Down-regulation of cell surface CXCR4 by HIV-1

Abstract

Background: CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled chemokine receptor family, can serve as a co-receptor along with CD4 for entry into the cell of T-cell tropic X4 human immunodeficiency virus type 1 (HIV-1) strains. Productive infection of T-lymphoblastoid cells by X4 HIV-1 markedly reduces cell-surface expression of CD4, but whether or not the co-receptor CXCR4 is down-regulated has not been conclusively determined.

Results: Infection of human T-lymphoblastoid cell line RH9 with HIV-1 resulted in down-regulation of cell surface CXCR4 expression. Down-regulation of surface CXCR4 correlated temporally with the increase in HIV-1 protein expression. CXCR4 was concentrated in intracellular compartments in H9 cells after HIV-1 infection. Immunofluorescence microscopy studies showed that CXCR4 and HIV-1 glycoproteins were co-localized in HIV infected cells. Inducible expression of HIV-1 envelope glycoproteins also resulted in down-regulation of CXCR4 from the cell surface.

Conclusion: These results indicated that cell surface CXCR4 was reduced in HIV-1 infected cells, whereas expression of another membrane antigen, CD3, was unaffected. CXCR4 down-regulation may be due to intracellular sequestering of HIV glycoprotein/CXCR4 complexes.

Figure 1

Flow cytometry analysis demonstrating reduced CXCR4 expression in HIV-1 infected RH9 cells. Panel A: RH9 T-lymphoblastoid cells infected with HIV-1_LA1. On days 1, 4, and 7 postinfection cells were fixed with 4% paraformaldehyde, stained with mouse MAb 12G5 anti-CXCR4 (10 μg/ml) or isotype-matched control antibody followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G, and analyzed by flow cytometry. Median fluorescence intensity was calculated as an indicator of the level of cell surface CXCR4 expression. Data are presented as single-color histograms with FITC fluorescence (CD3 expression) along the horizontal axis and relative cell number along the vertical axis. RH9 cells (control cells), heavy solid line: H9 cells infected with HIV, dotted line; H9 with an isotype-matched control antibody, thin solid line. Panel B: Analysis of surface CD3 expression in HIV-1 and mock infected RH9 cells by FACS analyzed on day 7 post-infection.

Figure 2

Immunofluorescence microscopy demonstrating reduced cell surface expression of CXCR4 in HIV-1 infected RH9 cells. Panel A: Immunofluorescence staining control with isotype-matched monoclonal antibody. Panel C: CXCR4 immunofluorescence staining of H9 cells. Panels E and G: CXCR4 immunofluorescence staining of H9 cells acutely infected by HIV-1. Panels B, D, F and H show phase contrast images of the same fields of cells shown in left panels. The fluorescent syncytial cell in panel G is representative of a minor population of cells in the infected culture (<10%) with a CXCR4 surface distribution similar to uninfected cells.

Figure 3

Immunofluorescence microscopy analysis of CXCR4 expression in permeabilized HIV-1 and mock infected RH9 cells. Four days after HIV-1 infection, cells were fixed, permeabilized with saponin and labeled with a mouse monoclonal antibody to CXCR4 (12G5) and a secondary, FITC-conjugated anti-mouse antibodies for observation with a fluorescence microscopy. Panel A: Immunofluorescence staining control with isotype-matched monoclonal antibody. Panel C: CXCR4 immunofluorescence staining of H9 cells. Panels E and G, CXCR4 immunofluorescence staining of HIV-1 infected H9 cells. Panels B, D, F and H show phase contrast images of the same fields of cells shown in left panels.

Figure 4

Co-localization CXCR4 and HIV-1 glycoprotein in HIV-1 infected H9 cells. Four days after HIV-1 infection, cells were fixed and permeabilized with saponin. Cells were then labeled with a human monoclonal antibody that interact with SU and then rhodamine-conjugated goat anti-human antibodies (Panel C: red fluorescence) and with 12G5 mAb followed by fluorescein-conjugated goat anti-mouse antibodies (Panel D:green fluorescence). Panel A: phase contrast image. Panel B represents a superposition of green and red fluorescence, with costained regions appearing in yellow. Yellow regions in panel B indicate the colocalization of chemokine receptor CXCR4 and HIV-1 proteins.

Figure 5

CXCR4 expression is reduced in Jurkat cells after induction of HIV-1 Env expression. After 4 days induction of HIV-1 Env proteins, non-induced and induced cells were fixed and labeled with a mouse MAb to CXCR4 (12G5) and a secondary FITC-conjugated anti-mouse antibodies for observation with a fluorescence microscopy. Panels A and C: CXCR4 staining of non-induced Jurakt cells. Panel E and G: CXCR4 staining of induced Jurkat cells. The fluorescent cell in panel G is representative of a minor population of cells in the induced culture (<5%) with a CXCR4 surface distribution similar to uninduced cells. Panels B, D, F and H show phase contrast images of the same fields of cells shown in left panels.