CD4 Down-regulation by HIV-1 and Simian Immunodeficiency Virus (SIV) Nef Proteins Involves Both Internalization and Intracellular Retention Mechanisms
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m409420200
ISSN1083-351X
AutoresJeremy J. Rose, Katy Janvier, Soundararajulu Chandrasekhar, Rafick‐Pierre Sékaly, Juan S. Bonifacino, Sundararajan Venkatesan,
Tópico(s)Immune Cell Function and Interaction
ResumoAmong the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells. Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells. The cytoplasmic domains of plasma membrane receptors contain sequence determinants that regulate orderly protein sorting during anterograde transport, specify organelle targeting, and determine the intracellular fate of internalized receptors after agonist binding or cell activation (1Bonifacino J.S. Traub L.M. Annu. Rev. Biochem. 2003; 72: 395-447Crossref PubMed Scopus (1632) Google Scholar, 2Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar, 3Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (437) Google Scholar). Animal viruses have evolved several strategies to subvert mechanisms of protein sorting, resulting in aberrant trafficking, intracellular trapping, and degradation of targeted receptors.Among the viral modulators of receptor expression and trafficking, the 27–32 kDa myristoylated Nef proteins of HIV 1The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; AP, adaptor protein; APC, allophycocyanin; siRNA, small interfering RNA; PBL, peripheral blood lymphocytes; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; EYFP, enhanced YFP; WT, wild type; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; MFV, mean fluorescence value; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PE, phosphatidylethanolamine; mAb, monoclonal antibody; CMV, cytomegalovirus; NX, NefXho; Tfn, transferrin; TfnR, Tfn receptor.1The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; AP, adaptor protein; APC, allophycocyanin; siRNA, small interfering RNA; PBL, peripheral blood lymphocytes; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; EYFP, enhanced YFP; WT, wild type; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; MFV, mean fluorescence value; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PE, phosphatidylethanolamine; mAb, monoclonal antibody; CMV, cytomegalovirus; NX, NefXho; Tfn, transferrin; TfnR, Tfn receptor. and SIV have been most extensively characterized for their ability to down-regulate CD4 and HLA-I receptors (reviewed in Refs. 4Geyer M. Fackler O.T. Peterlin B.M. EMBO Rep. 2001; 2: 580-585Crossref PubMed Scopus (316) Google Scholar, 5Wei B.L. Arora V.K. Foster J.L. Sodora D.L. Garcia J.V. Curr. HIV Res. 2003; 1: 41-50Crossref PubMed Scopus (41) Google Scholar, 6Oldridge J. Marsh M. Trends Cell Biol. 1998; 8: 302-305Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These receptors are modulated by different mechanisms and require distinct subdomains of Nef (7Piguet V. Wan L. Borel C. Mangasarian A. Demaurex N. Thomas G. Trono D. Nat. Cell Biol. 2000; 2: 163-167Crossref PubMed Scopus (248) Google Scholar, 8Mangasarian A. Piguet V. Wang J.K. Chen Y.L. Trono D. J. Virol. 1999; 73: 1964-1973Crossref PubMed Google Scholar, 9Greenberg M.E. Iafrate A.J. Skowronski J. EMBO J. 1998; 17: 2777-2789Crossref PubMed Scopus (271) Google Scholar, 10Craig H.M. Pandori M.W. Guatelli J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11229-11234Crossref PubMed Scopus (237) Google Scholar, 11Bresnahan P.A. Yonemoto W. Ferrell S. Williams-Herman D. Geleziunas R. Greene W.C. Curr. Biol. 1998; 8: 1235-1238Abstract Full Text Full Text PDF PubMed Google Scholar). Nef reduces the steady-state levels of CD4 apparently by accelerating receptor endocytosis and misdirecting the internalized CD4 to lysosomes for breakdown. A membrane-proximal dileucine motif in the cytoplasmic tail of CD4 is both necessary and sufficient for the HIV-1 Nef-mediated down-regulation (12Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 13Pitcher C. Honing S. Fingerhut A. Bowers K. Marsh M. Mol. Biol. Cell. 1999; 10: 677-691Crossref PubMed Scopus (134) Google Scholar). Specific interactions of Nef with the components of heterotetrameric adaptor complexes are critical determinants of this process. Mutations at the 2 leucines in the highly conserved (D/E)XXXL(L/I) dileucine motif in the solvent-exposed, C-terminal flexible loop of both HIV and SIV Nef proteins abolish AP-1 and AP-3 adaptor binding and CD4 down-regulation (10Craig H.M. Pandori M.W. Guatelli J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11229-11234Crossref PubMed Scopus (237) Google Scholar, 11Bresnahan P.A. Yonemoto W. Ferrell S. Williams-Herman D. Geleziunas R. Greene W.C. Curr. Biol. 1998; 8: 1235-1238Abstract Full Text Full Text PDF PubMed Google Scholar, 14Greenberg M. DeTulleo L. Rapoport I. Skowronski J. Kirchhausen T. Curr. Biol. 1998; 8: 1239-1242Abstract Full Text Full Text PDF PubMed Google Scholar, 15Janvier K. Kato Y. Boehm M. Rose J.R. Martina J.A. Kim B.Y. Venkatesan S. Bonifacino J.S. J. Cell Biol. 2003; 163: 1281-1290Crossref PubMed Scopus (183) Google Scholar). Furthermore, CD4-Nef or CD8-Nef chimeras fusing the ecto- and transmembrane domains of CD4 or CD8 to Nef undergo rapid endocytosis in a dileucine motif-dependent manner (11Bresnahan P.A. Yonemoto W. Ferrell S. Williams-Herman D. Geleziunas R. Greene W.C. Curr. Biol. 1998; 8: 1235-1238Abstract Full Text Full Text PDF PubMed Google Scholar, 16Mangasarian A. Foti M. Aiken C. Chin D. Carpentier J.L. Trono D. Immunity. 