Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes

Abstract

Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.

FIG. 1.

Analysis of the intracellular variation of calcium concentration by flow cytometry. PBMCs were loaded with Fluo-4/AM and Fura Red/AM and stained with monoclonal anti-CD14 antibodies conjugated to APC and were treated with 50 nM Tat at the time points indicated. (A to F) Both CD14⁺ cells (predominantly monocytes) and CD14⁻ cells (predominantly lymphocytes) were analyzed for calcium mobilization. Only HIV-1 subtype B Tat HXB2 was able to induce calcium mobilization in monocytes. (G) Cells were pretreated with either nimodipine or TMB-8. Data are shown for CD14⁺ cells. Nimodipine caused the marked inhibition of calcium flux in monocytes treated with subtype B Tat HXB2, whereas TMB-8 did not substantially affect the [Ca²⁺]_i.

FIG. 2.

Monocyte migration induced by Tat. (A) Monocyte migration induced by 50 nM Tat proteins by themselves or in the presence of calcium channel and calcium store inhibitors. Cells were treated with Tat by itself (black bars), Tat in the presence of 1 μM nimodipine (striped bars), Tat in the presence of 100 nM FS-2 (dark gray bars), and Tat in the presence of 100 μM TMB-8 (white bars). The control wells contained 10 nM subtype B Tat in each compartment. The error bars represent the standard deviations measured in three independent experiments carried out in triplicate. Tat 93In (93In) showed no chemotactic activity, as it failed to induce significant chemotaxis of monocytes. Both nimodipine and FS-2 caused the marked inhibition of chemotaxis to HIV-1 subtype B Tat HXB2 (HXB2) and subtype C Tat 96Bw (96Bw), whereas TMB-8 did not affect migration. (B and C) Morphological changes induced by Tat HXB2 (B) and Tat 93In (C) along a gradient treatment.

FIG. 3.

Equilibrium competition of ¹²⁵I-radiolabeled cytokines binding to monocytes. Radiolabeled cytokines were mixed with unlabeled Tat proteins and incubated with transfected cells for 40 min at 20°C. ¹²⁵I-CCL2 and CCR2⁺ cells (A) and ¹²⁵I-CXCL12 and CXCR4⁺ cells (B) were used. HIV-1 subtype C Tat 93In is represented by the open circles, and HIV-1 subtype B Tat HXB2 is represented by the closed circles. Cells were washed with binding buffer and fixed to a filter mat, and the amount of radiolabeled cytokine bound was determined by liquid scintillation. Error bars indicate standard deviations of two experiments carried out in duplicate. Both Tat proteins displaced CXCL12 from CXCR4 with the same 50% inhibitory concentration, while only Tat HXB2 displaced CCL2 from CCR2.

FIG. 4.

TNF and IL-6 expression in Tat-stimulated monocytes. (A) PBMCs were treated with 2 μM monensin and Tat and left to incubate for 4 hours. Only cells bearing the CD14 receptor (predominantly monocytes) were analyzed for the presence of these cytokines (APC-conjugated TNF [TNF APC] and phycoerythrin-conjugated IL-6 [IL-6 PE]). Examples of fluorescence-activated cell sorting analysis of monocytes stained for IL-6 and TNF are shown. The percentages in each quadrant are the average seen for each quadrant from three different experiments using PBMCs from three different donors carried out in triplicate. (B) Results of the three independent experiments are quantified in a histogram. Cells were treated with Tat HXB2 (black bars), Tat 93In (white bars), and buffer alone (gray bars). The error bars represent the standard deviations measured in three independent experiments carried out in triplicate. Tat HXB2 induces significantly more monocytes to produce both TNF and IL-6 than Tat 93In does (P = 0.0001 and 0.0052, respectively). Purified monocytes were also treated with 50 nM Tat HXB2 and 50 nM Tat 93In and incubated for 2 and 4 hours. (C) mRNA was extracted from treated cells and subjected to quantification using the Roche LightCycler. Results are represented as histograms of three independent experiments. IL-6 mRNA production is represented as a log scale, while TNF mRNA production is depicted on a linear scale. Cells were treated with Tat HXB2 (light gray bars), Tat 93In (white bars), Tat 96Bw (hatched bars) and buffer alone (black bars). The error bars represent the standard deviations of three independent experiments carried out in triplicate. B/B0, quantity of mRNA measured by the LightCycler/quantity of background mRNA expressed by monocytes in a 2:1 mixture of AIM-V and Iscove's media supplemented with 1% (vol/vol) fetal calf serum without Tat. Tat HXB2 induces significantly more TNF mRNA and IL-6 mRNA than Tat 93In does. (D) The supernatants of cultured monocytes were analyzed by ELISA for the presence of IL-6 and TNF. Results of three independent experiments are represented by histograms. Cells were treated with HXB2 (light gray bars), Tat 93In (white bars), and by buffer alone (black bars). The error bars represent the standard deviations measured in three independent experiments carried out in triplicate. Tat HXB2 induces significantly more secreted TNF and IL-6 than Tat 93In does.

FIG. 5.

CCL2 expression in Tat-stimulated monocytes and microglia. (A) Tat HXB2 and Tat 96Bw treatment of purified monocytes causes a significant upregulation of the expression of CCL2, whereas Tat 93In upregulated significantly less CCL2 (P = 0.0001). In the presence of anti-TNF neutralizing antibodies, there was significantly less upregulation after 4 h for both Tat 96Bw and Tat HXB2 (P values of 0.049 and 0.017, respectively). Controls included cultures with either isotype-matched monoclonal antibodies (BD Pharmingen, San Diego, CA) or with FcR blocking reagent (Miltenyi Biotec, Auburn, CA). No significant difference was observed between these cells and those with no reagents (control). (B) Both Tat 93In and Tat 96Bw were able to upregulate the expression of indoleamine 2,3-dioxygenase. (C) Tat HXB2 and Tat 96Bw treatment of microglia causes a significant upregulation of the expression of CCL2, whereas Tat 93In upregulated significantly less (P < 0.05). (D) Tat HXB2 treatment significantly upregulated the expression of CD40 mRNA. Cells were treated with Tat HXB2 (light gray bars), Tat 93In (white bars), Tat 96Bw (hatched bars), and buffer alone (black bars). B/B0, quantity of mRNA measured by the LightCycler/quantity of background mRNA expressed by monocytes in a 2:1 mixture of AIM-V and Iscove's media supplemented with 1% (vol/vol) fetal calf serum without Tat. The error bars represent the standard deviations measured in three independent experiments carried out in triplicate.