CD4 coexpression regulates DC-SIGN-mediated transmission of human immunodeficiency virus type 1

Abstract

Dendritic cells (DCs) potently stimulate the cell-cell transmission of human immunodeficiency virus type 1 (HIV-1). However, the mechanisms that underlie DC transmission of HIV-1 to CD4(+) T cells are not fully understood. DC-SIGN, a C-type lectin, efficiently promotes HIV-1 trans infection. DC-SIGN is expressed in monocyte-derived DCs (MDDCs), macrophage subsets, activated B lymphocytes, and various mucosal tissues. MDDC-mediated HIV-1 transmission to CD4(+) T cells involves DC-SIGN-dependent and -independent mechanisms. DC-SIGN transmission of HIV-1 depends on the donor cell type. HIV-1 Nef can upregulate DC-SIGN expression and promote DC-T-cell clustering and HIV-1 spread. Nef also downregulates CD4 expression; however, the effect of the CD4 downmodulation on DC-mediated HIV-1 transmission has not been examined. Here, we report that CD4 expression levels correlate with inefficient HIV-1 transmission by monocytic cells expressing DC-SIGN. Expression of CD4 on Raji B cells strongly impaired DC-SIGN-mediated HIV-1 transmission to T cells. By contrast, enhanced HIV-1 transmission was observed when CD4 molecules on MDDCs and DC-SIGN-CD4-expressing cell lines were blocked with specific antibodies. Coexpression of CD4 and DC-SIGN in Raji cells promoted the internalization and intracellular retention of HIV-1. Interestingly, internalized HIV-1 particles were sorted and confined to late endosomal compartments that were positive for CD63 and CD81. Furthermore, in HIV-1-infected MDDCs, significant downregulation of CD4 by Nef expression correlated with enhanced viral transmission. These results suggest that CD4, which is present at various levels in DC-SIGN-positive primary cells, is a key regulator of HIV-1 transmission.

FIG. 1.

CD4 expression levels correlate with monocytic cell restriction of DC-SIGN-mediated HIV-1 transmission. (A) Monocytic cell lines restrict DC-SIGN-mediated HIV-1 transmission. The HIV-1 transmission assay was performed as described previously (59). Immature MDDCs and DC-SIGN-expressing donor cell lines were pulsed separately with single-cycle luciferase HIV-1 (R5 Env_JRFL) and cocultured with Hut/CCR5 target cells. Donor cells alone were used as controls. The data show the means ± standard deviations (SD) of triplicate wells of cell samples. One representative experiment out of four is shown. cps, counts per second. (B) Surface expression levels of DC-SIGN and CD4 of immature MDDCs and DC-SIGN-expressing cell lines. (Top) Staining of DC-SIGN with mAbs against DC-SIGN (solid peaks) or isotypic controls (open peaks). (Bottom) Staining of CD4 with mAbs against CD4 (gray peaks, arrowheads) or isotypic controls (black peaks). Antibody staining (FL2-H) is depicted by the histogram plots along the x axis.

FIG. 2.

CD4 expression impairs DC-SIGN-mediated HIV-1 transmission. (A) Cell surface expression of DC-SIGN and CD4 of Raji-derived cells. Cells were double stained with DC-SIGN and CD4 mAbs and then analyzed by flow cytometry. Antibody staining (FL1-H and FL2-H) is depicted by the dot plots along the x and y axes. (B) Coexpression of CD4 and DC-SIGN impairs HIV-1 transmission. The HIV-1 transmission assay was performed using single-cycle luciferase HIV-1 (R5 Env_JRFL) as previously described (59). The Hut/CCR5 T-cell line or CD4⁺ primary T cells were used as target cells. The data show the means ± SD of triplicate wells of infected cells. One representative experiment out of four is shown. cps, counts per second.

FIG. 3.

Coexpression of CD4 and DC-SIGN promotes X4 HIV-1 infection. (A) Cell surface expression of CCR5 and CXCR4. Cells were double stained with mAbs against CCR5 and CXCR4 and then analyzed by flow cytometry. Antibody staining (FL1-H and FL2-H) is depicted by the dot plots along the x and y axes. (B) HIV-1 direct infection. R5- and X4-tropic single-cycle luciferase (Luc) HIV-1 was used separately to infect Raji cells and derivatives. Cells were washed after viral exposure and cultured for 3 days before the detection. Hut/CCR5 cells were used as positive controls. The data show the means ± SD of duplicate wells of infected cells. One representative experiment out of three is shown. cps, counts per second.

