CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD4 Yong-Jiu Jin, Xiaoping Zhang, J. Gildade Boursiquot and Steven J. Burakoff This information is current as of September 24, 2021. J Immunol 2004; 173:5495-5500; ; doi: 10.4049/jimmunol.173.9.5495 http://www.jimmunol.org/content/173/9/5495 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD41

Yong-Jiu Jin,2* Xiaoping Zhang,‡ J. Gildade Boursiquot,* and Steven J. Burakoff*†

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (ϳ95%), whereas that induced by PMA was partial (ϳ40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell

membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef Downloaded from interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regu- lation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface. The Journal of Immunology, 2004, 173: 5495–5500.

D4 is a coreceptor of the TCR in TCR-mediated T cell down-regulation of CD4 (21, 22), as is the membrane association

activation and signaling. Cell surface CD4 can be down- of Nef through its N-terminal myristoylation (25). In Nef, W57 http://www.jimmunol.org/ regulated by PMA treatment (1, 2). PMA treatment ac- and L58 are the residues critical for the Nef-CD4 interaction (26, C 3 tivates protein kinase C (PKC), which, in turn, induces the phos- 27). Replacing the CD4 tail with Nef results in a chimeric protein phorylation of Ser408 and Ser415 in the cytoplasmic tail of CD4 (3, that down-regulates CD4 in trans as well as the chimeric protein 4). As a result, CD4 dissociates from Lck and is recruited into the itself in cis (23). Several studies have shown that CD4 interacts clathrin-coated pits for endocytosis (5, 6). After endocytosis, some directly with Nef in vitro and in insect cells and that the dileucine of the internalized CD4 molecules are delivered to lysozomes for motif in the CD4 tail is essential for the interaction (25, 26, 28, 29). degradation, whereas others recycle back to the T cell surface (1, However, the direct interaction between CD4 and Nef in mamma- 5). Therefore, PMA-induced down-regulation of CD4 is incom- lian cells has yet to be demonstrated (24). plete. In T cells, which express Lck, PMA treatment removes 40– Available data also indicate that Nef links CD4 to the clathrin- by guest on September 24, 2021 50% of CD4 molecules from the cell surface; in CD4-transfected coated pits for endocytosis, but how Nef does this remains con- HeLa cells, which do not express Lck, PMA removes 75% of the troversial. Nef contains a highly conserved dileucine motif (L164- surface-expressed CD4 molecules (4, 6). L165) in its C-terminal flexible loop, which is essential for Nef CD4 is also the primary receptor for HIV to enter cells. HIV Nef down-regulation of CD4 (30–32). The dileucine motif, including protein, expressed early in viral replication (7), is a major deter- the one in the CD4 tail (2), has the consensus sequence of minant of HIV virulence (8–11). One