The presence of HIV-1 Tat protein second exon delays fas protein-mediated apoptosis in CD4+ T lymphocytes: a potential mechanism for persistent viral production

Abstract

HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1-72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4(+) T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4(+) T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.

Figures

FIGURE 1.

FasL-mediated apoptosis was reduced in PBLs expressing intracellular Tat101.A, resting PBLs were transiently transfected with pCMV-Tat72 or pCMV-Tat101 expression vectors or with pcDNA3 as negative control. pEYFP-C1 was used as control of transfection efficiency. PBLs were maintained for 3 days without stimulus, and then were treated with FasL for 18 h. Viability was measured by chemiluminescence. The bar diagram represents the media of three independent experiments, and lines on top of the bars correspond to the mean ± S.E. Two-way ANOVA with Bonferroni pos-test analysis was performed for statistical analysis; ** indicates p < 0.01. B, Tat expression and nuclear localization were confirmed by immunofluorescence. DAPI was used for nuclear staining. C, resting PBLs were transiently transfected with pCMV-Tat72 or pCMV-Tat101 expression vectors or with pcDNA3 as negative control. pEYFP-C1 was used as control of transfection efficiency. PBLs were maintained for 3 days without stimulus and then were treated with FasL for 18 h. Cells were stained with a monoclonal antibody against CD4 conjugated with PE and then with PI. Signals corresponding to EYFP, PE, and PI were analyzed by flow cytometry in FL1, FL2, and FL3 channels, respectively. SSC/FSC dot plot was used to select living (PI−) cells (region R1) as follows: red events correspond to PI−, living cells; magenta events correspond to PI+, dead cells; black events correspond to cellular debris. The numbers in the PI−/EYFP+/CD4+ dot plots show the percentage of CD4+/EYFP+ cells within the PI− region (region R3). The bar diagram represents the media of three independent experiments, and the lines on top of the bars correspond to the mean ± S.D.

FIGURE 2.

Tat101 reduced FasL-mediated apoptosis in HIV-1-transfected PBLs.A, PBLs were transfected with pNL4.3-TatM1I and pCMV-pTat72 or pCMV-Tat101 expression vectors. pcDNA3 was used as a negative control. pEYFP-C1 was used as a control of transfection efficiency. HIV-1 replication was allowed for 3 days before treatment with FasL for 18 h. Viability was measured by chemiluminescence. The bar diagram represents the media of three independent experiments, and lines on top of the bars correspond to S.E. Two-way ANOVA with Bonferroni pos-test analysis was performed for statistical analysis (* and ** indicates p < 0.05 and p < 0.01, respectively). B, expression of HIV-1 genes was monitored by quantifying the expression of the env/nef genes by quantitative RT-PCR using β-ACTIN as internal control. C, resting PBLs were transiently transfected with pNL4.3-TatM1I and pCMV-pTat72 or pCMV-Tat101 expression vectors. pcDNA3 was used as negative control. pEYFP-C1 was used as control of transfection efficiency. PBLs were maintained for 3 days without stimulus and then were treated with FasL for 18 h. Cells were stained with a monoclonal antibody against CD4 conjugated with PE and then with PI. Signals corresponding to EYFP, PE, and PI were analyzed by flow cytometry in FL1, FL2, and FL3 channels, respectively. SSC/FSC dot plot was used to select living (PI−) cells (region R1) as follows: red events correspond to PI−, living cells; magenta events correspond to PI+, dead cells; black events correspond to cellular debris. The numbers in the PI−/EYFP+/CD4+ dot plots show the percentage of CD4+/EYFP+ cells within the PI− region (region R3). The bar diagram represents the media of three independent experiments, and the lines on top of the bars correspond to S.D.

FIGURE 3.

