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, 85 (12), 6060-4

Disulfiram Reactivates Latent HIV-1 in a Bcl-2-transduced Primary CD4+ T Cell Model Without Inducing Global T Cell Activation

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Disulfiram Reactivates Latent HIV-1 in a Bcl-2-transduced Primary CD4+ T Cell Model Without Inducing Global T Cell Activation

Sifei Xing et al. J Virol.

Abstract

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4(+) cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4(+) T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.

Figures

Fig. 1.
Fig. 1.
Screening of small-molecule libraries identifies disulfiram as an agent that reactivates latent HIV-1. (A) Summary of screening results from the MicroSource Spectrum library. The results were normalized to the response to 10 ng/ml phorbol myristate acetate. (B) Chemical structures of disulfiram and its metabolites, DDTC and DDTC-Me. (C) Representative flow cytometry data sets for untreated, disulfiram-treated, and anti-CD3 plus anti-CD28 antibody-treated cells. The dot plots in the first row are forward scatter-side scatter (FSC-SSC) plots. Histographs in the second row show cells gated in R1 in FSC-SSC plots. The GFP-FL2 dot plots in the third row show viable cells gated in R2 in propidium iodide histographs. Cells that appear in R3 (GFP+) are reactivated latently infected cells. The percentage of GFP+ cells was calculated based on the number of cells in R3 divided by the number of cells in R2.
Fig. 2.
Fig. 2.
(A) Effects of disulfiram on latently infected Bcl-2-transduced CD4+ T cells from different donors. (B) Kinetic profile for the reactivation of latent HIV-1 by different concentrations of disulfiram. (C) Effects of disulfiram and costimulation on levels of HIV-1 transcripts in latently infected Bcl-2-transduced cells after 16 h of treatment. The fold change is shown relative to levels in untreated cells. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments, both of which produced similar results. (D) Kinetic profile of the effect of 0.5 μM disulfiram on levels of HIV-1 transcripts in latently infected Bcl-2-transduced cells. The fold change is shown relative to levels in untreated cells. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments, both of which produced similar results. (E) Comparison of disulfiram, DDTC, and DDTC-Me for reactivating latent HIV-1. (F) Toxicity of disulfiram and DDTC treatment, measured in a propidium iodide exclusion assay.
Fig. 3.
Fig. 3.
Disulfiram does not cause CD4+ T cell activation. (A) Effects of disulfiram and anti-CD3 plus anti-CD28 treatment on the sizes of resting CD4+ T cells. Cell size was measured by flow cytometry based on forward scatter at 24 h and 48 h. (B) Effects of disulfiram and DDTC on the expression of activation markers in primary resting CD4+ T cells, compared to the effect of anti-CD3 plus anti-CD28 costimulation. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments. (C) Effects of disulfiram and DDTC on global DNA and RNA levels in primary resting CD4+ T cells, compared to the effect of anti-CD3 plus anti-CD28 costimulation. Hoechst 33342 and pyronin Y were used to stain DNA and RNA, respectively, and results were read using a flow cytometer.

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