Mechanism for down-regulation of CD28 by Nef
2001; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês
10.1093/emboj/20.7.1593
ISSN1460-2075
Autores Tópico(s)T-cell and B-cell Immunology
ResumoArticle2 April 2001free access Mechanism for down-regulation of CD28 by Nef Tomek Swigut Tomek Swigut Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Nadim Shohdy Nadim Shohdy Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Jacek Skowronski Corresponding Author Jacek Skowronski Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Tomek Swigut Tomek Swigut Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Nadim Shohdy Nadim Shohdy Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Jacek Skowronski Corresponding Author Jacek Skowronski Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Search for more papers by this author Author Information Tomek Swigut1, Nadim Shohdy1 and Jacek Skowronski 1 1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1593-1604https://doi.org/10.1093/emboj/20.7.1593 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event. Introduction Nef, a multifunctional regulatory protein of human and simian immunodeficiency viruses (HIV and SIV), is required for optimal virulence in vivo (Kestler et al., 1991; Deacon et al., 1995; Alexander et al., 1999; Kirchhoff et al., 1999). Nef increases virion infectivity and the replication of SIV and HIV viruses in primary T cells and model cell lines (Spina et al., 1994; Alexander et al., 1997). Nef has multiple independent effects on normal T-cell function: it modulates signal transduction pathways in T cells; it disrupts the sorting of proteins important for antigen-specific responses in major histocompatibility complex (MHC) class II-restricted T cells; and it compromises the antiviral response of the host to HIV/SIV-infected cells (for recent reviews see Oldridge and Marsh, 1998; Collins and Baltimore, 1999; Piguet et al., 1999; Skowronski et al., 1999; Renkema and Saksela, 2000). Nef disrupts several aspects of the T-cell receptor (TCR) machinery in CD4-positive T cells. It accelerates the endocytosis of the CD4 co-receptor (Garcia and Miller, 1991) by promoting the recruitment of CD4 to the AP-2 clathrin adaptor at the cell membrane (for recent reviews see Oldridge and Marsh, 1998; Piguet et al., 1999; Skowronski et al., 1999). Both HIV-1 and SIV Nefs interact directly with the AP-2 adaptor complex, and this interaction is mediated in HIV-1 Nef by a di-leucine sorting signal located in the C-terminal disordered loop of the Nef molecule (Bresnahan et al., 1998; Craig et al., 1998; Greenberg et al., 1998a), and in SIV Nef by two non-di-leucine-based elements located in the N-terminal loop of the molecule (Piguet et al., 1998; Lock et al., 1999). HIV-1 Nef also interacts directly with CD4 (Grzesiek et al., 1996; Hua and Cullen, 1997), possibly forming a ternary complex that stabilizes the normal interaction between the di-leucine-based sorting signal in the CD4 cytoplasmic domain and the AP-2 clathrin adaptor. Nef proteins also disrupt normal TCR-initiated signaling by interfering with the CD3-TCR complex (Iafrate et al., 1997). HIV-1 Nef blocks a membrane-proximal event in the CD3-TCR signaling cascade (Luria et al., 1991; Iafrate et al., 1997), while SIV Nef interacts directly with the ζ-subunit of CD3 and down-regulates expression of the CD3-TCR complex at the cell surface by an as yet unknown pathway (Bell et al., 1998; Howe et al., 1998; Swigut et al., 2000). The observation that HIV-1 and SIV Nefs use different mechanisms to disrupt TCR signaling and to down-regulate CD4 expression is evidence that Nef-mediated disruption of antigen-specific signaling in infected T cells provides a distinct survival advantage for the virus. Additional effects of Nef involve the modulation of several effectors and signaling pathways in T cells such as PAK and PKC kinases (Lu et al., 1996; Smith et al., 1996), activation of NFAT1 (Manninen et al., 2000) and modulation of