibitors for 4 days and then cultured without drug for an additional 4 days. On day 8, HDAC inhibitors were added back to the culture for another 4 days. The percentage of GFP-positive cells was measured at days 4, 8 and 12. The concentrations of HDAC inhibitors used for the experiments are as follows: vorinostat, 1 μM; TsA, 200 nM; oxamflatin, 1 μM; scriptaid, 1 μM; belinostat, 200 nM; and givinostat, 200 nM. The fraction of GFP-positive cells was measured by flow cytometry at the indicated timepoints. The effect of HDAC inhibitors or anti-CD3 plus anti-CD28 antibodies over time was normalized to the effect of anti-CD3 plus anti-CD28 antibodies at day 2. Error bars represent SEM, n = 3. APHA, 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxypropenamide.
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