Cooperative interactions of simian immunodeficiency virus Nef, AP-2, and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis

Abstract

The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-zeta subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-zeta protein containing only the CD3-zeta cytoplasmic domain (8-zeta), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-zeta likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-zeta, both proteins colocalize with AP-2, indicating that Nef induces 8-zeta internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-zeta endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-zeta colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-zeta cytoplasmic domain demonstrated that SIV Nef and CD3-zeta cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis.

FIG. 1.

SIV Nef accelerates endocytosis of TCR-CD3. Two-color flow cytometric analysis of TCR-CD3 cell surface expression in Jurkat T cells (JJK subline) (panels 1 to 4) and J.CaM1 cells (panels 5 to 8) transiently expressing 239.Nef together with GFP from a bicistronic transcription unit or GFP alone (vector) (panels 1, 2, 5, and 6) is shown. Histograms of CD3 cell surface expression for populations of cells with identical levels of GFP fluorescence, gated as indicated in panels 1 and 5, are shown for cells expressing 239.Nef together with GFP or GFP alone (panels 3 and 7). The rates of CD3 endocytosis in the absence or presence of 239.Nef, determined by a fluorescence-activated cell sorting-based endocytosis assay, are shown in panels 4 and 8.

FIG. 2.

SIV Nef associates with the CD3 complex at the cell surface. Jurkat T cells expressing 239.Nef (Nef) or not (−) were surface biotinylated with NHS-biotin and lysed. (A) Cytoplasmic extracts were immunoprecipitated (IP) with anti-CD3-ζ (lanes 1 and 2) or sequentially with anti-HA and anti-AU1 MAbs (lanes 3 and 4), and the immunoprecipitates were immunoblotted with streptavidin-horseradish peroxidase. Approximately five times more extract from 239.Nef-expressing cells than from control cells was used in this experiment to compensate for lower steady-state CD3-ζ levels in 239.Nef-expressing cells. Bands corresponding to CD3-ζ chains are indicated. (B) Identification of biotinylated protein bands that correspond to CD3-ζ and CD3-ɛ subunits. Jurkat T cells were surface biotinylated and lysed in the presence of 1% NP-40, which disrupts the association between CD3-ζ and CD3-ɛ (lanes 1, 2, 5, and 6), or in the presence of 1% digitonin, which does not affect the association of the two CD3 subunits (lanes 3, 4, 7, and 8). The extracts were immunoprecipitated with anti-CD3-ζ (lanes 2, 4, 6, and 8) or with anti-CD3-ɛ (lanes 1, 3, 5, and 7), and the immune complexes were immunoblotted with streptavidin-horseradish peroxidase (lanes 1 to 4) or anti-CD3-ζ (lanes 5 to 8). Protein bands corresponding to CD3-ɛ and CD3-ζ are indicated.

FIG. 3.

SIV Nef induces endocytosis of the 8-ζ chimeric receptor in nonlymphoid cells. Cell surface expression of chimeric protein comprising the CD3-ζ cytoplasmic domain peptide fused to the ecto- and transmembrane domain of CD8-α (8-ζ) in HeLa cells transiently coexpressing 8-ζ and GFP reporter protein from the same bicistronic transcription unit in the absence (panel 1) or presence (panel 2) of 239.Nef is shown in panels 1 and 2. The histograms of 8-ζ expression in populations of cells with comparable GFP expression, gated as indicated in panel 1, for cells expressing 239.Nef together with GFP or GFP alone are shown in panel 3. The rates of 8-ζ endocytosis in the absence or presence of 239.Nef, determined by a fluorescence-activated cell sorting-based endocytosis assay, are shown in panel 4.

FIG. 4.

In SIV Nef-expressing cells, Nef and 8-ζ colocalize with the AP-2 clathrin adaptor. (Upper panels) SIV Nef redistributes the 8-ζ chain to the AP-2 clathrin adaptor. Dual-color immunofluorescence detection of clathrin adaptors and 8-ζ-GFP fusion proteins (panels 1 to 8) expressed in IMR90 fibroblasts in the presence (panels 5 to 8) or absence (panels 1 to 4) of SIV Nef is shown. The overlay of β-adaptin and 8-ζ-GFP patterns is shown in the two leftmost panels. The three rightmost panels are the magnified images. (Lower panels) 8-ζ colocalizes with SIV Nef. Localization of 8-ζ chimeras in IMR90 fibroblasts expressing 239.Nef-GFP fusion protein (panels 13 to 16) or GFP only (panels 9 to 12) is shown. The overlay of 8-ζ distribution, revealed by indirect fluorescence with an anti-CD8 MAb, and of GFP fluorescence is shown in the two leftmost panels. The three rightmost panels are magnified images.

