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. 2013 Jan 11:10:6.
doi: 10.1186/1742-4690-10-6.

TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse

Fabien P Blanchet  1 , Romaine Stalder, Magdalena Czubala, Martin Lehmann, Laura Rio, Bastien Mangeat, Vincent Piguet
Affiliations

TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse

Fabien P Blanchet et al. Retrovirology. .

Abstract

Background: Dendritic cells and their subsets, located at mucosal surfaces, are among the first immune cells to encounter disseminating pathogens. The cellular restriction factor BST-2/tetherin (also known as CD317 or HM1.24) potently restricts HIV-1 release by retaining viral particles at the cell surface in many cell types, including primary cells such as macrophages. However, BST-2/tetherin does not efficiently restrict HIV-1 infection in immature dendritic cells.

Results: We now report that BST-2/tetherin expression in myeloid (myDC) and monocyte-derived dendritic cells (DC) can be significantly up-regulated by IFN-α treatment and TLR-4 engagement with LPS. In contrast to HeLa or 293T cells, infectious HIV-1 release in immature DC and IFN-α-matured DC was only modestly affected in the absence of Vpu compared to wild-type viruses. Strikingly, immunofluorescence analysis revealed that BST-2/tetherin was excluded from HIV containing tetraspanin-enriched microdomains (TEMs) in both immature DC and IFN-α-matured DC. In contrast, in LPS-mediated mature DC, BST-2/tetherin exerted a significant restriction in transfer of HIV-1 infection to CD4+ T cells. Additionally, LPS, but not IFN-α stimulation of immature DC, leads to a dramatic redistribution of cellular restriction factors to the TEM as well as at the virological synapse between DC and CD4+ T cells.

Conclusions: In conclusion, we demonstrate that TLR-4 engagement in immature DC significantly up-regulates the intrinsic antiviral activity of BST-2/tetherin, during cis-infection of CD4+ T cells across the DC/T cell virological synapse. Manipulating the function and potency of cellular restriction factors such as BST-2/tetherin to HIV-1 infection, has implications in the design of antiviral therapeutic strategies.

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Figures

Figure 1
Figure 1
Vpu-dependent BST-2/tetherin cell surface down-regulation but lack of BST-2/tetherin restriction activity in DC. (A). 2 × 105 DC (A, upper panel) and Vpx-transduced DC (A, lower panel) were infected or not (NI) with HIV-WT or HIV-ΔVpu viruses. Infection of DC was scored by FACS analysis of BST-2/tetherin+/p24gag+ cells, three days post-infection. Percentage of cell surface BST-2/tetherin+ in infected DC from 7 independent experiments was calculated (right graph panels). (B) TZM-bl cells were incubated for 48 hours with 1/10 volume of supernatant from HIV-WT or HIV-ΔVpu infected DC or myDC harvested three days post-infection. Infectious units (IU/ml) were quantified using the β-gal assay obtained from 5 and 3 experiments in DC and myDC respectively. (C) Similar experiments (as above) were performed with supernatant obtained from HIV-WT or HIV-ΔVpu infected Hela cells (results from duplicate experiments). Error bars represent the mean ± SD of indicated experiment numbers. (D) Lysates and supernatants of uninfected (NI), HIV-WT or HIV-ΔVpu infected DC were subjected to immunoblotting with anti-Gag and anti-BST-2. The presence of Vpu was assessed by immunoblotting with anti-Vpu and anti-actin served as loading control.
Figure 2
Figure 2
LPS-induced BST-2/tetherin restriction activity in HIV-1 infected DC. (A) 2 × 105 DC, previously treated (or not) with IFN-α or LPS, were left uninfected (NI) or infected for 3 days with HIV-WT, HIV-ΔVpu or HIV-F522Y viruses in parallel with SIV3 (+Vpx) transduction. Infection rates in DC were scored by FACS analysis of BST-2/tetherin+/p24gag+ cells, three days post-infection. (B) TZM-bl cells were incubated for 48 hours with 1/10 volume of supernatant from HIV-WT or HIV-ΔVpu infected DC, previously untreated (NT) or treated with IFN-α or LPS, and harvested three days post-infection. Infectious units (IU/ml) were quantified as above in NT DC and treated DC (from 5 and 3 experiments respectively). (C) Supernatants from HIV-WT or HIV-ΔVpu infected DC, previously untreated (NT) or treated with IFN-α or LPS, were harvested three days post-infection and HIV-p24 levels were quantified by ELISA. (D) Graph represents the percentage of HIV-ΔVpu / HIV-WT infectivity ratios from treated DC as indicated and compared to Hela cells.
Figure 3
Figure 3
LPS-induced relocalization of BST-2/tetherin in HIV-containing tetraspanin-enriched compartment. (A) Confocal immunofluorescence analysis of HIV-Gag (green), BST-2/tetherin (red) and CD81 (blue) of DC pre-treated (or not) with IFN-α or LPS and left uninfected or challenged with HIV-WT or HIV-ΔVpu for 3 days. Data are representative of four independent experiments. Scale bars correspond to 5 μm. (B) Graphs represent the quantification of BST-2/tetherin and CD81 co-localisation with HIV-Gag from at least 24 cells per condition from 3 independent experiments.
Figure 4
Figure 4
BST-2/tetherin impact on DC-mediated HIV-1 transfer in trans. (A). 1 × 105 DC, previously transfected (or not) with siTetherin or si Control (siCtr) for 48 hours, were left untreated (NT) or treated with IFN-α or LPS and challenged for six hours with 200 ng P24gag of HIV-ΔVpu or HIVF522Y viruses. DC, extensively washed, were then co-cultured with 1 × 105 autologous CD4+ T cells pre-treated 30 minutes before co-culture with Indinavir. HIV-1 transfer on autologous CD4+ T cells was scored by FACS analysis of CD3+p24gag+FITC cells, four days post-transfer. Data shown are representative of three independent experiments. (B). Fold enhancement of HIVWT and HIV-ΔVpu transfer in CD3+/CD4+ T cells. A value of 1 was assigned to the percentage of transfer in the non-treated (HIV) conditions. Error bars indicate standard error mean (SEM). Data are representative of four different experiments. P-values of paired bilateral Student Test were not significant (p>0.18). (C). DC were transfected with siTetherin or siControl (siCtr) and left untreated (NT) or treated with IFN-α for 24 hours. Tetherin expression was analyzed by western blot. Data are representative of four experiments.
Figure 5
Figure 5
BST-2/tetherin-mediated restriction on cis-infection from matured infected DC. (A) 1 × 105 DC, previously treated (or not) with IFN-α or LPS, were left uninfected (NI) or infected for 3 days with HIV-WT, HIV-ΔVpu or HIVF522Y viruses in parallel with Vpx-VLPs transduction. Cells were then co-cultured with 1 × 105 autologous CD4+T lymphocytes. HIV-1 transfer on T lymphocytes was scored by FACS analysis of CD3+p24gag+FITC cells, three days post-transfer. (B) Quantification of viral transfer from treated DC as indicated was obtained from 5 different experiments for NT and IFN-α conditions and 3 independent experiments for LPS condition. (C) Percentage of DC-mediated HIV-ΔVpu transfer for each condition was then measured and depicted in the lower graph.
Figure 6
Figure 6
BST-2/tetherin polarization and enrichment at the Virological Synapse. (A) Confocal immunofluorescence analysis of HIV-Gag (green) and BST-2/tetherin (red) upon DC/T cell contacts from experiments above. (B) Virological Synapse (VS) signal intensity / total signal was then quantified from 3 different experiments (n= 15 for each condition) for each marker.
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