FIG. 4.

Blockade of cell surface CD4 with specific mAbs promotes HIV-1 transmission. (A) Raji/DC-SIGN/CD4 cells, (B) THP-1/DC-SIGN cells, or (C) immature MDDCs were preincubated separately with the CD4 mAbs or anti-DC-SIGN mAbs before HIV-1 incubation. Mouse IgG (mIgG) was used as a control. The HIV-1 transmission assay was performed as described previously (59). Hut/CCR5 cells were used as target T cells. HIV-1 trans infection was determined 2 days postinfection by measuring the luciferase activity of cell lysates. (D) Preincubation with purified mouse anti-human CD4 mAbs blocked HIV-1 infection of Hut/CCR5 cells. The data show the means ± SD of triplicate cell samples. One representative experiment out of four is shown. cps, counts per second. Asterisks indicate significant differences compared with mouse IgG controls (*, P < 0.05; **, P < 0.01).

FIG. 5.

Coexpression of DC-SIGN and CD4 facilitates HIV-1 internalization and alters viral trafficking. (A) Coexpression of DC-SIGN and CD4 enhances HIV-1 binding and internalization. Raji/DC-SIGN cells or Raji/DC-SIGN/CD4 cells were separately incubated with AT-2-inactivated R5 HIV-1 for 2 h. Cells were washed and treated with trypsin or medium and lysed for HIV-1 p24 quantification. The data represent the means ± SD of triplicate cell samples. One representative experiment out of 10 is shown. *, significant differences compared with Raji/DC-SIGN cells (P < 0.01). (B) Raji/DC-SIGN/CD4 cells internalize HIV-1 in intracellular vesicles. Cells were washed after exposure to AT-2-inactivated HIV-1 and were prepared immediately for electron microscopy. Scale bars, 100 nm. (C and D) HIV-1 colocalized with the late endosomal markers CD63 (C) and CD81 (D) in Raji/DC-SIGN/CD4 cells. Cells were pulsed with AT-2-inactivated HIV-1, coimmunostained, and analyzed by confocal microscopy. Green, HIV-1 p24; red, CD63 or CD81. Magnification, ×60.

FIG. 6.

Raji/DC-SIGN/CD4 cells retain intracellular HIV-1 particles for at least 24 h. Cells were incubated with AT-2-inactivated R5 HIV-1 at 37°C for 2 h, washed, and cultured for an additional 24 h. Cells were then analyzed by electron microscopy. (A) Raji/DC-SIGN cells. Arrows indicated cell surface-associated HIV-1 particles. (B) Raji/DC-SIGN/CD4 cells. (C) Higher-magnification image of the boxed area from panel B. Arrows indicate the intracellular compartments that trapped intact HIV-1 particles. Scale bars, 100 nm.

FIG. 7.

Nef-induced CD4 downregulation in HIV-1-infected DCs promotes viral transfer to CD4⁺ T cells. (A) WT and nef-inactivated (Δnef) HIV-1 replicate similarly in Hut/CCR5 cells. Cells were pulsed with R5 (NLAD8) or X4 (NL4-3) HIV-1 (5 ng p24 each), and cell-free supernatants were harvested 3 dpi for p24 quantification. (B) Comparable WT and Δnef HIV-1 infections of DCs. Immature MDDCs were pulsed with HIV-1 (50 ng p24 per inoculum) and cultured. Cell-free supernatants were measured for p24 at the indicated times. (C) Nef expression facilitates DC-mediated HIV-1 transmission. At 3, 5, and 7 dpi, MDDCs were washed and cocultured with Hut/CCR5 cells for an additional 3 days. Cell-free supernatants of cocultures were measured for p24. The data show the means ± SD of triplicate cell samples. One representative experiment out of five is shown. (D) Comparison of infection and transmission between WT and Δnef HIV-1-infected MDDCs at 5 dpi. The average ratio of p24 production of DCs alone and DC-T-cell cocultures is shown. Data are the means ± SD of results of DCs derived from different donors in independent experiments. (E) Nef modulates DC-SIGN and CD4 expression of HIV-1-infected MDDCs. The cell surface expression of DC-SIGN and CD4 was measured by flow cytometry at 5 dpi. MFI, mean fluorescence index. Relative expression levels versus Δnef HIV-1-infected MDDCs are shown. One representative experiment out of five is shown. Asterisks indicate a significant difference (*, P < 0.05 compared with DC alone; **, P < 0.01 compared with the WT).