FIG. 5.

Effect of mutations in CAIDs in SIV Nef on the downmodulation of TCR-CD3 via accelerated endocytosis. (A) Schematic representation of wild-type and mutant SIV Nef proteins and location of selected protein-protein interaction sites. The nomenclature of mutant proteins is shown on the right. The locations of the CAIDs in the N-terminal region of 239.Nef (N-proximal CAID and N-distal CAID) and of the C-terminal adaptor interaction determinant (C-terminal) and the ability of the mutant Nef proteins to bind CD3-ζ are also indicated. (B) CAIDs in 239.Nef are not required for its ability to downmodulate cell surface CD3. Flow cytometry analysis of steady-state CD3, CD4, and class I MHC expression in Jurkat T cells transiently expressing wild-type or mutant 239.Nef proteins and GFP reporter from bicistronic transcription units is shown. (C and D) Effect of mutations disrupting CAIDs on rates of endocytosis of TCR-CD3 in the Jurkat T cell subline J.CaM1 (C) and of 8-ζ in HeLa cells (D) expressing wild-type and mutant 239.Nef proteins. The rates of TCR-CD3 and 8-ζ endocytosis in the absence or presence of 239.Nef were determined by a flow cytometry-based endocytosis assay.

FIG. 6.

8-ζ and SIV Nef mutants lacking N- and C-terminal AP-2 adaptor binding elements are corecruited to AP-2 in vivo. The distributions of 239.Nef_{(Δ23-74, LM194AA)}-GFP (panels 1 to 8), with mutated constitutive N- and C-terminal CAIDs, and of the HSIV.Nef-GFP chimera (panels 9 to 16) and clathrin adaptors in IMR90 fibroblasts in the absence (panels 1 to 4 and 9 to 12) or presence (panels 5 to 8 and 13 to 16) of 8-ζ molecules are shown.

FIG. 7.

SIV Nef and the CD3-ζ subunit cytoplasmic domain cooperatively bind the AP-2 clathrin adaptor in vitro.(Upper panel) Recombinant CD3-ζ cytoplasmic domain peptide ζ_(52-161) was loaded in vitro onto wild-type and mutant 239.Nef proteins fused to GST. Aliquots of various GST-Nef-ζ_(52-161) complexes or GST-Nef fusion proteins alone were incubated with extracts containing clathrin adaptors. The bound AP-2 and AP-1 were quantitated by immunoblot analysis with α-adaptin-specific MAb 100/2 (α), γ-adaptin-specific MAb 100/3 (γ), and β-adaptin-specific MAb 100/1 (β). A GST fusion protein containing CD3-ζ_(52-161) cytoplasmic domain peptide [GST-ζ_(52-161)] and GST alone were used as negative controls (lanes 1 to 3). Aliquots of adaptor extract corresponding to 0.25 to 8% of the amount used for each binding reaction were used as standards for quantitation (lanes 11 to 16). (Lower panel). Three percent aliquots from the binding reactions were resolved by electrophoresis on SDS-polyacrylamide gels and stained with Coomassie brilliant blue to demonstrate that equivalent amounts of various GST-Nef proteins were used in the binding reactions and to visualize the presence of ζ_(52-161) peptide in appropriate complexes (lanes 5, 7, and 9).

FIG. 8.

Models for the cooperative assembly of heterotrimeric complexes containing SIV Nef, AP-2, and the CD3-ζ subunit of the TCR-CD3 complex as cargo. (A) A likely model for the induction of TCR-CD3 endocytosis by Nef. SIV Nef and the CD3-ζ cytoplasmic domain bind with high affinity to form a stable 239.Nef-CD3-ζ complex. Complexed together, 239.Nef and CD3-ζ produce a high-affinity binding site for AP-2. This complex is competent to recruit CD3-ζ to clathrin-coated pits. Connector model (B) and cooperative binding model (C) for the recruitment of cell surface receptors by Nef to the AP-2 clathrin adaptor. A connector model in which Nef contacts AP-2 and the target site in the membrane receptor, such as CD4, CD28, or CD3-ζ, by independent interactions is illustrated in panel B. Data from the studies on the mechanism of CD3-ζ recruitment to the AP-2 by SIV mac239 Nef provide strong support for an alternative model called a cooperative binding model, in which Nef and the target receptor bind cooperatively AP-2, as shown in panel C.