HIV-1 Vpu mediated downregulation of CD155 requires alanine residues 10, 14 and 18 of the transmembrane domain

Abstract

HIV-1 NL4-3 Vpu induces downregulation of cell surface CD155, a ligand for the DNAM-1 activating receptor of NK and CD8(+) T cells, to evade NK cell mediated immune response. Here we show that the conserved alanine residues at positions 10, 14 and 18 in the TM domain of Vpu are required for the efficient downregulation of cell surface CD155. In contrast, the CK-2 phosphorylation sites and the second α-helix in the cytoplasmic Vpu domain have no influence on the surface expression of CD155. Thus, compared to Vpu׳s effect on CD4, NTB-A and tetherin, the Vpu mediated downregulation of CD155 is an independent Vpu function. We finally show that in contrast to other lentiviral strains, only Vpu and Nef from HIV-1 M NL4-3 potently interfere with CD155 surface expression. Thus, Vpu seems to subvert NK cell responses against HIV-1 infected T cells by modulation of receptors necessary for NK cell activation.

Keywords: CD155; HIV-1; Nef; SIV; Vpu.

Fig. 1

Schematic representation of wt Vpu (A) or Vpu mutants KKDQ (B), RD (C), A7N (D), A10N (E), A14N (F), A18N (G), A18H (H), m26 (I) or Δ23 (J).

Fig. 2

The TM domain, particularly A10N, A14N and A18N, of Vpu is required for the efficient downregulation of cell surface CD155. HeLa cells were transfected with either Vpu-IRES-GFP or mutants thereof. 24 h post transfection, cells were harvested and either stained for surface CD155 (A) or intracellular CD155 (B) using a CD155 specific antibody. Subsequently, cells were analyzed by flow cytometry. Summary of relative CD155 surface (A) and intracellular (B) expression from 3 independent experiments is given below. Mock indicates non-infected and unstained control cells. Values from three independent experiments were plotted and assessed for statistical significance by using the GrapPad Prism V5.0 software package (***; p≤0.0005, 1 way- ANOVA with Dunnett’s test). In addition, cells were lysed and analyzed by Western blot using anti-β-actin, anti-GFP and anti-Vpu antibodies (C).

Fig. 3

Downregulation of cell surface CD155 by Vpu in CD4⁺ Sup-T1 T cells. CD4⁺ Sup-T1 T cells were infected with either VSVG-pseudotyped HIV-1 NL4-3 Vpu Del-1, HIV-1 NL4-3 wt, HIV-1 NL4-3 Vpu RD or HIV-1 NL4-3 Vpu A18H. 2 days after infection the cells were harvested and stained for surface CD155, using a CD155-specific antibody. Afterwards the cells were stained intracellularly for HIV-1 p24 antigen, using anti-p24-FITC specific antibody and analyzed by flow cytometry. Values from three independent experiments were plotted and assessed for statistical significance by using the GrapPad Prism V5.0 software package (***; p≤0.0005, 1 way- ANOVA with Dunnett’s test), which is given below.

Fig. 4

Sub-cellular distribution of CD155 in presence of NL4-3 wt Nef, wt Vpu or mutants. (A) HeLa cells were transfected with either YFP, wt Nef, wt Vpu-, RD-, A10N, A14N or A18N-YFP fusion proteins (green). 24 h post transfection, cells were fixed, permeabilized and stained for CD155 (red) using anti-CD155 specific antibody. (B) Values for the ratio of CD155 levels at the plasma membrane (PM)/perinuclear area of 10 analyzed cells were plotted and assessed for statistical significance by using the GrapPad Prism V5.0 software package (***; p≤0.0002, 1 way-ANOVA with Dunnett’s test). Scale bars: 4 μm.

Fig. 5

NL4-3 HIV-1 wt induces an accumulation of CD155 in perinuclear compartments. (A) HeLa cells were transfected with either pBR HIV-1 NL4-3 wt-IRES-GFP, pBR HIV-1 NL4-3 ΔNef-IRES-GFP, pBR HIV-1 NL4-3 ΔVpu-IRES-GFP or pBR HIV-1 NL4-3 ΔNefΔVpu-IRES-GFP (green). 24 h post transfection, cells were fixed, permeabilized and stained for CD155 (red) using anti-CD155 specific antibody. (B) Values for the ratio of CD155 levels at the plasma membrane (PM)/perinuclear area of 10 analyzed cells were plotted and assessed for statistical significance by using the GrapPad Prism V5.0 software package (***; p≤0.0002, 1 way- ANOVA with Dunnett’s test). Scale bars: 4 μm.

Fig. 6

Effect of lentiviral Vpu and Nef proteins on the cell surface expression of CD155. (A) HeLa cells were transfected with either AU1-tagged Vpu or Nef of HIV-1 M NL4-3, SIVgsn 166, SIVcpz EK505, SIVgor CP2139 or HIV-1 N CK1.62 HIV-1 P RBF168. 24 h post transfection, cells were harvested and stained for surface CD155 using a CD155 specific antibody and analyzed by flow cytometry. Summary of relative CD155 surface expression in presence of lentiviral Vpu (A) or Nef (B) proteins is given below. Values from three independent experiments were plotted and assessed for statistical significance by using the GrapPad Prism V5.0 software package (***; p≤0.0005, 1 way- ANOVA with Dunnett’s test). In addition, cells were lysed and analyzed by Western blot using anti-β-actin and anti-AU1 antibodies.