well-characterized Nef func- [DE]XXXL[LI]. The dileucine motifs are known sorting signals, tion is the down-regulaton of CD4 from the cell surface (12), recognizable by adaptor protein complexes, including AP-1, AP-2, which correlates with Nef-dependent enhancement of viral patho- genicity (13–20). AP-3, and AP-4 (33). The prevailing view is that the adaptor pro- The Nef down-regulation of CD4 is independent of the phos- tein-2 (AP-2) is involved in endocytosis from plasma membranes, phorylation of CD4 (12). The available data indicate that Nef whereas AP-1 and AP-3 are involved in the trans-Golgi network down-regulation of CD4 requires its interaction with CD4 on the T and lysosome vesicular trafficking, respectively (34). It has been cell plasma membrane (21–24). The CD4 tail is necessary for Nef shown that the colocalization of Nef with the AP-2 complex is correlated with Nef down-regulation of CD4 (35). However, yeast two- or three-hybrid studies show that the dileucine motif in HIV † *Skirball Institute of Biomedical Research, Department of Medicine, New York Nef interacts mainly with AP-1 and AP-3 and only weakly with University School of Medicine, New York, NY 10016; and ‡Department of Pharma- ceutics, College of Pharmacy, Rutgers University, Piscataway, NJ 08854 AP-2 (36, 37). A GST-tagged HIV Nef binds to AP-1, but not Received for publication June 29, 2004. Accepted for publication August 20, 2004. AP-2 (38). Apparently, CD4-Nef interacts with an AP complex, The costs of publication of this article were defrayed in part by the payment of page but the specific AP complex involved needs to be further defined. charges. This article must therefore be hereby marked advertisement in accordance In an effort to elucidate the differential pathways of CD4 inter- with 18 U.S.C. Section 1734 solely to indicate this fact. nalization induced by Nef and PMA, we found that Nef down- 1 This work was supported in part by grants from National Institutes of Health, the regulation of CD4 was partially reversed in Jurkat T cells by treat- Association for International Cancer Research (United Kingdom), and a pilot AIDS funding from New York University. ment of PMA. Further analysis suggested that phosphorylation of 2 Address correspondence and reprint requests to Dr. Yong-Jiu Jin, Skirball Institute CD4 induced by PMA disrupted the interaction between Nef and of Biomedical Research, 540 1st Avenue, Lab 5-1, New York University School of the CD4 tail. A model is proposed in which the Nef down-regu- Medicine, New York, NY 10016. E-mail address: [email protected] lation of CD4 is mediated through a CD4-Nef-AP interaction, 3 Abbreviations used in this paper: PKC, protein kinase C; AP, adaptor protein com- plex; BIM1, bisindolylmaleimide 1; DTSSP, 3,3-dithiobis[sulfosuccinimidyl-propi- whereas the PMA down-regulation of CD4 is through a direct onate]; wt, wild type. interaction of the phosphorylated CD4 with an AP complex. PMA