calcium signaling (Skowronski et al., 1993; Baur et al., 1994). The biological consequences and roles of these effects are not clear. Nef down-regulates expression of class I MHC at the cell surface, and may thereby compromise immune surveillance of HIV/SIV-infected cells (Schwartz et al., 1996). The abnormally low class I MHC expression on the surface of HIV-1-infected cells increases the probability that these cells will not be detected and eliminated by cytotoxic T cells that recognize epitopes derived from viral proteins (Collins et al., 1998). Decreased class I MHC expression on the surface of Nef-expressing cells reflects the accelerated endocytosis of MHC (Schwartz et al., 1996) and sorting to a Golgi subcompartment that contains the AP-1 clathrin adaptor (Greenberg et al., 1998a; Le Gall et al., 1998). Nef-induced class I MHC down-regulation possibly involves an interaction between Nef and the trans-Golgi network (TGN) sorting protein PACS-1 (Piguet et al., 2000), but does not appear to require Nef's interaction with AP-2 clathrin adaptor (Greenberg et al., 1998a; Lock et al., 1999; Le Gall et al., 2000). Notably, induction of CD4 endocytosis by Nef requires Nef's ability to interact with the AP-2 (Greenberg et al., 1998a,b; Lock et al., 1999), but not with PACS-1. Thus, Nef probably exploits distinct sorting pathways to induce CD4 and class I MHC endocytosis. Here we report that HIV-1 as well as SIV Nef proteins down-regulate cell surface expression of the CD28 molecule. The CD28-initiated co-stimulatory signal is critical for normal antigen-specific T-cell responses, and interference with the CD28 signaling pathway could result in suppression of the immune response and anergy (reviewed by Schwartz, 1992; Lenschow et al., 1996). CD28 down-regulation by Nef involves direct molecular interactions between Nef and CD28 molecules. Genetic and functional evidence suggests that the mechanism that Nef uses to down-regulate CD28 expression is similar to that used to down-regulate CD4, since in both instances Nef accelerates the rate of endocytosis via the AP-2 clathrin adaptor pathway. The likely consequence of the concerted disruption of antigen-specific signaling machineries in HIV-1-infected T cells by Nef is to uncouple T-cell activation from the antigen-specific interactions of T cells with antigen-presenting cells (APCs) and thus facilitate the spread of the infected T cells. Results Nef accelerates the rate of CD28 endocytosis A transient expression assay in human Jurkat T cells was used to study the effect of HIV-1 and SIV Nef proteins on CD28 expression at the cell surface (Iafrate et al., 1997). Jurkat T cells were transfected with plasmids expressing Nef and a green fluorescent protein (GFP) reporter from the same bi-cistronic transcription unit, and CD28 cell surface expression and GFP expression were detected by flow cytometry. As shown Figure 1A, expression of SIV Nef from the pathogenic SIV mac239 strain resulted in a dose-dependent decrease in the steady-state CD28 surface expression, with a maximal 10-fold decrease in cells expressing the highest levels of GFP (compare panel 3 with panel 1). This result confirmed an earlier observation that the SIV mac239 nef allele can decrease CD28 surface expression (Bell et al., 1998). Expression of a natural HIV-1 Nef protein encoded by the NA7 allele (Mariani and Skowronski, 1993) also resulted in a similar decrease in the steady-state level of CD28 on the cell surface (panel 2). Figure 1.Nef down-regulates surface CD28 expression. (A) Dose-response analysis of surface CD28 antigen down-regulation by HIV-1 and SIV Nef proteins. Jurkat T cells were transfected with 10 μg of the bi-cistronic vectors expressing HIV-1 Nef (panel 2) or SIV Nef (panel 3) and GFP reporter, and a control vector expressing GFP alone (panel 1). Surface CD28 and GFP were detected simultaneously by two-color flow cytometry and are shown on the logarithmic scale on the ordinate and