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. 2013;8(1):e54295.
doi: 10.1371/journal.pone.0054295. Epub 2013 Jan 23.

Down-regulation of CTLA-4 by HIV-1 Nef protein

Mohamed El-Far  1 , Catherine Isabelle, Nicolas Chomont, Martin Bourbonnière, Simone Fonseca, Petronela Ancuta, Yoav Peretz, Younes Chouikh, Rabih Halwani, Olivier Schwartz, Joaquín Madrenas, Gordon J Freeman, Jean-Pierre Routy, Elias K Haddad, Rafick-Pierre Sékaly
Affiliations

Down-regulation of CTLA-4 by HIV-1 Nef protein

Mohamed El-Far et al. PLoS One. 2013.

Abstract

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nef-mediated CTLA-4 down-regulation.
Transient co-expression of CTLA-4 and Nef in 293T cells. a) Left panels: Surface expression of CTLA-4 analyzed by FACS following staining of 293T cells co-transfected with CTLA-4 and either Nefneg or Nefwt vectors. Right panel: Western blotting analysis on total cell lysates from 293T cells mock-transfected or co-transfected with CTLA-4 and either Nefneg or Nefwt vectors (Arbitrary Densitometric units normalized to ß-actin are shown above corresponding lanes. b) Co-transfection of 293T cells with CD4 and either Nefneg or Nefwt vectors (Left and Right Panels as shown in a). * Refers to the corresponding MW on the SDS gel.
Figure 2
Figure 2. The cytoplasmic tail of CTLA-4 is dispensable for Nef-induced CTLA-4 down-regulation.
a) Accumulation of CTLA-4 on the cell surface after transfection with CTLA-4 Y201AY218G and CTLA-4ΔCT mutants and lower expression after transfection with CTLA-4 LL181AA. Black solid histograms represent the isotype controls. Grey solid histograms represent the expression of CTLA-4 Wt. Black empty histograms represent CTLA-4 mutants. Numbers in inset represent the MFI of CTLA-4 mutant/CTLA-4 Wt. b) Surface expression levels of the different mutants of CTLA-4 with (empty histograms) or without (filled histograms) Nef expression as measured by flow cytometry. The numbers in inset represent the MFI of CTLA-4 under Nefwt/Nefneg co-expression. c) MFI of surface CTLA-4 analyzed by FACS under Nefneg or Nefwt co-expression (mean of three independent experiments). The p values were calculated comparing values of CTLA-4 MFI under Nefwt co-transfection relative to Nefneg co-transfection. d) Western blot analysis showing the total expressions of CTLA-4 Wt and CTLA-4 mutated forms after co-transfection with Nefneg (−) or Nefwt (+) in 293T cells. * Refers to the corresponding MW on the SDS gel.
Figure 3
Figure 3. CTLA-4 down-regulation by Nef requires motifs in Nef involved in the interaction with the vacuolar ATPase, β-COP and the AP-1 sorting complexes.
a) Total expression levels of CTLA-4 in 293T cells after co-transfection with Nefwt or Nef mutants by Western blot. Numbers above the Nef blot represent the Nef densitometric units normalized to the ß-actin and showing similar expression levels for the different Nef constructs. b) Densitometric results, normalized to the ß-actin, are presented as mean percentages of CTLA-4 expression after co-transfection with Nefwt or Nef mutants and normalization to the negative control (Nefneg) from 3 independent experiments. The p values are calculated using the percentage of CTLA-4 expression under co-transfection with each of the Nef mutants relative to CTLA-4 expression under Nefwt co-transfection. * Refers to the corresponding MW on the SDS gel.
Figure 4
Figure 4. Lysosomal function is required for Nef-induced down-regulation of CTLA-4.
a) CTLA-4 surface expression levels on 293T cells co-transfected with Nefneg (upper panels) or Nefwt (lower panels) after treatment with increasing concentrations of Concanamycin A. Grey solid histograms represent the unstained controls. Grey empty histograms represent Mock-transfected cells (stained with anti-CTLA-4 antibody) and Solid Black histograms represent CTLA-4 and either Nefneg (upper panels) or Nefwt (lower panels) co-transfected cells. b) Percentage of CTLA-4+ cells (left panel) and CD4+ cells (right panel) from the same experiment at different concentrations of Concanamycin A (0–20 nM). Solid line represents CTLA-4 or CD4 under Nefneg and dotted line represents CTLA-4 or CD4 under Nefwt co-transfection. c) Rescue of total CD4 protein levels by Concanamycin A treatment. Total lysates from 293T cells co-transfected with CD4 and Nefneg or Nefwt and treated with increasing concentrations of Concanamycin A (1–20 nM). Numbers on top of CD4 blot represent arbitrary densitometric units after normalization to the β-actin. d) Western blot analysis for total lysates from 293T cells co-transfected with CTLA-4 and either Nefneg or Nefwt vectors and treated with increasing concentrations of NH4Cl (0–20 mM) (n = 2). Numbers on the CTLA-4 Western blot represent arbitrary units for CTLA-4 protein expression levels after normalization to β-actin. e) FACS analysis on 293T cells showing CTLA-4 surface levels under Nefneg (upper panels) and Nefwt (lower panels) co-expression in the presence or absence of NH4Cl. * Refers to the corresponding MW on the SDS gel.
Figure 5
Figure 5. Nef and CTLA-4 co-localize in early and recycling endosomes.
a) HeLa cells transfected only with a FLAG-Nef expression vector (upper panels) or co-transfected (lower panels) with CTLA-4 expressing vector were incubated with Alexa 633 labeled transferrin (blue), CTLA-4 specific antibody (green) and FLAG-Nef antibody (red). The extreme left panels show a transmission light field. The middle lower panel shows the co-localization between Nef and CTLA-4 (yellow color results from merging green (CTLA-4) and red (Nef)). The extreme right lower panel shows the co-localization of the three proteins; CTLA-4, Nef and transferrin together (the white color is a combination of green (CTLA-4), red (Nef) and blue (transferrin)), Bar = 20 µm. b) Scatter diagram showing the intensity of the CTLA-4-Alexa-fluor 488 (Y-axis) and Nef-Alexa-fluor 546 (X-axis). c) Levels of co-localization between CTLA-4 and Nef is represented by the mean Pearson's correlation coefficient (Rr) (n = 16, 9 fields).
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