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 5496 REVERSIBILITY OF Nef-DOWN-REGULATION OF CD4 switches the connection of CD4 from the former route to the later, Results resulting in a net gain of CD4 on the T cell surface. PMA treatment partially restored the surface CD4 staining in T cells expressing Nef-GFP Materials and Methods Cells, Abs, and chemicals Because both Nef and PMA can down-regulate CD4, apparently by two independent mechanisms, we started this study by asking The Jurkat T cell line J77, a variant of clone E6-1 (from American Type the question whether Nef and PMA were synergistic in CD4 down- Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium regulation. We transfected Jurkat T cells with Nef-GFP. The cells containing 10% FBS at 37°C in a 5% CO2 humidified atmosphere. PE- conjugated anti-CD4 (Leu 3A) was purchased from BD Immunocytometry were previously stably transfected with wt CD4, 2C CD4 (a non- (San Diego, CA), and polyclonal anti-CD4 Ab was obtained from Santa Lck binding CD4), or tail-less CD4 as previously described (39). Cruz Biotechnology (Santa Cruz, CA). mAb OKT4D was prepared from a After PMA treatment, cells were surface-stained with the PE-con- hybridoma purchased from American Type Culture Collection. Anti-HIV-1 jugated anti-CD4 for two-color cytometric analysis (Fig. 1a). FL1 Nef rabbit serum was obtained from the National Institutes of Health AIDS Research and Reference Reagent Repository provided by Dr. R. Swan- shows the GFP fluorescence intensity of Nef-GFP expression; FL2 strom. The PKC inhibitor bisindolylmaleimide1 (BIM1) was purchased shows the CD4 surface staining. Without PMA treatment from Calbiochem (San Diego, CA), and 3,3-dithiobis[sulfosuccinimidyl- (ϪPMA), Nef-GFP down-regulated wt CD4 and 2C CD4 in a propionate] (DTSSP) was obtained from Pierce (Rockford, IL). dose-dependent manner. In cells expressing high levels of Nef- Generation of mutant CD4 and HIV Nef GFP (FL1, Ͼ500), CD4 was almost completely down-regulated. Unexpectedly, with PMA treatment (ϩPMA), the Nef down-reg- The construction and the stable expression of wild-type (wt) CD4, 2C CD4, ulated wt CD4 and 2C CD4 were partially restored to the cell and tail-less CD4 in Jurkat T cells have been described previously (39). Ͼ Downloaded from The serine mutant of CD4 used in this study was similarly constructed. The surface in Nef-GFP-positive cells (FL1 10; right to the vertical PCR product containing Ser to Ala substitutions at residues 408 and 415 line). In contrast, CD4 was down-regulated by PMA in Nef-GFP- was digested with SfoI/BamHI, and the resultant fragment was used to negative cells (FL1, Ͻ10). replace the corresponding wt CD4 fragment in wt CD4 cDNA originally For a better comprehension, Fig. 1b quantitatively compared the cloned in pcDNA3 vector (Invitrogen Life Technologies, Carlsbad, CA). The CD4 Ser mutant plasmid was then stably transfected into Jurkat T effects of PMA on the surface CD4 expression in Nef-positive or cells. Nef (NA7)-GFP plasmid was provided by Dr. J. Skowronski (Cold Nef-negative cells. The left panel shows the percentage of surface Spring Harbor Laboratory, Cold Spring Harbor, NY: Ref. 35). The non- CD4 expression in Nef-negative cells. PMA treatment reduced the http://www.jimmunol.org/ GFP-tagged Nef was constructed by PCR subcloning of Nef (NA7) into surface CD4 of wt CD4 and 2C CD4 by 40 and 75%, respectively pcDNA3 vector (Invitrogen Life Technologies). Nef , Nef , Nef - LL WL LL (converting the results in Fig. 1a from a log scale to a linear scale). GFP, and NefWL-GFP were generated by PCR mutagenesis using Nef or Nef-GFP as the template following the protocol of the QuikChange Site- The right panel shows the percentage of surface CD4 in Nef-pos- Directed Mutagenesis kit (Stratagene, La Jolla, CA). All mutations gener- itive cells. Without PMA treatment, Nef-GFP down-regulated wt ated in this study were confirmed by DNA sequencing. CD4 and 2C CD4 to 5 and 3% of the steady state level (medium PMA treatment and two-color cytometric analysis CD4 staining without PMA treatment). With PMA treatment, the surface wt CD4 and 2C CD4 were restored from 5 to 35% and For transient expression, 20 ␮g of double CsCl gradient-purified plasmid