abscissa, respectively. (B) CD28 internalization is accelerated by HIV-1 and SIV Nef proteins. The percentage fraction of CD28 molecules internalized in Jurkat T cells expressing HIV-1 Nef (squares) or SIV Nef (triangles) together with GFP, or a control vector expressing GFP alone (circles), determined as described in Materials and methods, is shown as a function of time. Download figure Download PowerPoint We next measured the rates of CD28 endocytosis in Jurkat T cells transiently expressing Nef and GFP, or GFP alone as a control. Cells were reacted with anti-CD28 monoclonal antibody (mAb) labeled with phycoerythrin (PE), and the rates of CD28 internalization in Nef-expressing cells and in control cells were determined for populations of cells showing identical levels of GFP fluorescence using a flow cytometry-based endocytosis assay (Greenberg et al., 1997). As shown in Figure 1B, expression of SIV or HIV-1 Nef proteins resulted in an ∼5-fold increase in the rate of CD28 internalization over that seen with cells expressing GFP alone. Thus, the accelerated endocytosis of CD28 is probably responsible for down-regulation of CD28 expression on the surface of Nef-expressing cells. Mutations that abolish the interaction of Nef with the AP-2 clathrin adaptor disrupt CD28 down-regulation To address how Nef induces CD28 endocytosis, we determined which surfaces of SIV mac239 Nef protein (239.Nef) are required for this effect. Previous studies have revealed that Nef uses different surfaces and mechanisms to induce endocytosis of CD4 and class I MHC, and that defined mutations in Nef disrupt molecular interactions selectively required for these different functions (Iafrate et al., 1997; Lock et al., 1999; Le Gall et al., 2000; Swigut et al., 2000). We tested the effects of these Nef mutations on down-regulation of CD28 in a transient expression assay in Jurkat T cells. We first tested mutations that disrupt the interaction of SIV Nef with the AP-2 clathrin adaptor. As shown in Figure 2 and Table I, one such mutation in 239.Nef, namely deletion of amino acids Gln64-Asn67 [239(Δ64–67)], abolished CD28 down-regulation. This deletion disrupts the N-distal element in the N-terminal region of 239.Nef that mediates interaction with the AP-1/AP-2 clathrin adaptors and that is required for down-modulation of CD4 cell surface expression by Nef (Lock et al., 1999). Since HIV-1 Nef and SIV Nef are related proteins but use different surfaces to interact with the clathrin adaptors (Bresnahan et al., 1998; Lock et al., 1999; Skowronski et al., 1999; Swigut et al., 2000), we performed the same studies with HIV-1 Nef. HIV-1 Nef requires Leu164 and Leu165 to bind to clathrin adaptors (Bresnahan et al., 1998; Greenberg et al., 1998a). As shown in Table I, alanine substitutions for Leu164 and Leu165 [NA7(LL164AA)] disrupted the ability of HIV-1 Nef to down-regulate CD28 surface expression. Thus, both HIV-1 and SIV Nef are likely to induce CD28 endocytosis via a pathway involving the AP-2 clathrin adaptor. Figure 2.Effect of mutations in 239.Nef that disrupt CD4, CD3 and class I MHC down-regulation from the cell surface on CD28 down-regulation. Jurkat T cells were transfected with 10 μg of bi-cistronic plasmids expressing wild-type or mutant 239.Nef proteins and GFP reporter, or a control vector expressing GFP alone. Surface CD28, class I MHC, CD4 or CD3 and GFP were detected simultaneously by two-color flow cytometry and are shown in logarithmic scale as indicated. Download figure Download PowerPoint Table 1. Effect of mutations in SIV and HIV-1 Nef on their ability to down-regulate surface CD3, CD4 and class I MHC expression Nef allele Down-regulation of CD4 CD28 MHC I SIV 239.Nef +++ +++ +++ 239(Δ23–74) - - ++++ 239(Δ23–43) +++ ++ +++ 239(Δ42–45) +++ +++ +++ 239(Δ46–49) +++ +++ +++ 239(Δ54−57) +++ +++ +++ 239(Δ64–67) - - +++ 239(D155L) +++ +++ - 239(D204R) - - +++ 239(G238*) +++ +++ - 239(Δ96–103) ++ ++++ + HIV-1 NL43 +++ +++ +++ NL43(WL57AA) - - +++ NL43(WLE57AAA) - - +++ NL43(E59A) +++ +++ +++ NL43(R106A) ++ ++ ++ NA7(LL164AA) - - +++ The relative ability of mutant SIV Nef and HIV-1 Nef proteins to down-regulate CD4, CD28 and class I MHC expression is shown. All determinations were performed as shown in Figure 2. −, 90% of the activity seen with wild-type 239.Nef. CD4 and CD28 down-regulation are related functions of Nef We then tested additional mutations that selectively disrupt other Nef functions. As shown in Figure 2 and summarized in Table I, mutations in the C-terminal region of 239.Nef that disrupt interactions required for the down-regulation of class I MHC and have some negative effect on the down-regulation of CD3 surface expression, such as a deletion of the C-terminal region of the 239(G238*), mutation of Asp155 to leucine [239(D155L)] or mutation of Leu20 and Leu21 to alanines [239(LL20AA)], had no detectable effect on CD28 down-regulation. In contrast, mutation of Asp204 to arginine in 239.Nef, which disrupts down-regulation of CD4 expression without affecting the ability of 239.Nef to interact with the AP-1/AP-2 clathrin adaptors [239(D204R); Lock et al., 1999; Iafrate et al., 2000], abolished 239.Nef's ability to down-regulate CD28 surface expression. These observations suggest that induction of CD28 and CD4 endocytosis could share a common molecular interaction in addition to that with the AP-2 clathrin adaptor. Nef also interacts directly with an element in the cytoplasmic domain of CD4. The putative surface of HIV-1 Nef required for this interaction was defined previously by solution NMR in the presence of CD4 cytoplasmic domain peptides, and is comprised of Trp57, Leu58, Glu59 and Arg106 (Grzesiek et al., 1996). We therefore tested the effects of substitutions at these residues on CD28 and CD4 down-regulation by HIV-1 Nef. As summarized in Table I and shown in Figure 3A, mutation of Glu59 or Arg106 alone had little detectable effect on the ability of Nef to down-regulate CD28 and CD4 expression, indicating that these residues contribute little to the interaction with CD4 and CD28. Triple alanine substitution of Trp57, Leu58 and Glu59 [NL43(WLE57AAA)] disrupted both CD4 and CD28 down-regulation. Also, a double alanine substitution of Trp57 and Leu58 had a similar effect [NL43(WL57AA); see Table I and Figure 3A]. Thus, HIV-1 Nef probably uses similar and overlapping surfaces to interact with CD4 and CD28, which probably contact determinants other than di-leucine residues in CD4 and CD28 cytoplasmic domains. Figure 3.(A) Mutations of the putative CD4-binding surface in HIV-1 Nef have similar effects on CD4 and CD28 down-regulation. Jurkat T cells were transfected with bi-cistronic vectors expressing wild-type HIV-1 Nef protein, or HIV-1 Nef with mutations of amino acid residues that form the putative CD4-binding site, and GFP reporter. Histograms of CD28 and CD4 cell surface expression on populations of cells with identical GFP fluorescence recorded by two-color flow cytometry are shown. (B) Mutation in SIV 239.Nef separates CD4 and CD28 down-regulation. Experiments were performed as described in (A). Download figure Download PowerPoint Mutation separates the effect of SIV Nef on CD4 and CD28 expression To assess whether the effects of Nef on CD4 and CD28 expression are separable functions, we screened a large number of previously characterized mutations in HIV-1 and SIV Nefs (data not shown). In this search, we found that deletion of amino acids 96–103 in 239.Nef enhances CD28 down-regulation but suppresses CD4 down-regulation [239(Δ96–103); Table I and Figure 3B). The separation of CD4 and CD28 down-regulation by the Δ96–103 deletion in SIV Nef is evidence that CD28 and CD4 down-regulation are genetically separable functions, even though both link to the AP-2 clathrin adaptor pathway. Evidence for direct interaction of Nef with the CD28 cytoplasmic domain To understand further the interactions between Nef, CD28 and the AP-2 clathrin adaptor, we studied the amino