Binding of human immunodeficiency virus type 1 to immature dendritic cells can occur independently of DC-SIGN and mannose binding C-type lectin receptors via a cholesterol-dependent pathway

Abstract

Interactions of human immunodeficiency virus type 1 (HIV-1) with immature dendritic cells (DC) are believed to be multifactorial and involve binding to the CD4 antigen, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose binding C-type lectin receptors (MCLR), and heparan sulfate proteoglycans (HSPG). In this study we assessed the relative contributions of these previously defined virus attachment factors to HIV binding and accumulation in DC and the subsequent transfer of the bound virus particle to CD4(+) T cells. Using competitive inhibitors of HIV-1 attachment to DC, we have identified the existence of DC-SIGN-, MCLR-, and HSPG-independent mechanism(s) of HIV attachment and internalization. Furthermore, virus particles bound by DC independently of CD4, DC-SIGN, MCLR, and HSPG are efficiently transmitted to T cells. Treatment of virus particles with the protease subtilisin or treatment of immature DC with trypsin significantly reduced virus binding, thus demonstrating the role of HIV envelope glycoprotein interactions with unidentified DC-surface factor(s). Finally, this DC-mediated virus binding and internalization are dependent on lipid rafts. We propose that pathways to HIV-1 attachment and uptake in DC exhibit functional redundancy; that is, they are made up of multiple independent activities that can, at least in part, compensate for one another.

Figures

FIG. 1.

HIV-1 binding to DC can occur in the presence of competitive inhibitors of MCLR and CD4. Cells (4 × 104 cells/well) were incubated with increasing concentrations of mannan at 4°C and then exposed to either Lai (A) or NL4-3/Ba-L env (B) virus particles (30 ng of p24gag) for 2 h at 4°C. See Materials and Methods for experimental details. Cell-associated virus binding was determined by a p24gag ELISA. The mean level of Lai or NL4-3/Ba-L env binding to DC in the presence of increasing amounts of mannan was normalized to the level of virus binding to DC in the absence of mannan (set at 100%) and reported as relative virus binding. Infections with HIV/Lai (A) and NL4-3/Ba-L (B) were performed at least five independent times with DC derived from five independent donors. The results shown here are the averages of the mean percent binding observed for all the experiments. (C) A representative experiment of Lai virus binding to DC in the presence of a constant amount of anti-CD4-neutralizing monoclonal antibodies (Sim.4; 1 μg/ml) and increasing amounts of mannan. The mean level of virus binding to DC in the presence of either CD4-neutralizing antibodies or mannan was normalized to the level of virus binding to DC in the absence of mannan and CD4-neutralizing antibody (set at 100%) and reported as relative virus binding. Note that presence of anti-CD4 monoclonal antibodies had no synergistic effect with mannan in inhibiting Lai binding to DC. One representative experiment out of two is shown. (D) DC, THP1, or THP1/DC-SIGN cells (4 ×104) were incubated with Lai virus particles in the presence or absence of mannan (50 μg/ml) for 2 h at 4°C, and the cell-associated virus fraction was determined by p24gag ELISA. The experiment was performed at least three independent times, and each experiment was performed in triplicate. The mean level of virus binding to THP1 cells and THP1/DC-SIGN cells in the presence of mannan (50 μg/ml) was normalized to the level of virus binding to THP1/DC-SIGN cells in the absence of mannan (set at 100%) and reported as relative virus binding. Also, in each experiment, the mean level of virus binding to DC in the presence of mannan (50 μg/ml) was normalized to the level of virus binding to DC in the absence of mannan.

FIG. 2.

Monomeric gp120 binding to DC is dependent on MCLR. DC were untreated or incubated with mannan prior to incubation with either (A) 250 ng of SF162 monomeric gp120 or (B) HIV/SF162 virus particles (30 ng of p24gag) at 4°C. The mean level of gp120 binding to DC in the presence of increasing amounts of mannan was normalized to the level of gp120 binding to DC in the absence of mannan (set at 100%) and reported as relative gp120 binding. The mean level of HIV/SF162 virus particle binding to DC in the presence of increasing amounts of mannan was normalized to the level of virus binding to DC in the absence of mannan (set at 100%) and reported as relative virus binding. The results from one representative experiment out of two are shown, and each experiment was performed in triplicate.

FIG. 3.