Tat101 reduced FasL-mediated apoptosis in HIV-1-transfected Jurkat cells and in Jurkat cells with stable expression of intracellular Tat101.A, Jurkat E6-1 cells were transfected with pNL4.3-TatM1I together with pCMV-Tat72 or pCMV-Tat101 expression vectors. pcDNA3 was used as negative control. pEYFP-C1 was used as control of transfection efficiency. After 3 days in culture, cells were treated with FasL for 4 h. Viability was measured by chemiluminescence. The bar diagram represents the media of three independent experiments, and lines on top of the bars correspond to S.E. Two-way ANOVA with Bonferroni pos-test analysis was performed for statistical analysis (* indicates p < 0.05). HIV-1 replication was monitored by quantifying the expression of the env/nef genes by quantitative RT-PCR using β-ACTIN as internal control. B, Jurkat-Tat101, Jurkat-Tat72, and control cells were treated with FasL for 2, 4, 6, and 18 h and then stained with annexin-V-FITC to measure by flow cytometry the percentage of apoptotic cells. C, Jurkat-Tat101, Jurkat-Tat72, and control cells were treated with FasL during 4 h and then stained with PI. The induction of apoptosis was measured by flow cytometry. Region R1 was used to select the living PI− cells in the groups of untreated cells, displayed as SSC/FSC dot plots. The percentage of apoptotic cells (PI+) was determined within R1 in treated cells and then represented as histograms where the basal cell death (continuous line) was compared with the apoptosis induced after treatment with FasL (discontinuous line). The numbers shown correspond to the percentage of apoptotic cells within R1 after subtracting the basal death. D, Jurkat-Tat101, Jurkat-Tat72, and control cells were stained with a monoclonal antibody against Fas receptor (FasR/CD95) and a secondary antibody conjugated with FITC. The percentage of cells expressing Fas was measured by flow cytometry. The histograms compare the isotype control (discontinuous line) with the expression of FasR (continuous line) on the cell surface. Numbers shown are the percentage of cells expressing CD95 after subtracting the isotype control. All data shown are media or representative of three independent experiments.

FIGURE 4.

Activation of caspase-3 and -8 was impaired in Jurkat-Tat101 treated with FasL.A, procaspase-3 cleavage was analyzed by immunoblotting using an antibody against procaspase-3 (p32) and active fragments p17/p11 in protein extracts obtained from Jurkat-Tat101, Jurkat-Tat72, and control cells treated or not with FasL for 4 or 18 h. β-Actin was used as internal loading control. Gel bands were quantified by densitometry, and the background noise was subtracted from the images. The relative ratio of the optical density units corresponding to each sample was calculated regarding the internal control (β-actin) per each lane. B, cleavage of PARP-1 was analyzed by using a monoclonal antibody against the cleaved fragment p89. β-Actin was used as loading control. C, caspase-3/-7 activation was measured by chemiluminescence in cells treated or not with FasL. D, caspase-8 activation was measured by chemiluminescence in cells treated or not with FasL. Levels of procaspase-8 were analyzed by immunoblotting using an antibody against procaspase-8 (p55) in protein extracts obtained from Jurkat-Tat101, Jurkat-Tat72, and control cells treated with FasL for 18 h. β-Actin was used as internal loading control. The bar diagrams show the media of relative RLUs fold from three independent experiments, and the lines on the top of the bars represent the S.D. Two-way ANOVA with Bonferroni post-test analysis was performed for statistical analysis. * and *** indicate p < 0.05 and p < 0.001, respectively.

FIGURE 5.

Release of cytochrome c induced by FasL was reduced in Jurkat-Tat101.A, release of cytochrome c from the mitochondria was quantified by flow cytometry in Jurkat-Tat101, Jurkat-Tat72, and control cells treated or not with FasL during 4 or 18 h. The histograms correspond to one representative experiment out of three and compare the percentage of cytochrome c that was retained in the mitochondrial intermembrane space (continuous line; marker M1) versus the cytochrome c released after treatment with FasL (discontinuous line; marker M2). B, release of cytochrome c (Cyt. c) was analyzed by immunofluorescence. After staining with the Mitotracker probe, the cells were fixed and stained with a monoclonal antibody against cytochrome c and a secondary antibody conjugated with Alexa 488. The nucleus was stained with DAPI.

FIGURE 6.