acid residues in CD28 required for constitutive and Nef-induced endocytosis. First, we tested the effect of deletions in the CD28 cytoplasmic domain on the rate of constitutive CD28 endocytosis. To permit an accurate and sensitive comparison of the wild-type and mutant CD28 proteins, we constructed a vector co-expressing CD28 and GFP, which is used as a marker of transfected cells, from the same bi-cistronic transcription unit. This design results in a constant ratio of CD28 and GFP, thus allowing reliable comparison of the properties of the wild-type and mutant CD28 proteins. HeLa cells were transiently transfected with bi-cistronic plasmids co-expressing wild-type or mutant forms of CD28 together with GFP. The rates of endocytosis of different forms of CD28 were determined for populations of cells showing identical GFP fluorescence. As summarized in Figure 4A, column 1, and shown in Figure 4B, deleting all but the first four membrane-proximal amino acids of the CD28 cytoplasmic domain (CD28.R185*) decreased the rate of constitutive CD28 endocytosis by ∼5-fold. A truncation at Asn193 (CD28.N193*) showed a wild-type level of constitutive endocytosis, suggesting that important sorting signals are located between Arg185 and Asn193. Next, the effects of amino acid substitutions for amino acid residues located proximal to Asn193, including two putative sorting signals located in this region, namely Tyr191 and Leu186 and Leu187, were tested. The Y191A substitution disrupted constitutive CD28 endocytosis to an extent similar to that seen with the R185* deletion (CD28.Y191A). In contrast, substitutions of Leu186 and Leu187 (CD28.LL186AA), or other amino acid residues in this membrane-proximal region, had little effect (CD28.D190A and CD28.S189A). Thus, Tyr191 is critical for the normal interaction of CD28 with the endocytic machinery in HeLa cells. These observations are consistent with previous data from experiments with T cells (Cefai et al., 1998). Figure 4.Summary of mutations in CD28 and their effects on CD28 endocytosis. (A) Alignment of amino acid sequences of a wild-type and mutant CD28 cytoplasmic domains is shown. Dots indicate amino acid identity with the wild-type protein, letters identify amino acid substitutions in the single letter code and the asterisks reflect stop codons. The nomenclature of mutant CD28 proteins is shown on the left. The relative ability of mutant CD28 molecules to undergo endocytosis in the absence or presence of HIV-1 or SIV Nef proteins is indicated on the right. (B) Effect of mutations in the CD28 cytoplasmic domain on steady-state cell surface expression and CD28 endocytosis. CD28-negative HeLa cells were co-transfected with plasmids expressing wild-type, or mutant CD28 proteins and GFP from the same bi-cistronic transcription unit, and a plasmid expressing HIV-1 Nef (NA7 allele) or SIV Nef (mac239), or an empty control vector. Histograms of CD28 expression on cells co-transfected with an empty control vector (black line), or on cells co-expressing HIV-1 Nef (red line) or SIV Nef (blue line) are shown for populations of cells with identical levels of GFP expression and therefore comparable CD28 expression rates (left panels). The percentage fraction of CD28 internalized from the surface cells expressing CD28 alone, or co-expressing CD28 and HIV-1 Nef, or SIV Nef, as a function of time is also shown (right panels). The internalization rates were measured for populations of cells with identical levels of GFP expression. Download figure Download PowerPoint To assess whether the induction of CD28 endocytosis by Nef involves an interaction of CD28 with the sorting machinery, we determined the effect of the same set of mutations on CD28 endocytosis induced by HIV-1 Nef and SIV Nef. HeLa cells were co-transfected with bi-cistronic plasmids expressing wild-type or mutant CD28 molecules together with GFP and with a plasmid expressing HIV-1 Nef or SIV Nef proteins. The effect of Nef on cell surface