from 3 to 20%, respectively. The tail-less CD4 was not down- by guest on September 24, 2021 DNA was electroporated into 5 ϫ 106 CD4 T cells at 800 ␮F and 250 V. Sixteen to 18 h after transfection, cells were treated with 100 ng/ml PMA regulated by PMA treatment or by Nef expression (Fig. 1, a and b), in RPMI 1640 medium containing 1 mg/ml BSA at 37°C for 15 min. For in agreement with the observations by others that CD4 tail was CD4 staining, cells were incubated with PE-conjugated anti-CD4 Ab di- necessary for the PMA-induced (3) and the Nef-dependent CD4 luted 1/100 in PBS on ice for 45 min. Cells were then subjected to FACS down-regulation (21, 22). The surface expression of tail-less CD4 using a FACScan (BD Biosciences, Mountain View, CA). In FACS anal- ysis, vertical and horizontal lines were drawn to demarcate CD4- and Nef- in Nef-expressing cells was also not affected by PMA treatment, GFP-positive and -negative cell populations. The line settings were based suggesting that the CD4 tail may be the part in CD4 that partici- on the cell surface staining intensities of CD4 and GFP, which varied pated in the restoration. somewhat among transfected cell lines. The relative cell surface CD4 ex- The dose of PMA and the time required for the restoration were pression (percentage) was the ratio of the medium CD4 staining of PMA- determined (Fig. 1c). PMA at 10–20 ng/ml was sufficient, and the treated cells (ϩPMA) to that of PMA-untreated cells (ϪPMA). The 100% level (steady state) was the CD4 expression in mock-transfected control process started right after the addition of PMA and completed cells without PMA treatment. Each value was the average of three inde- within 10 min, with kinetics similar to those of PMA activation pendent experiments (mean Ϯ SD). of PKC. Subcellular fractionation, chemical cross-linking, and immunoblotting Partial restoration of surface CD4 by PMA may require the phosphorylation of CD4 at Ser408 and Ser415 The subcellular fractionation of Jurkat cells and the immunoblotting were 408 conducted as previously described (39). After washing with PBS, cells It is known that the PMA-induced phosphorylation of Ser and were incubated in hypotonic buffer (10 mM Tris (pH 7.5) and 330 mM Ser415 in the CD4 tail is necessary for the down-regulation of CD4 sucrose) for 10 min on ice. Cells were then passed 10 times through a (3, 4). To determine whether the phosphorylation of Ser408 and 30-gauge needle while on ice. The extract was centrifuged at 250 ϫ g for 415 10 min to remove the nuclei and intact cells. To separate the cytosolic Ser was also involved in the partial restoration of the surface fraction from membrane, the postnuclear supernatant was centrifuged at CD4 in Nef-expressing T cells, we generated a serine CD4 mutant 50,000 rpm for 1 h using a TL-100 centrifuge and a TLS-55 rotor (Beck- (Ser CD4), in which Ser408 and Ser415 were replaced by alanine. T man Coulter, Fullerton, CA). cells stably expressing Ser CD4 were transfected with Nef-GFP. 7 For chemical cross-linking, the membrane fraction from 10 T cells was Serine mutation did not affect the Nef-induced down-regulation of resuspended in 100 ␮l of PBS, to which 100 ␮l of 2 mM DTSSP (Pierce) freshly made in PBS was added. Cross-linking reactions were allowed to CD4 (Fig. 2a), in agreement with the previous report (12). The proceed for 15 min on ice and terminated by the addition of 1 ml of 1% extent of the down-regulation of Ser CD4 by Nef-GFP was similar Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris to that of wt CD4 (Fig. 2a, left panel). When treated with PMA (pH 7.4), 0.5% sodium deoxycholate, 50 mM NaF, and 1 mM PMSF). The (Fig. 2a, right panel), however, unlike wt CD4, the surface stain- lysates were used in immunoprecipitation and immunoblotting as previ- ously described (39). Samples were boiled in SDS sample buffer without or ing of Ser CD4 was not restored. Moreover, the restoration of the with 1% ␤-ME (Sigma-Aldrich, St. Louis, MO) for 4 min before loading surface staining of wt CD4 by PMA was abolished when Jurkat T to the gel for SDS-PAGE. cells were preincubated with a PKC inhibitor, BIM1, at 50–100 The Journal of Immunology 5497 Downloaded from http://www.jimmunol.org/

