HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production

Abstract

HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.

FIGURE 1.

a, DEAE-Sephacel chromatography and PG-ELISA. HSPGs were extracted from HeLa cells with TTU buffer. The extract was chromatographed over a DEAE-Sephacel column equilibrated with TTU buffer. Elution was performed by stepwise salt gradient (NaCl in TTU buffer). Eluted fractions were diluted 1:2 in water and coated on microtiter wells. Fractions were assayed for HSPG presence by an indirect ELISA using anti-CD44v3, anti-Sy-2, anti-Sy-4 antibodies or an unrelated antibody as negative control (K⁻). The data are from a representative experiment of three independent experiments. b, capture ELISA. To define which HSPG interacts with the matrix protein, specific antibodies to Sy-2, Sy-4, CD44v3, and unrelated antibody (anti-CD41) were adsorbed on microtiter plate wells. Positive and negative fractions were pooled and reacted with the four solid phases. Captured antigens were reacted with the matrix protein. Complexes were detected by MK-1 antibody and by rabbit anti-mouse IgG-HRP conjugate. Data are representative of three independent experiments.

FIGURE 2.

UFH inhibits the interaction between HSPGs and p17. The matrix protein (0.25 μg/ml) was mixed with increasing concentrations of UFH (0, 0.2, 1, 5, 20 μg/ml) for 1 h before the addition to HSPGs on capture ELISA solid phases. An assay was then performed as described for the capture ELISA assay. Data are representative of three independent experiments.

FIGURE 3.

Co-localization analysis on activated CD4⁺ T cells. The matrix protein was incubated on fixed cells and detected with anti-p17 monoclonal antibody (MK-1). Complexes were stained with rabbit anti-mouse IgG Texas Red conjugate (red, 1a, 2a, and 3a). CD44v3 (1b), Sy-2 (2b), and Sy-4 (3b) were detected with a primary rabbit polyclonal antibody and stained with FITC-labeled secondary antibody (green). 1c, 2c, and 3c are merged red and green channels. Where the red and green of the image overlap, the pixels appear yellow. 4a, 4b, and 4c images are from unmatched antibodies. Data are representative of three individual experiments.

FIGURE 4.

Effect of HIV-1 p17 on TNF-α and IL-2 production by activated CD4⁺ T cells. The cells were activated for 72 h with PHA and PMA in the presence or absence of the matrix protein. Supernatants were collected, and TNF-α and IL-2 production was measured by ELISA. The data are representative of three independent experiments with the error bars indicating the ± S.D. #, p < 0.05 compared with PHA/PMA cell activation.