expression and the rate of endocytosis of different forms of CD28 was determined for a population of cells with identical GFP expression. As summarized in Figure 4A, columns 2 and 3, and shown in Figure 4B, deletion of the cytoplasmic domain abolished the accelerated rate of CD28 endocytosis induced by either SIV or HIV-1 Nef (CD28.R185*). In contrast, deletions of sequences distal to Ser189 had no detectable effect on the rate of endocytosis seen with HIV-1 Nef, and only a minor negative effect on that seen with SIV Nef. Notably, the Y191A substitution, which disrupted constitutive CD28 endocytosis, had little effect on endocytosis induced by SIV and HIV-1 Nefs. Interestingly, mutations of Leu186 and Leu187 to alanines disrupted the effect of SIV Nef, but had only a marginal effect on HIV-1 Nef-induced or constitutive CD28 endocytosis. The observation that the amino acid residues required for the induction of CD28 endocytosis by Nef are different from those critical for constitutive CD28 endocytosis suggests that Nef modifies normal interactions between the CD28 cytoplasmic domain and the endocytic machinery. Furthermore, the observation that the effects of HIV-1 and SIV Nef require different amino acid residues in the CD28 cytoplasmic tail suggests that these two Nef molecules make direct but different contacts with CD28, and that these interactions are critical for the acceleration of the CD28 endocytosis rate. Nef redistributes CD28 to the AP-2 clathrin adaptor and to endosomes Evidence from genetic experiments predicted that Nef is likely to interact with the AP-2 clathrin adaptor to facilitate the recruitment of CD28 to the endocytic machinery in a manner similar to that proposed previously for CD4 (reviewed in Piguet et al., 1999; Skowronski et al., 1999). To test this model, we studied the cellular localization of CD28 in cells expressing 239.Nef. We used a chimeric protein comprised of 239.Nef joined at the C-terminal end to a strongly fluorescing variant of the GFP reporter (239-GFP) to visualize 239.Nef directly (Lock et al., 1999). As shown in Figure 5A, all Nef functions necessary for the down-regulation of CD28 surface expression are retained in this fusion protein. Figure 5.CD28 co-localizes with the 239-GFP fusion and AP-2 clathrin adaptor in IMR90 fibroblasts. (A) 239-GFP down-regulates CD28 expression on the cell surface. Jurkat T cells were transfected with 10 μg of plasmids expressing GFP alone (left panel) or 239-GFP (right panel), and CD28 expression on the cell surface and GFP expression were analyzed by two-color flow cytometry. (B) CD28 co-localizes with 239-GFP. IMR90 fibroblasts co-transfected with plasmids expressing CD28 and GFP (panels 1–3) or with plasmids expressing CD28 and 239–GFP (panels 4–9) were fixed, and CD28 was detected with mAb CD28.2 and visualized by indirect immunofluorescence (panels 2, 3, 5, 6, 8 and 9). GFP and 239–GFP were revealed by direct fluorescence (panels 1, 3, 4, 6, 7 and 9). Panels 7–9 are magnifications of a fragment of the cell shown in panels 4–6, respectively. The overlays of GFP and CD28 images were produced using Oncor imaging software (panels 3, 6 and 9). The bar in the lower left corner of panel 1 represents a distance of ∼20 μm in panels 1–6 and 10–15, and ∼2 μm in the remaining panels. (C) CD28 co-localizes with AP-2 clathrin adaptor in cells expressing 239.Nef. IMR90 fibroblasts were transfected with a plasmid expressing CD28 (panels 10–12), or CD28 and 239.Nef (panels 13–18). Cells were fixed and the CD28 pattern was revealed by indirect fluorescence with mAb CD28.2 (panels 10, 12, 13, 15, 16 and 18). Subsequently, cells were permeabilized and the β-adaptin subunit of AP-1 and AP-2 clathrin adaptors was revealed by indirect fluorescence with mAb 100/1 (panels 11, 12, 14, 15, 17 and 18). The overlays of CD28 and β-adaptin patterns are also shown (panels 12, 15 and 18). Panels 16–18 are magnifications of images shown in panels 13–15, respectively. Download