FIGURE 2. The phosphorylation of CD4 at Ser408 and Ser415 is required for the restoration of the surface CD4 in Nef-GFP-expressing T cells. a, Restoration of wt CD4 and Ser CD4 in cells treated with PMA. Cells

expressing wt CD4 or Ser CD4 were transfected with Nef-GFP for 18 h. by guest on September 24, 2021 After PMA treatment, cells were stained with CD4-PE for FACS. b, Inhi- bition of the CD4 restoration by PKC inhibitor BIM1. Nef-GFP-transfected cells were incubated with BIM1 at different concentrations for 5 min at 37°C. Cells were then treated with PMA and stained with CD4-PE. The percentage of CD4 staining was calculated from the FACS results as de- scribed in Fig. 1b.

FIGURE 1. PMA treatment partially restored the surface CD4 expres- sion in Jurkat T cells expressing Nef-GFP. Jurkat T cells stably expressing sembly step of the CD4-Nef-AP complex. Therefore, we examined wt CD4, 2C CD4, or tail-less CD4 were transfected with Nef-GFP. Eigh- the effects of PMA on CD4 down-regulation in cells expressing teen hours after transfection, cells were treated with PMA and then stained Nef-GFP mutants defective for linking CD4 to the AP complex. In with anti-CD4 PE for FACS. a, Two-color cytometric analysis of the sur- NefWL-GFP mutant, residues W57 and L58 essential for Nef in- face CD4 staining. The x-axis (FL1-H) shows the fluorescence intensity of teraction with CD4 were substituted by alanines. In NefLL-GFP Nef-GFP; the y-axis (FL2-H) shows the CD4-PE staining. See Materials mutant, the dileucine motif (L164-L165) essential for Nef inter- and Methods for the line settings in FACS. b, Effects of PMA on relative action with an AP complex was similarly substituted. FACS anal- surface expression of CD4 in Nef-GFP positive cells (FL1, Ͼ10) or Nef- ysis indicated that, in contrast to wt Nef-GFP (Fig. 3, upper left GFP negative cells (FL1, Ͻ10). The percentage is calculated from the FACS results shown in a as described in Materials and Methods. c, Time panel), NefWL-GFP and NefLL-GFP did not down-regulate CD4 course and dose dependence of the surface CD4 staining treated with PMA. (Fig. 3, middle left and lower left panels), excluding a few highly Ͼ Nef-GFP-transfected cells were treated with different doses of PMA for 20 NefWL-GFP-positive cells (FL1, 500) where there was a low min at 37°C or treated with 100 ng/ml PMA for different time periods. The level of CD4 down-regulation. Unlike the case with wt Nef-GFP percentage of CD4 staining was calculated from the FACS results. (upper right panel), PMA treatment did not increase surface CD4

that was not down-regulated in cells expressing NefWL-GFP and NefLL-GFP (Fig. 3, middle right and lower right panels), exclud- nM (Fig. 2b). The results thus suggested that the PMA-induced ing a few highly NefWL-GFP-positive cells where the surface CD4 phosphorylation of Ser408 and Ser415 was required for the resto- staining was partially restored. The results indicated that only ration of the surface CD4 in Nef-GFP-expressing T cells. when CD4 was down-regulated by Nef-GFP was PMA able to partially restore the surface staining of CD4, suggesting that PMA PMA triggered a reversal of the Nef down-regulation of CD4 triggered a reversal of the Nef down-regulation of CD4, probably A simple explanation of the partial restoration of CD4 by PMA is by affecting the CD4-Nef interaction, because PMA-induced phos- the reversal of Nef down-regulation of CD4, possibly at the as- phorylation of the CD4 tail may alter its conformation (3, 4). 5498 REVERSIBILITY OF Nef-DOWN-REGULATION OF CD4 Downloaded from

FIGURE 3. Effects of the expression of NefLL and NefWL mutants on

PMA-induced CD4 internalization. Cells expressing wt CD4 were trans- http://www.jimmunol.org/ fected with Nef-GFP, NefLL-GFP, and NefWL-GFP for 18 h; treated with PMA; then stained with CD4-PE for FACS.

