HIV-1 clade B Tat, but not clade C Tat, increases X4 HIV-1 entry into resting but not activated CD4+ T cells

Abstract

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4+ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4+ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4+ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4+ T cells, and both proteins inhibited the infection of activated CD4+ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4+ T cells more susceptible to X4 HIV-1 infection.

FIGURE 1.

Tat proteins from different clades up-regulate CXCR4 expression of resting CD4⁺ T cells. Purified resting CD4⁺ T cells from HIV-negative subjects were incubated with 2 nm Tat from different clades. After 4 h, the cells were harvested and stained for surface CXCR4. All of the Tat proteins significantly up-regulated CXCR4 surface expression except the clade C Tat_93In and Tat_92Br. *, p < 0.001.

FIGURE 2.

B Tat induces up-regulation of surface CXCR4 expression on resting CD4⁺ T cells through a CCR2-dependent mechanism that does not require de novo protein synthesis. A, purified resting CD4⁺ T cells from HIV-negative subjects were incubated with increasing concentrations of Tat. After 4 h, the cells were harvested and stained for surface CXCR4. B Tat, but not C Tat, induced a dose-dependent increase in surface expression of CXCR4. The data are expressed as the means ± S.E. of the mean fluorescence intensity calculated from three independent experiments. B, purified resting CD4⁺ T cells were cultured in the presence or absence of 20 μm RS102895 and 1 nm CCL2 or CXCL12. After 4 h, the cells were harvested and analyzed for the phosphorylation of SAPK/JNK and p38 by immunoblotting. The blots were then stripped and reprobed for the expression of total SAPK/JNK or p38. RS102895 successfully inhibited the CCL2/CCR2-mediated phosphorylation events but not those through CXCL12/CXCR4, indicating CCR2 antagonism specificity. C, purified resting CD4⁺ T cells were pretreated with 20 μm RS102895 and then cultured for 4 h in the presence of 2 nm B Tat (black line histogram) or with vehicle control (solid gray histogram). The cells were then harvested and stained for surface CXCR4. RS102895 inhibited the up-regulation of surface CXCR4 expression. Histograms are shown from a representative donor. D–F, purified resting CD4⁺ T cells were incubated with 2 nm Tat for 4 h, lysed, subjected to electrophoresis, and immunoblotting (D). Alternatively, the cells were stained for surface CXCR4 or permeabilized and stained for total CXCR4 (E) or evaluated for CXCR4 mRNA content using real time PCR and summarized in F. The histograms and blots for a representative donor are shown. Neither Tat protein induced de novo protein synthesis. *, p < 0.001.

FIGURE 3.

Expression of CXCR4 on IL-2 stimulated CD4⁺ T cells. A, surface expression of CXCR4 and CD25 (as an indicator of activation) was assessed over 96 h after stimulation with 20 units/ml IL-2 and 5 μg/ml PHA. As the cells became activated, CXCR4 expression was down-regulated. B, at 24 h post-IL-2 or IL2 plus PHA treatment, the ability of B Tat to up-regulate CXCR4 was assessed by flow cytometry. The data are expressed as the means ± S.E. of the mean fluorescence intensity calculated from three or more independent experiments performed in triplicate. B Tat had no effect on the CXCR4 surface expression of activated CD4⁺ T cells.

FIGURE 4.

Effect of B Tat on CXCR4 internalization and on plectin content. A, CXCR4 internalization was quantified by flow cytometric assessment of anti-CXCR4 antibody binding following acid stripping of cell surface-bound antibody. Acid-resistant binding (internalized CXCR4) is expressed as a fraction of total antibody binding over 2 h of incubation at 37 °C. B Tat reduced the CXCR4 internalization by 65% across four independent experiments (p = 0.008). B, effect of B Tat on plectin expression. Immunoblotting was used to quantify plectin levels in resting CD4⁺ T cells cultured for 4 h in the presence of 0 (U), 2 nm C Tat (C), or 2 nm B Tat (B). Two donors are shown. Parallel determination of β-actin verified equivalent protein loading. C, densitometric analysis of the immunoblots shown in B revealed that B Tat, but not C Tat, conditioning of resting CD4⁺ T cells significantly increased plectin content (p = 0.001). *, p = 0.008; **, p = 0.001.

FIGURE 5.

Effect of plectin RNA interference on B Tat-mediated CXCR4 surface up-regulation. Purified CD4⁺ T cells were transiently transfected with a scramble siRNA as a control (S) or plectin-specific siRNA (P) and subjected to immunoblot analysis as described above (A) or treated with B Tat, C Tat, or vehicle control (B). After 4 h, the cells were harvested and stained for surface CXCR4. Plectin siRNA almost completely abrogated the B Tat-mediated up-regulation of CXCR4 surface expression (p < 0.0001).

FIGURE 6.

