Interplay between Selenium, Selenoproteins and HIV-1 Replication in Human CD4 T-Lymphocytes

Abstract

The infection of CD4 T-lymphocytes with human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), disrupts cellular homeostasis, increases oxidative stress and interferes with micronutrient metabolism. Viral replication simultaneously increases the demand for micronutrients and causes their loss, as for selenium (Se). In HIV-infected patients, selenium deficiency was associated with a lower CD4 T-cell count and a shorter life expectancy. Selenium has an important role in antioxidant defense, redox signaling and redox homeostasis, and most of these biological activities are mediated by its incorporation in an essential family of redox enzymes, namely the selenoproteins. Here, we have investigated how selenium and selenoproteins interplay with HIV infection in different cellular models of human CD4 T lymphocytes derived from established cell lines (Jurkat and SupT1) and isolated primary CD4 T cells. First, we characterized the expression of the selenoproteome in various human T-cell models and found it tightly regulated by the selenium level of the culture media, which was in agreement with reports from non-immune cells. Then, we showed that selenium had no significant effect on HIV-1 protein production nor on infectivity, but slightly reduced the percentage of infected cells in a Jurkat cell line and isolated primary CD4 T cells. Finally, in response to HIV-1 infection, the selenoproteome was slightly altered.

Keywords: HIV-1; Jurkat; SELENOO; SELENOS; SupT1; glutathione peroxidase; primary T cells; selenoproteome; thioredoxin reductase; translational control; viral infection.

Figure 1

Analysis of selenoprotein mRNA levels in Jurkat and SupT1 in control conditions and in response to 100 nM selenium supplementation of culture media (three days). The geometrical mean of four housekeeping genes (HPCB, RPS13, HRPT, and GAPDH) was used to normalize mRNA abundance. In all panels, mRNA levels are represented in logarithmic scales. The values are given in Table S1. Selenoprotein mRNA levels in control medium (Ctrl) are represented for Jurkat (a) and SupT1 (b), from most to least abundant (from left to right). To evaluate the impact of selenium supplementation on steady state levels of selenoprotein mRNAs, the values obtained in selenium supplemented conditions (100 nM) were plotted as a function of values obtained with unsupplemented ones (Ctrl) for Jurkat (c) SupT1 (d) cells (±standard deviation). The experiments were done in biological triplicate and in technical triplicate. The selenoprotein genes with significant changes are labeled in red and the statistical analyses are given in Table S1.

Figure 2

Analysis of selenoprotein mRNA levels in CD4-T cells isolated from donors in control conditions (Ctrl) and selenium-supplemented (100 nM) conditions after three days. The levels are represented in logarithmic scales. The transcripts from four different donors were measured and normalized similarly to what has been done for Jurkat and SupT1 extracts. The mRNAs expressed in control conditions were represented as histograms (a–d) and arbitrarily separated in high, medium and low abundance by dashed lines. Additionally, the values obtained in selenium-supplemented conditions (100 nM) were plotted as a function of values obtained with unsupplemented ones for every donor (±standard deviation) (e–h). The values are given in Table S2. The experiments were done in biological duplicates and in technical triplicates. The selenoprotein genes with significant changes are labeled in red and the statistical analyses are given in Table S2.

Figure 3

Heatmap representation of selenoprotein mRNA levels (in logarithmic scales) as a function of T-cell types (donors 1 to 4, Jurkat and SupT1) and growth conditions (Ctrl or supplemented with 100 nM of selenium). The values are given in Tables S1 and S2.

Figure 4

Analysis of selenoprotein expression in Jurkat and SupT1 cells in response to different selenium supplementations of the culture media (in nM) after three days. The results from a representative experiment are shown in (a). The values are given in Table S4. The fold-increase in expression of each selenoprotein in response to selenium supplementation was calculated between the value at 100 nM over that at Ctrl. These ratios were plotted from the highest to the lowest for Jurkat (b) and SupT1 (c) cells. The dotted line represents a fold-increase value of one.

Figure 5

Analysis of selenoprotein expression in CD4 T cells isolated from four different donors in control (Ctrl) and selenium-supplemented (100 nM) conditions over three days. The results from a representative experiment are shown in (a). The values are given in Table S4. (b) The fold-increase in expression of each selenoprotein in response to selenium supplementation was calculated between the value at 100 nM over that at Ctrl. The average value (±standard deviation) of the four donors was represented by a bar, but the individual value of each donor was indicated by a square. The dotted line represents a fold-increase value of one.

Figure 6

Evaluation of GPX (a–c) and TXNRD (d–f) enzymatic activities in protein extracts from various T cell types (Jurkat, SupT1 and the four donors) cultured with different concentrations of supplemented selenium. The GPX activities were represented for Jurkat (a) (n = 1), SupT1 (b) (n = 1) and the four donors (c) (n = 2) for the different growth conditions. The TXNRD activities were represented for Jurkat (d) (n = 1), SupT1 (e) (n = 1) and the four donors (f) (n = 2) for the different growth conditions. The experiments were done in technical triplicates. The differences between the lowest and highest values were indicated by an arrow or a bar (±standard deviation), with the corresponding fold-change factor beside or above.

Figure 7

Kinetics of HIV-1 infection of Jurkat cells in control (Ctrl) or 100 nM supplemented conditions. (a) Schematic of the experimental procedure used to follow the different parameters of viral production and cell infection during eleven days. The culture media were collected at different time points and analyzed for the levels of HIV-1 p24 capsid protein by Western blot (WB) (b) and infectivity of TZM-bl cells (c). The anti-p24 Western blots were assessed with biological duplicates, and levels of p24 were expressed relative to the maximum value, arbitrarily set at 100%. The infectivity assays were assessed with biological duplicates in technical triplicates. The maximum value was arbitrarily set as 100%. (d) The ratio between infectivity and p24 values were calculated and plotted as a function of time, the maximum value was arbitrarily set as 100%. (e) The percentage of infected cell was evaluated in one experiment by immuno-labeling of fixed cells using anti-HIV-1 p24 antibodies coupled with FITC followed by flow cytometry analysis.

Figure 8

Kinetics of HIV-1 infection of T-cells isolated from donors in Ctrl or 100 nM supplemented conditions (a–e). (a) Schematic of the experimental procedure used to follow the different parameters of viral production and cell infection over four days. Similar to what was done with Jurkat in Figure 7, the media were collected at different time points and evaluated for the levels of p24 (b), infectivity of TZM-bl cells (c) and the ratio of infectivity over p24 levels (d) (n = 1). The infectivity assays were assessed with technical triplicates. For panels (b–d), the maximum value was arbitrarily set as 100%.

Figure 9

Analysis of selenoprotein expression in response to HIV-1 infection, in control (Ctrl) and 100 nM supplemented conditions. NI, non-infected cell extracts; INF, infected cell extracts. Donor 3 and Donor 4 were analyzed (n = 1) for the expression of SELENOO, GPX1, GPX4 and TXNRD1 (a) and SELENOS (b). Non-specific bands are indicated by an asterisk. The hash indicates an unknown band for GPX1. The arrows indicate the migration of two isoforms for SELENOS. The values are given in Table S5.