HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells

Abstract

The interaction between HIV and dendritic cells (DCs) is an important early event in HIV-1 pathogenesis that leads to efficient viral dissemination. Here we demonstrate a HIV gp120-independent DC capture mechanism that uses virion-incorporated host-derived gangliosides with terminal α2-3-linked sialic acid linkages. Using exogenously enriched virus and artificial liposome particles, we demonstrate that both α2-3 gangliosides GM1 and GM3 are capable of mediating this interaction when present in the particle at high levels. In the absence of overexpression, GM3 is the primary ligand responsible for this capture mechanism, because siRNA depletion of GM3 but not GM1 from the producer cell and hence virions, resulted in a dramatic decrease in DC capture. Furthermore, HIV-1 capture by DCs was competitively inhibited by targeting virion-associated GM3, but was unchanged by targeting GM1. Finally, virions were derived from monocytoid THP-1 cells that constitutively display low levels of GM1 and GM3, or from THP-1 cells induced to express high surface levels of GM1 and GM3 upon stimulation with the TLR2/1 ligand Pam3CSK4. Compared with untreated THP-1 cells, virus produced from Pam3CSK4-stimulated THP-1 cells incorporated higher levels of GM3, but not GM1, and showed enhanced DC capture and trans-infection. Our results identify a unique HIV-1 DC attachment mechanism that is dependent on a host-cell-derived ligand, GM3, and is a unique example of pathogen mimicry of host-cell recognition pathways that drive virus capture and dissemination in vivo.

Fig. 1.

Gangliosides with α2–3 NeuNAc linkages are important for HIV-1 capture by mDCs. (A) Gag-eGFP VLPs were mock treated or treated with 0.5 units/μL α2–3, 2–6, 2–8 NA. (B) Gag-eGFP VLPs were derived from siRNA-treated HEK293T cells. NT, nontargeting; UGT8, galactosyl transferase; CERT, ceramide transfer protein; UGCG, glucosyltransferases, ST3, GM3 transferase. Capture of VLPs by mDCs was analyzed by FACS (A and B). Data are reported as percentage of eGFP⁺ mDCs normalized to NT-treated VLPs. (C and D) Ganglioside-deficient HIV_Lai was derived from HEK293T cells knocked down for NT, UGCG, or ST3. (C) Virions were labeled for p24^gag (green) and GM3 (red). Representative fields are shown and the average mean fluorescent intensity (MFI) of GM3 normalized to p24^gag ± SD is reported, *P < 0.001, one-way ANOVA with Dunnett’s multiple comparison. (D) Fold decrease of ganglioside-depleted HIV_Lai capture relative to NT-treated viruses by mDCs is reported. (E) Fold decrease in HIV_LaiΔEnv virus capture treated with 0.5 units/μL α2–3, 2–6, 2–8 NA or α2–3-specific NA relative to mock-treated viruses by mDCs is reported. All capture assays represent averaged data from a minimum of three donors, ±SEM, one-sample t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Enrichment of HIV-1 particles with α2–3-linked gangliosides results in enhanced capture by mDCs. HIV_Lai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) (A–C), or exogenous GM1 (+GM1) (D–F). (A and D) Enriched GSLs were detected on the cell membrane using (A) α-GM3 mAb or (D) CtxB and analyzed by FACS. Histograms for mock (gray line) and soluble (A) +GM3 or (D) +GM1 (black line) HeLa cells are shown. Gray fill shows secondary Ab-only control for A and unstained control for D. (B and E) GSL enrichment of virions was analyzed by staining for p24^gag (green) and (B) GM3 (red) or (E) GM1 (red). Representative fields are shown and the average MFI normalized to p24^gag ± SD is reported, **P < 0.001, Student’s t test. (C and F) Capture of HIV_Lai enriched with (C) GM3 or (F) GM1 by mDCs was analyzed by detecting p24^gag content in cell lysates by ELISA. Values represent the average from three donors and are reported as fold enhancement relative to mock-treated virus, ±SEM, *P < 0.05, **P < 0.001, one-sample t test.

Fig. 3.

Liposomes with α2–3-linked gangliosides are captured by mDCs and compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of Cer, Gal, GM3 (α2–3 linked), GM1 (α2–3 linked), or GQ1b (α2–8 linked). (A) Representative transmission electron microscope negative stain of 1% GM3 liposomes at 25k magnification. (B) Liposomes were incubated with mDCs and analyzed for MFI by FACS. Data are normalized to blank liposomes and represent the average from at least three donors, ±SEM, *P < 0.001, one-sample t test. (C and D) Competition of virus (C) or VLP (D) capture by mDCs by increasing amounts of liposomes was analyzed by detecting p24^gag content in cell lysates by ELISA (C) or eGFP⁺ cells by FACS (D). Data shown represent the mean of three donors.

Fig. 4.

HIV-1 derived from Pam3CSK4-stimulated monocytoid cells have increased levels of GM3 and display enhanced capture by mDCs. (A and B) Cell surface expression of GSLs on THP1 cells was determined using (A) α-GM3 mAb or (B) CtxB and analyzed by FACS. Histograms for untreated (gray line) and Pam3CSK4-stimulated (black line) cells are shown. Gray fill shows secondary Ab-only control for A and unstained control for B. (C and D) HIV_Lai/YU2 derived from untreated or Pam3CSK4-treated THP-1 cells were incubated with mDCs and (C) analyzed by p24^gag ELISA for virus capture or (D) washed and cocultured with autologous CD4⁺ T cells. Cocultures were stained for cell surface expression of CD3 and intracellular p24^gag, and the number of dual-positive T cells was determined by FACS. Values were normalized to those observed with virus derived from untreated THP-1 cells and are reported as average fold increase from a minimum of four donors. ±SEM, *P < 0.05, **P < 0.01, one-sample t test. (E and F) HIV_Lai/YU2 derived from untreated (mock) or Pam3CSK4-stimulated (+Pam3CSK4) THP-1 cells were labeled for p24^gag and (E) GM3 or (F) GM1. A minimum of 10 fields were quantified for MFI of (E) GM3 or (F) GM1 normalized to that of p24^gag; average MFI ± SD is shown, *P < 0.0001, Student’s t test.

Fig. 5.

Impairment of GM3-dependent interactions of HIV-1 particle results in decreased capture by mDCs. (A) HIV_Lai or (B) Gag-eGFP VLPs produced from siRNA transfected HEK293T cells were analyzed for mDC capture by (A) p24^gag ELISA or (B) % eGFP⁺ cells by FACS. NT, nontargeting; UGCG, glucosyltransferases; ST3, GM3 transferase; and B4, GM2 transferase. Data are normalized to NT-treated particles and reported as fold decrease in mDC capture. Values represent averaged data from at least two donors, ±SEM, *P < 0.05, one-sample t test. (C) HIV_LaiΔEnv or (D) Gag-eGFP VLPs were preincubated with increasing concentrations of α-GM3 Fab (solid gray lines) or CtxB (dashed black lines), before addition to mDCs. Isotype control Fab was tested at the highest concentration for each assay (open circles). Virion capture was quantified by (C) p24^gag ELISA or (D) % eGFP⁺ cells by FACS. All values were normalized to the (C) virus or (D) VLP-only condition. Average data from a minimum of two donors ±SEM are reported.