B Tat, but not C Tat, augments CXCR4-tropic gp120_MN chemotactic and F-actin response. A, CD4⁺ T cell migration induced by 200 nm gp120_MN or gp120_SF162 alone or with prior conditioning with 2 nm Tat and/or 20 μm RS102895, 1 μm AMD3100, 1 μg/ml PTX, or 10 μg/ml anti-CD3 antibody. The bars represent the means ± S.E. of the percentage of migrated cells from three independent experiments performed in triplicate. Chemokinesis was minimal when equal concentrations of gp120 was added to both the apical and basal chambers and has been subtracted from these histograms. CXCR4-tropic gp120_MN induced chemotaxis of CD4⁺ T cells and was augmented by preconditioning with 2 nm B Tat but not C Tat. The augmentation was abrogated in the presence of RS102895 (p < 0.0001). Treatment with AMD3100, PTX, or anti-CD3 antibody inhibited gp120_MN-induced chemotaxis. R5 gp120_SF162 induced minimal chemotaxis and was unaffected by all inhibitor and stimulus treatments. B, effect of B Tat on CXCR4-tropic gp120_MN-induced F-actin polymerization in resting CD4⁺ T cells. Using FITC-phalloidin as a probe for intracellular F-actin, the effects of Tat preconditioning on F-actin polymerization in resting CD4⁺ T cells was assessed by flow cytometry. The results show the kinetics of F-actin polymerization following stimulation of unconditioned cells with R5 gp120_SF162 (open symbols) or X4 gp120_MN (closed symbols) at time 0 with or without inhibitors. All of the data are representative of three independent experiments. *, p < 0.0001.

FIGURE 7.

Effect of B Tat on gp120 induced Erk1/2 phosphorylation in primary resting CD4⁺ T cells. A, primary resting CD4⁺ T cells pretreated with B Tat or C Tat for 4 h and then stimulated with gp120 for 2 min in the presence of 1 μm AMD3100, 1 μg/ml PTX, or vehicle control, fixed, permeabilized, and stained with a fluorescently tagged antibody specific for phosphorylated Erk1/2 and then analyzed by flow cytometry. The bars represent the means and S.E. of the relative change in fluorescence intensity indicative of Erk1/2 phosphorylation after stimulation with CXCR4-tropic gp120_MN (black bars) or CCR5-tropic gp120_SF162 (gray bars). White bars represent resting cells treated with 40 nm 12-O-tetradecanoylphorbol-13-acetate (TPA) or with vehicle control in the absence of gp120 stimulation. The change in Erk1/2 phosphorylation is expressed as the relative fold change in the mean fluorescence intensity, with the base-line fluorescence intensity before the addition of gp120 expressed as 1.00. Alternatively, cells were lysed, and Erk1/2 phosphorylation was determined through immunoblotting. B, B Tat, but not C Tat, preconditioning of resting CD4⁺ T cells significantly increased gp120_MN-induced phosphorylation of Erk1/2 (p < 0.0001) but had no effect on gp120_SF162-induced events. Both AMD3100 and PTX inhibited Erk1/2 phosphorylation by gp120_MN. C, CD4⁺ T cell migration induced by 200 nm gp120_MN or gp120_SF162 after preconditioning with Tat and/or 10 μm U0126. The bars represent the means ± S.E. of the number of migrated cells from three independent experiments performed in triplicate. Chemokinesis was minimal when equal concentrations of gp120 were added to both the apical and basal chambers and has been subtracted from these histograms. *, p < 0.0001.

FIGURE 8.

X4 HIV-1 infection of primary resting CD4⁺ T cells. A, purified resting CD4⁺ T cells were conditioned with 2 nm Tat, 1 nm CXCL12, or left unconditioned for 4 h before infection with X4 HIV-1_IIIB (black bars) or the R5 HIV-1_93In905 (gray bars) at a multiplicity of infection of 0.01 for 3 h. The cells were then cultured for 14 h without further stimulation. Entry of HIV-1 was evaluated by real time PCR using primers specific for the LTR R/U5 region as described under “Experimental Procedures.” B, cells infected with HIV-1_IIIB as described above were further cultured in the presence of 20 units/ml IL-2. After 96 h, the supernatants were collected and p24 quantified. C, purified CD4⁺ T cells were stimulated for 72 h in the presence of 20 units/ml IL-2, conditioned with 2 nm Tat for 4 h and then subjected to infection with HIV-1_IIIB (black bars) or the HIV-1_93In905 (gray bars) at a multiplicity of infection of 0.01 for 3 h. The cells were then cultured for a further 14 h before the HIV-1 LTR DNA quantity was evaluated. D, cells infected with HIV-1_IIIB from C were further stimulated with 20 units/ml IL-2 for 96 h before supernatants were collected, and p24 quantified. B Tat, but not C Tat, conditioning significantly augmented X4 HIV-1_IIIB but not R5 HIV-1_93In905 infection of resting CD4⁺ T cells. Conversely, both B Tat and C Tat significantly inhibited X4 HIV-1_IIIB but not R5 HIV-1_93In905 infection of activated CD4⁺ T cells. *, p < 0.05.