HSPG do not play a role in HIV-1 binding to DC. DC (4 ×104 cells/well) were incubated with mannan (50 μg/ml), treated with heparinase I (10 U/ml), or exposed to a combination of both reagents and then exposed to Lai (A), NL4-3/Ba-L (B), or 93BR019 (C) virus particles (30 ng of p24gag) for 2 h at 4°C. (D) HeLa cells (4 × 104 cells/well) were left untreated or treated with heparinase I (10 U/ml) and then exposed to either Lai or NL4-3/Ba-L. The percent virus (p24gag) binding represents the mean ± standard deviation of triplicate cultures. The results from one representative experiment out of three with DC derived from three independent donors are shown.

FIG. 4.

DC can mediate HIV-1 transmission in a MCLR-independent manner. DC (2 × 104 cells/well) were preincubated with increasing concentrations of mannan and then incubated with Lai (A) or NL4-3/Ba-L env (B) at 4°C, extensively washed, and then cocultured with Jurkat-CCR5 T cells (2 × 105 cells/well). (C) THP1 or THP1/DC-SIGN cells (2 × 104 cells/well) in the presence or absence of mannan were incubated with Lai, extensively washed, and then cocultured with Jurkat-CCR5 T cells. Culture supernatants were assayed for p24gag content 3 days after the start of the coculture. Shown here are the results from one representative experiment out of three; each infection was performed in triplicate, and the mean amounts of p24gag content with standard deviations are shown here. The three independent experiments were performed with DC from three independent donors.

FIG. 5.

Protease treatment of immature DC or virus particles reduces virus particle binding to DC surface. (A) DC (4 × 104/well) were treated with 0.25% trypsin at 37°C for 30 min and then washed with cold complete RPMI medium to stop the protease digestion. The cell pellet was then incubated with mannan (100 μg/ml) for 30 min prior to virus exposure (30 ng of p24gag) for 2 h at 4°C. Virus binding to trypsin-treated and untreated DC was assessed as described in Materials and Methods. (B) Immunoblot analysis of Lai (lanes 1 and 2) and LaiΔenv (lanes 3 and 4) virus preparations isolated and purified from HEK293T cell supernatants that were either left untreated (lanes 1 and 3) or treated with the protease subtilisin (lanes 2 and 4). The blots were reacted with an antibody specific for the Lai gp120 glycoprotein. (C) DC (4 × 104 cells) were incubated with untreated or subtilisin-treated virus preparations (30 ng of p24gag) at 4°C for 2 h. The percent virus (p24gag) binding in panels A and C represents means ± standard deviations of triplicate cultures. The results from one representative experiment out of three are shown.

FIG. 6.

Binding of HIV-1 to DC is cholesterol dependent. DC were exposed to the endocytosis inhibitors and then incubated with FITC-labeled transferrin at 4°C. Cells were also stained with DAPI to help in the visualization of the cellular architecture. The images are the composite of a series of Z sections collected through the entire thickness of the cell monolayer and projected onto a two-dimensional plane. (A) At 4°C, transferrin-FITC can bind to its receptor but is not internalized, as evident from the staining observed predominantly at the plasma membrane. (B) Cells were shifted to 37°C for a period of 15 min and then monitored again for transferrin localization. Note the prominent intracellular green fluorescence indicative of uptake of transferrin-FITC. (C) Treatment with 500 μM amantadine prior to incubation with transferrin-FITC for 1 h at 37°C resulted in transferrin-FITC staining at the plasma membrane (indicated by white arrowheads) and completely abolished the intracellular staining, while treatment with 5 mM MβCD (D) had no effect on the intracellular accumulation of transferrin. (E) DC (4 × 104 cells) treated with the endocytosis inhibitors were exposed to Lai virus particles (30 ng of p24gag) for 2 h at 37°C. The mean level of virus (Lai) binding in the presence of endocytosis inhibitors was normalized to the level of virus binding in the absence of inhibitors (set at 100%). This experiment was performed at least five independent times with DC derived from five independent donors, and each experiment was performed in triplicate. The results shown are the averages of the mean percent binding (± standard deviations) observed for all the experiments. (F) DC (4 × 104 cells) were treated with amantadine (500 μM) and then exposed to Lai virus particles (30 ng of p24gag) for 2 h either at 4 or 37°C. (G) DC (4 × 104 cells) were treated with MβCD (5 mM) and then exposed to Lai virus particles (30 ng of p24gag) for 2 h either at 4 or 37°C. The percent virus (p24gag) binding values shown in panels F and G represent the means ± standard deviations of triplicate cultures. The results from one representative experiment out of three with DC derived from three independent donors are shown in panels F and G.