Jurkat-Tat101 showed a reduced cleavage of Bid and procaspase-9, overexpression of BCL2, and higher stability of the mitochondrial inner membrane potential.A, cleavage of procaspase-9 was analyzed by immunoblotting in cytosolic protein extracts obtained from Jurkat-Tat101, Jurkat-Tat72, and control cells treated or not with FasL during 4 or 18 h, using an antibody against the procaspase-9 (p46) and active caspase-9 (p37/p35) fragments. β-Actin was used as loading control. Gel bands were quantified by densitometry, and the background noise was subtracted from the images. The relative ratio of the optical density units corresponding to each sample was calculated regarding the internal control (β-actin) per each lane. B, caspase-9 activity was measured by chemiluminescence. The bar diagram shows the media of relative RLUs fold from three independent experiments, and the lines on the top of the bars represent the S.D. Two-way ANOVA with Bonferroni post-test analysis was performed for statistical analysis, and *, **, and *** correspond to p < 0.05, p < 0.01, or p < 0.001, respectively. C, mitochondrial membrane potential gradient (ΔΨm) was measured by flow cytometry using the lipophilic dye JC-1. Red fluorescent aggregates in the undamaged mitochondria were represented in a dot plot versus the green fluorescent monomers dispersed through the cytosol. The numbers upper left in the diagrams represent the cells with stable mitochondrial membrane integrity; the numbers lower right represent the loss of mitochondrial membrane integrity. A representative experiment out of three is shown. D, expression of BCL2 and Bid (p22) was analyzed by immunoblotting, and β-actin was used as loading control.

FIGURE 7.

Network of predicted interactions between proteins related to apoptosis and mitochondrial membrane integrity, as specified in Table 1, the expression of which was modified in Jurkat-Tat101 and Jurkat-Tat72 versus control cells. Medium confidence score level was 0.400. Caspase-3 was included to evaluate its central point in the network. Data supporting protein-protein interactions were derived from experimental studies (dark purple lines), homology (light purple lines), databases (light blue lines), text mining (light green lines), concurrence (dark blue lines), and co-expression (black lines). Node color is arbitrary.

FIGURE 8.

NF-κB-dependent proteins involved in the control of apoptosis were up-regulated in Jurkat-Tat101.A, expression of c-FLIPL (55 kDa), c-FLIPR (43 kDa), and c-FLIPS (28 kDa) isoforms was analyzed by immunoblotting in cytosolic protein extracts from Jurkat-Tat101, Jurkat-Tat72, and control cells treated or not with FasL for 4 or 18 h. β-Actin was used as loading control. B, NF-κB activity was analyzed in basal conditions by using DNA affinity immunoblotting assay. Gel bands were quantified by densitometry, and the background noise was subtracted from the images. The relative ratio of the optical density units corresponding to each sample was calculated regarding the internal control (β-actin) per each lane. C, Jurkat-Tat72, Jurkat-Tat101, and control cells were treated with FasL during 4 h and then stained with an antibody against CD69 conjugated with PE. The percentage of cells expressing CD69 on the cell surface was measured by flow cytometry. The bar diagram represents the percentage of cells expressing CD69 after subtracting the isotope control. D, NF-κB-dependent transactivation activity was measured by transient transfection of vector p3κB-LUC. Fold mean of RLUs corresponding to three independent experiments is represented, and lines on the top of the bars correspond to S.D. E, network of proteins related to apoptosis and identified by antibody-based microarray (see Table 2), expression of which was modified in Jurkat-Tat101 and Jurkat-Tat72 versus control cells. Predicted protein-protein interaction was obtained using a medium confidence score level (0.400). Data supporting protein-protein interactions derived from experimental studies (reddish purple lines), homology (light purple lines), databases (light blue lines), text mining (light green lines), and co-expression (black lines). The color of the nodes is arbitrary. F, Jurkat Tet-Off cells were transiently co-transfected with p3κB-LUC vector and pTRE2hyg-Tat101 ESKKKVE (wild type), pTRE2hyg-Tat101 ESRVNVV (mutated), or pTRE2hyg empty vector as negative control. After 18 h, NF-κB activity was measured by chemiluminescence. Fold of relative RLUs regarding the control cells are represented (upper bar diagram). Cells were then treated with FasL for 4 h, and apoptosis was measured by PI staining and analyzed by flow cytometry (lower bar diagram). The bar diagrams show the media obtained from three independent experiments, and lines on the top of the bars represent the S.D. Two-way ANOVA with Bonferroni post-test analysis was performed for statistical analysis. Symbols *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

FIGURE 9.

Model summarizing the steps in Fas apoptotic pathway that were thwarted by the intracellular expression of Tat. See Table 1, Table 2, and text for further explanation.