Cell fractionation and chemical cross-linking showed that PMA treatment disrupted the interaction between CD4 and Nef To determine whether PMA-induced phosphorylation of the CD4 tail disrupts the CD4-Nef interaction, we set out to show that there was an association between CD4 and Nef in T cells. Before our by guest on September 24, 2021 study, attempts to coimmunoprecipitate wt Nef and CD4 in sam- ples from mammalian cells had failed (24). Therefore, we tried to coimmunoprecipitate CD4 and Nef , because Nef interacts LL LL FIGURE 4. Effects of PMA on membrane distribution and chemical with, but does not down-regulate, CD4. To our disappointment, cross-linking of CD4 and Nef mutants. Cells were transfected with non- like wt Nef, NefLL too was not coimmunoprecipitated with CD4 tagged wt Nef (WT), NefLL(LL), or NefWL(WL) or were mock-transfected (complete data not shown; cf., Fig. 4b, lane 2). We then resorted (Ϫ), treated with PMA, and then fractionated into membrane and cytosolic to cell fractionation and chemical cross-linking. Cells were co- fractions. a, Anti-CD4 and anti-Nef immunoblotting of cytosol and mem- transfected with constructs expressing GFP and each of the three brane fractions. The amounts of cytosol and membrane fractions repre- non-GFP-tagged Nef (wt Nef, NefLL, and NefWL). The use of the sented 1/30th and 1/6th of each treated cells, respectively. Cells were ex- nontagged Nef was necessary to avoid a possible nonspecific pressing wt CD4. After protein transfer, the polyvinylidene difluoride cross-linking between CD4 and the GFP-tag. Approximately 15– membrane was cut into two pieces. The top part was immunoblotted with 20% of the cells expressed Nef according to the expression of the polyclonal anti-CD4 (top panel); the bottom was immunoblotted with anti- Nef rabbit antiserum (bottom panel). b, The membrane fractions were cotransfected GFP (data not shown). The cells were then fraction- cross-linked by DTSSP, except in lane 2, and then lysed in Nonidet P-40 ated into membrane and cytosol fractions. The immunoblotting lysis buffer. After immunoprecipitation with anti-CD4, the samples were analysis showed that CD4 and all three Nef were predominantly immunoblotted with anti-CD4 or anti-Nef as indicated. Cells were mock- membrane-associated (Fig. 4a, ϪPMA). Anti-CD4 blotting (Fig. transfected (lane 1), transfected with NefLL (lanes 2–7, 8, 10, 12, and 14) 4a, upper panel) showed that the membrane-associated CD4 was or with wt Nef (lanes 9, 11, 13, and 15). T cells expressed wt CD4 (lanes ϳ20% less in cells transfected with wt Nef (lane 6) than in cells 1–3 and 5–11), tail-less CD4 (lane 4), or Ser CD4 (lanes 12–15). Twice the mock-transfected (lane 5), NefLL-transfected (lane 7), or NefWL- amount of membrane from PMA-treated wt CD4 cells (lanes 11 and 12)as transfected (lane 8; analyzed by densitometry). Considering that from NefLL was immunoprecipitated with anti-CD4. Samples were run in only 15–20% cells expressed Nef, the results indicated that CD4 nonreducing conditions, except in lane 7. c, Anti-Nef immunoblotting of was down-regulated by wt Nef, but not by Nef or Nef ,in the nontagged Nef protein (lanes 2–4) and Nef-GFP protein (lanes 5–7). LL WL Cells (107) were transfected with 20 ␮g of plasmid DNA and lysed in 100 agreement with the FACS results with expression of GFP-tagged ␮l of Nonidet P-40 lysis buffer. Lysates (25 ␮l) was resolved by SDS- Nef (Fig. 3). PMA treatment increased the CD4 level in the cytosol PAGE, then immunoblotted with anti-Nef. fractions (Fig. 4a, lanes 9–12 vs lanes 1–4) and decreased the CD4 level in the membrane fractions (Fig. 4a, lanes 5–8 vs lanes 13–16). Also, PMA treatment attenuated the difference in mem- the results by FACS (Fig. 1). PMA treatment essentially did not brane-associated CD4 amounts between cells transfected with wt affect the membrane distribution of Nef, including wt Nef, NefLL, Nef and Nef mutants (lanes 13–16). The results suggested a re- and NefWL (Fig. 4a, bottom panel). At least 3–4 times more NefLL versal of Nef down-regulation of CD4 by PMA in agreement with (lanes 3, 7, 11, and 15) than wt Nef (lanes 2, 6, 10, and 14) and The Journal of Immunology 5499

