Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5

Abstract

Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

Figure 1

Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4⁺ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as in a and b.

Figure 1

Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4⁺ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as in a and b.

Figure 1

Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4⁺ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as in a and b.

Figure 2

Tyrosine phosphorylation of Pyk2 after contact with HIV-1 envelope glycoprotein on the surface of transfected 293T cells and virions. (a) HL60 or DU6 cells were lysed 30 s after being mixed with 293T cells expressing M-tropic (JRFL), T-tropic (HXB2), or no (−) envelope. (b) DU6 cells were mixed with HIV–luc particles pseudotyped with either JRFL or VSV-G envelopes and lysed after 90 s. As a positive control, MIP-1β was incubated with DU6 cells for 30 s before lysis.

Figure 2

Tyrosine phosphorylation of Pyk2 after contact with HIV-1 envelope glycoprotein on the surface of transfected 293T cells and virions. (a) HL60 or DU6 cells were lysed 30 s after being mixed with 293T cells expressing M-tropic (JRFL), T-tropic (HXB2), or no (−) envelope. (b) DU6 cells were mixed with HIV–luc particles pseudotyped with either JRFL or VSV-G envelopes and lysed after 90 s. As a positive control, MIP-1β was incubated with DU6 cells for 30 s before lysis.

Figure 3

Tyrosine phosphorylation of Pyk2 mediated by T-tropic gp120 can be inhibited by pertussis toxin or a monoclonal antibody against CXCR4. (a) HL60 cells were pretreated with pertussis toxin or left untreated. Treated and untreated cells were incubated with T-tropic gp120 (BH10, 5 μg/ml) for the indicated times before lysis. (b) HL60 cells were resuspended in growth media/0.5% FCS either without antibody (no Ab) or containing monoclonal antibodies specific for CXCR4 (anti-CXCR4) or human CD8 (control Ab). Cells were incubated with antibody at 37°C for 15 min, then mixed with T-tropic SF-2 envelope for 30 s before lysis.

Figure 3

Tyrosine phosphorylation of Pyk2 mediated by T-tropic gp120 can be inhibited by pertussis toxin or a monoclonal antibody against CXCR4. (a) HL60 cells were pretreated with pertussis toxin or left untreated. Treated and untreated cells were incubated with T-tropic gp120 (BH10, 5 μg/ml) for the indicated times before lysis. (b) HL60 cells were resuspended in growth media/0.5% FCS either without antibody (no Ab) or containing monoclonal antibodies specific for CXCR4 (anti-CXCR4) or human CD8 (control Ab). Cells were incubated with antibody at 37°C for 15 min, then mixed with T-tropic SF-2 envelope for 30 s before lysis.

Figure 4

Tyrosine phosphorylation of Pyk2 in response to M-tropic gp120/gp41 requires expression of CCR5 on target cells. DU6 cells (5 × 10⁶) or the equivalent number of CD4⁺CCR5⁻ T cells (EU2) were incubated with MIP-1β (500 nM), SDF-1α (500 nM), or JRFL gp120/gp41 (5 μg/ml) for 30 s before lysis.