figure Download PowerPoint The relative distribution of CD28 and 239-GFP was studied in a CD28-negative human fibroblast line (IMR90) transiently expressing these proteins. As shown in Figure 5B, indirect immunofluorescence with a mAb specific for CD28 under conditions that block endocytosis revealed a uniform pattern of CD28 expression at the cell surface (panel 2). In contrast, in cells co-expressing CD28 and 239-GFP, CD28 was redistributed and displayed a characteristic punctate pattern at the cell surface (panel 5). Superposition of the 239-GFP and CD28 patterns revealed a large extent of co-localization (panels 6 and 9). In T cells and fibroblasts, Nef co-localizes with AP-2-containing clathrin coats (Greenberg et al., 1997, 1998a; Lock et al., 1999). To demonstrate directly that CD28 co-localizes with AP-2 coats in Nef-expressing cells, CD28 and AP-2 were visualized simultaneously by two-color fluorescent microscopy in IMR90 cells transiently co-expressing 239.Nef and CD28. As shown in Figure 5C, the CD28 fluorescent pattern co-localized with the AP-2 pattern in cells expressing 239.Nef (panels 15 and 18). In contrast, CD28 was distributed uniformly at the cell surface and was not detectable at AP-2 coats in the absence of 239-Nef (panels 10–12). The co-localization of CD28 with 239.Nef and AP-2 clathrin adaptor suggests that Nef redistributes CD28 to AP-2 coats at the plasma membrane. In Nef-expressing cells, class I MHC complexes internalized from the cell surface are redistributed to the TGN while the internalized CD4 is redistributed to endosomes and lysosomes (Schwartz et al., 1995; Greenberg et al., 1998b; Le Gall et al., 1998). Experiments were performed to determine which pathway is used with Nef-induced CD28 sorting from the plasma membrane. To visualize the internalized CD28, cells transiently co-expressing CD28 and 239.Nef, or expressing CD28 alone, were cultured in the presence of CD28.2 mAb (reacting with CD28). Fluorescein isothiocyanate (FITC)-labeled transferrin was also included in the culture medium to visualize the early compartments of the endocytic pathway. Cells were collected at various time points, fixed, permeabilized and the internalized CD28 was revealed by indirect fluorescence using Texas red (TxR)-labeled anti-mouse IgG antibody. Two-color fluorescence microscopy revealed rapid internalization of CD28 in cells expressing 239.Nef. Figure 6 shows that after 30 min of incubation with CD28.2 mAb and transferrin, the majority of cell surface CD28 redistributed into a punctate pattern similar to the pattern of transferrin (panel 13). Close examination of magnified images revealed a large extent of co-localization of the two patterns (compare panels 14–16). In cells expressing 239.Nef, the redistribution of CD28 and transferrin into punctate co-localizing patterns could be detected even as early as after 2 min culture with CD28.2 mAb (data not shown). In contrast, control cells expressing CD28 alone had a fairly uniform distribution of CD28. Furthermore, this uniform distribution persisted for >30 min, indicating much slower internalization of CD28 in the absence of Nef (compare panels 9 and 11 with 13 and 15). In contrast, transferrin was redistributed rapidly into a punctate pattern in both the absence and presence of Nef, which shows that the effect of Nef on CD28 sorting was specific. Finally, the co-localization of internalized CD28 and transferrin in early endosomes indicates that they are sorted along similar pathways. Figure 6.Localization of CD28 molecules internalized from the surface of Nef-expressing cells. IMR90 fibroblasts expressing CD28 alone (panels 1–4 and 9–12) or co-expressing CD28 and 239-Nef (panels 5–8 and 13–16) were cultured for 30 min in the presence of CD28.2 mAb and FITC-transferrin (time = 30; panels 9–16). As a control to reveal the initial distribution of CD28 and transferrin receptor at the cell surface, cells were
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