NefWL (lanes 4, 8, 12, and 16) was detected, even though cells each other in T cells, and the Ser phosphorylation of CD4 dis- were transfected with equal amounts of different plasmid DNAs. rupted the interaction. For chemical cross-linking experiments, we used the membrane It has been shown that the CD4-associated Nef binds to the fraction only because the CD4-Nef interaction occurred on the T ␤-coatomer protein on endosomes, targeting CD4 for lysosomal cell membrane (25). In addition to the membrane fraction from the degradation (40). As a result, Nef prevents down-regulated CD4 wt Nef-transfected cells, we used the membrane fraction from the molecules from recycling back to the cell surface. In the absence ␤ ϳ NefLL-transfected cells, because among wt Nef and two Nef mu- of Nef- -coatomer protein interaction, 45% of internalized CD4 tants, it was expressed at the highest level, and it was defective in molecules recycled back to the cell surface within 10 min (40). Nef-AP interaction, which would make the interpretation of results This explains why the magnitude of CD4 down-regulation by Nef less complicated than for the wt Nef. The wt Nef and NefLL were is more complete than that by PMA. In this study, for instance, transfected into cells expressing wt CD4, tail-less CD4, or Ser CD4 down-regulation by Nef was nearly complete (95%), whereas CD4. After fractionation, the membrane fractions were treated that by PMA was partial (40%; Fig. 1, a and b). with DTSSP, a thiol-cleavable cross-linker. The samples were then A postulated mechanism of the reversal is as follows. The Nef immunoprecipitated and analyzed by immunoblotting (Fig. 4b). down-regulation of CD4 is mediated through the CD4-Nef inter- Anti-CD4 recognized a protein band of ϳ80–85 kDa on nonre- action. PMA induces phosphorylation of the CD4 tail, which dis- ducing SDS gel from the NefLL-transfected samples treated with rupts the CD4-Nef interaction. The phosphorylated CD4 tail now DTSSP (lanes 3 and 5), but not from the mock-transfected samples bypasses Nef, binding directly to an AP complex, most likely to (lane 1) or sample not treated with DTSSP (lane 2). Anti-Nef also AP-2 (our preliminary data not shown). In other words, PMA treat- recognized the 80- to 85-kDa band on nonreducing SDS gel, but ment switches CD4 internalization from a Nef-dependent to a Nef- Downloaded from recognized a 27-kDa band on reducing SDS gel, which was the independent pathway, which allows some of the down-regulated cleaved product of the 80- to 85-kDa band by ␤-ME and corre- CD4 molecules to recycle back to the cell surface, resulting in a net sponded to the size of Nef (lane 7). The results thus indicated that gain in cell surface CD4 in Nef-expressing T cells. the 80- to 85-kDa band was the chemically cross-linked CD4-Nef Our hypothesis is consistent with the known facts about CD4 product. Tail-less CD4 was not cross-linked (lane 4), indicating and Nef. The CD4 tail comprises a single ␣-helix from Arg402 to 417 413 414 408 415 that the CD4 tail was required for the interaction. Lys (41). Leu , Leu , Ser , and Ser are all part of this http://www.jimmunol.org/ The effects of PMA on the chemical cross-linking are shown in ␣-helix. The dileucine motif (Leu413 and Leu414) involved in the the right panel of Fig. 4b. From samples transfected with wt CD4, CD4-Nef interaction and CD4 internalization is close to Ser408 and the 80- to 85-kDa cross-linking products (lanes 8 and 9) disap- Ser415. Therefore, the phosphorylation of Ser408 and Ser415 may peared upon PMA treatment (lanes 10 and 11). In contrast, from directly disrupt the CD4-Nef interaction or may indirectly do so by samples transfected with the nonphosphorylatable Ser CD4, the altering the conformation of the CD4 tail. In this respect, it is similar 80- to 85-kDa band was detected regardless of PMA treat- known that the phosphorylation of Ser408 and Ser415 of CD4 re- ment (lanes 12–15). The results thus suggested that the PMA- sults in the dissociation of Lck from the CD4 tail as a result of a induced serine phosphorylation of CD4 might disrupt the interac- conformational change in the CD4 tail (5). It is conceivable that tion between CD4 and Nef. Note that the 80- to 85-kDa band for the same conformational change that breaks the interaction be- by guest on September 24, 2021

NefLL (lanes 8, 12, and 14) was more intense than that for wt Nef tween CD4 and Lck may also break the interaction between CD4 (lanes 9, 13, and 15), consistent with the above observation that and Nef. Consistent with this is the observation that Lck and Nef more NefLL than wt Nef was present on T cell membrane (Fig. 4a). appear to complete for binding to CD4 (42).

