Integrase inhibitors effective against human T-cell leukemia virus type 1

Abstract

Drugs targeting the viral enzyme integrase have been in use for several years as part of the treatment regimen for patients with human immunodeficiency virus type 1 (HIV-1), and similar classes of compounds have been shown to inhibit human T-cell leukemia virus type 1 (HTLV-1) integration in vitro. The current study shows that the clinically approved HIV-1 integrase inhibitor, raltegravir, as well as the more recent diketo acid derivative, MK-2048, are active inhibitors of HTLV-1 infection in vitro. These agents were effective in inhibiting cell-free and cell-to-cell transmission of HTLV-1 in lymphoid and nonlymphoid cells. The drugs also inhibited HTLV-1 immortalization of human peripheral blood mononuclear cells. A novel adaptation of the Alu assay for viral integration was used to show that the drugs inhibit viral integration without affecting reverse transcription. These data support the administration of raltegravir and other integrase inhibitors as treatments for patients with HTLV-1-associated diseases.

Figures

Fig. 1.

Chemical structures of the integrase inhibitors MK-0518 (A), also known as raltegravir and Isentress, and MK-2048 (B).

Fig. 2.

Raltegravir and MK-2048 significantly inhibit cell-free infection by recombinant HTLV-1 in vitro and are not toxic to target cells. The cell-free infection of 293T cells with luciferase-encoding recombinant HTLV-1 in the presence of increasing concentrations of MK-2048 (A) and raltegravir (B) was assessed. (C) MTT assays for cell viability were performed in the presence of each of the two drugs and of the apoptosis-inducing drug staurosporine. Each data point represents the mean of three individual values (*, P < 0.05; **, P < 0.01 [error bars indicate the standard deviation]).

Fig. 3.

Raltegravir and MK-2048 significantly inhibit HTLV-1 cell-cell infection of B5 Luc cells. B5Luc cells (fetal rhesus lung cell line clone B5 containing a stably integrated luciferase gene under the control of a Tax-inducible LTR) were coincubated with lethally irradiated MT2 cells in the presence or absence of the indicated concentrations of MK-2048 and raltegravir. Each bar represents the mean of three values (*, P < 0.05; **, P < 0.01 [error bars indicate standard deviation]).

Fig. 4.

Raltegravir and MK-2048 reduce the number of viral integration events and total viral DNA levels per cell in cell-cell infections of Jurkat cells and is not toxic to the cells. (A) Jurkat cells were infected by coincubation with lethally irradiated MT2 cells for 48 h and passaged three times over a period of 10 days in the presence or absence of various concentrations of raltegravir and MK-2048. The number of copies of integrated DNA per cell was measured by Alu assay (A), while the total DNA per cell was measured by real-time PCR analysis using primers to tax (B). Each bar represents the mean of three values. (C) Jurkat cells were either untreated or incubated in medium containing 500 nM or 5 μM raltegravir and then passaged for 9 days. A 100-μl portion of a 1:5 dilution of each cell category was analyzed by using the CellTiter-Blue cell viability assay (Promega). The data represent the means of triplicate readings with background (medium only) subtracted. (D) Jurkat cells infected by cell-cell contact with irradiated MT2 cells were diluted in carrier DNA and subjected to analysis by Alu assay for viral integration. Each data point is the mean of three values (*, P < 0.05; **, P < 0.01 [error bars indicate the standard deviation]).

Fig. 5.

Raltegravir blocks infection of PBMC by HTLV-1 cell-to-cell transmission from irradiated MT2 cells. PBMC were extracted from whole blood and coincubated with irradiated MT2 cells for 90 days. (A) Cell viability was measured by trypan blue stain resistance at the indicated time points approximately 1 week apart. Each data point is the mean of three cell counts. (B) Viral production was measured from supernatants collected at day 41 after culture and quantified by p19 antigen ELISA (Zeptometrix). Each bar represents the mean of two measurements. The number of integrated proviral copies per cell was measured by Alu assay (C), and the total DNA measured by real-time PCR analysis (D) (*, P < 0.05; **, P < 0.01 [error bars indicate standard deviation]). The reported values are the means of three measurements; R-0, R-100, R-250, and R-500 represent infected PBMC cultured in 0, 100, 250, and 500 nM raltegravir, respectively.

Fig. 6.

Raltegravir- and MK-2048-mediated inhibition of HTLV-1 infection in vitro occurs at the stage of viral integration. Total DNA assays were performed on MT2 cells infected for 24 h by cell-cell contact with lethally irradiated MT2 cells in the presence of the indicated concentrations of inhibitors (*, P < 0.05; **, P < 0.01 [error bars indicate the standard deviation, and the values show the means of three measurements]).