By anti-Nef immunoblotting, the amount of nontagged NefLL in ϳ the membrane fraction was 3- to 4-fold more than that of wt Nef Acknowledgments or Nef (Fig. 4a), whereas by FACS on GFP fluorescence, sim- WL We thank J. Skowronski for the Nef-GFP constructs. We also thank ilar amounts of wt Nef-GFP, NefLL-GFP, and NefWL-GFP were John Hirst for FACS analysis. detected (Fig. 3). To verify that tagging GFP to Nef made the disappearance of the overexpression of NefLL relative to the other two Nef, we side-by-side compared the expression levels of non- References 1. Pelchen-Matthews, A., J. E. Armes, and M. Marsh. 1989. Internalization and tagged and GFP-tagged Nef by anti-Nef immunoblotting (Fig. 4c). recycling of CD4 transfected into HeLa and NIH3T3 cells. EMBO J. 8:3641. Again, no difference was found among Nef-GFP, whereas NefLL 2. Shin, J., C. Doyle, Z. Yang, D. Kappes, and J. L. Strominger. 1990. Structural ϳ features of the cytoplasmic region of CD4 required for internalization. EMBO J. was 3- to 4-fold more than the wt Nef and NefWL. Therefore, the phenomenon proved true. A plausible explanation might be that 9:425. 3. Shin, J., R. L. Dunbrack, Jr., S. Lee, and J. L. Strominger. 1991. Phosphorylation- the dileucine motif in Nef might be involved in Nef endocytosis dependent down-modulation of CD4 requires a specific structure within the cy- and/or degradation and that tagging GFP to Nef might slow down toplasmic domain of CD4. J. Biol. Chem. 266:10658. 4. Pitcher, C., S. Honing, A. Fingerhut, K. Bowers, and M. Marsh. 1999. Cluster of Nef degradation to the extent that the difference in the amounts of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require the three GFP-tagged Nef becomes greatly diminished. This pos- activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. sibility is currently under further investigation. Cell 10:677. 5. Pelchen-Matthews, A., I. J. Parsons, and M. Marsh. 1993. Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from Discussion p56lck, increased association with clathrin-coated pits, and altered endosomal In this study we found that HIV Nef-down-regulation of CD4 from sorting. J. Exp. Med. 178:1209. 6. Marsh, M., and A. Pelchen-Matthews. 1996. Endocytic and exocytic regulation of the Jurkat T cell surface was reversible by PMA treatment (Fig. 1). CD4 expression and function. Curr. Top. Microbiol. Immunol. 205:107. The reversal appeared to require the phosphorylation of CD4, be- 7. Cullen, B. R. 1994. The role of Nef in the replication cycle of the human and cause both mutating Ser408 and Ser415, the PMA phosphorylation simian immunodeficiency viruses. Virology 205:1. 8. Kestler, H. W., III, D. J. Ringler, K. Mori, D. L. Panicali, P. K. Sehgal, sites in CD4 (3, 4), and the use of a PKC inhibitor, BIM1, abol- M. D. Daniel, and R. C. Desrosiers. 1991. Importance of the nef gene for main- ished the reversal (Fig. 2). CD4 and Nef could be cross-linked by tenance of high virus loads and for development of AIDS. Cell 65:651. DTSSP in membranes isolated from T cells not treated with PMA, 9. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J. Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, et al. 1995. but not in membranes from PMA-treated cells (Fig. 4). Taken to- Genomic structure of an attenuated quasi species of HIV-1 from a blood trans- gether, the data suggested that CD4 and Nef were associated with fusion donor and recipients. Science 270:988. 5500 REVERSIBILITY OF Nef-DOWN-REGULATION OF CD4

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