1997; 6: 67-77Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). SIV Nef has an additional tyrosine-based sorting signal of the type YXXØ, which presumably binds to the AP-2 complex and is implicated in enhancing endocytosis of CD4 (17Bresnahan P.A. Yonemoto W. Greene W.C. J. Immunol. 1999; 163: 2977-2981PubMed Google Scholar, 18Piguet V. Chen Y.L. Mangasarian A. Foti M. Carpentier J.L. Trono D. EMBO J. 1998; 17: 2472-2481Crossref PubMed Scopus (187) Google Scholar). From these findings, Nef is presumed to serve as a "connector" between CD4 and the endocytic machinery (6Oldridge J. Marsh M. Trends Cell Biol. 1998; 8: 302-305Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16Mangasarian A. Foti M. Aiken C. Chin D. Carpentier J.L. Trono D. Immunity. 1997; 6: 67-77Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Such a role would necessitate a physical interaction between Nef and CD4. CD4-Nef complexes have been demonstrated in insect cells overexpressing both proteins (19Harris M.P. Neil J.C. J. Mol. Biol. 1994; 241: 136-142Crossref PubMed Scopus (73) Google Scholar), in a yeast two-hybrid system (20Rossi F. Gallina A. Milanesi G. Virology. 1996; 217: 397-403Crossref PubMed Scopus (65) Google Scholar), and in in vitro binding assays with Nef protein and peptides corresponding to the CD4 cytoplasmic domain (21Grzesiek S. Stahl S.J. Wingfield P.T. Bax A. Biochemistry. 1996; 35: 10256-10261Crossref PubMed Scopus (304) Google Scholar, 22Preusser A. Briese L. Baur A.S. Willbold D. J. Virol. 2001; 75: 3960-3964Crossref PubMed Scopus (49) Google Scholar).The objectives of this study were to inquire whether endocytic enhancement is the sole mechanism of Nef-induced CD4 down-modulation and to elucidate the differences, if any, in the mechanisms of HIV-1 and SIV Nef with respect to recruitment of different adaptor complexes and subunits in the process. Using a yeast three-hybrid assay, we demonstrated recently that (D/E)XXXL(L/I)-type signals from HIV and SIV Nefs interacted in a bipartite manner with the adaptor hemicomplexes, γ·σ1 of AP-1 and δ·σ3 of AP-3. The analogous α·σ2 of AP-2 and ϵ·σ4 subunits of AP-4 did not interact with Nef. Interaction of HIV-1 Nef protein with the individual subunits of AP complexes was much weaker or non-existent (15Janvier K. Kato Y. Boehm M. Rose J.R. Martina J.A. Kim B.Y. Venkatesan S. Bonifacino J.S. J. Cell Biol. 2003; 163: 1281-1290Crossref PubMed Scopus (183) Google Scholar).We have extended the above studies to show that the loss of CD4 at the cell surface of HIV-1 Nef-expressing cells did not result exclusively from accelerated endocytosis of CD4. Both the recycling CD4 and the nascent receptor in transit to the plasma membrane were susceptible to intracellular retention and degradation in HIV-1 Nef-expressing cells. In contrast, SIV Nef-induced CD4 down-regulation resulted mostly from enhanced endocytosis and was dramatically reversed by genetic inhibitors of endocytosis or small interfering RNA (siRNA)-driven knockdown of AP-2 complexes.EXPERIMENTAL PROCEDURESCells and Viruses—Human T cell lines used in this study included CD4-positive Jurkat cells or the A3.01 derivative of CEM cells and CD4-negative A2.01 cells. These T cell lines and a HeLa CD4 cell line, clone 6C (HT-6C), were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, MD and propagated. The T lymphocytes were propagated in RPMI with 10% fetal calf serum (FCS) and the HeLa CD4 cell line in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. The HeLa CD4 cells were monitored periodically for the inevitable outgrowth of CD4(–) cells, at which time they were selected on G418 (500 μg/ml) for two passages. Human T cell lines expressing wild type CD4 (line A2D8), or the tCD4 mutant (line T402), truncated at the 402nd residue and thus lacking the C-terminal domain, were constructed by retroviral transduction of the CD4-negative T cell line A2.01 using a retroviral vector and neomycin selection (23Gratton S. Yao X.J. Venkatesan S. Cohen E.A. Sekaly R.P. J. Immunol. 1996; 157: 3305-3311PubMed Google Scholar) and maintained in RPMI medium supplemented with 10% fetal bovine serum and periodically selected by G418 (500 μg/ml) as above. PBLs were isolated from whole blood, buffy coat, or lymphocyterich leukopaks provided by the Department of Transfusion Medicine at the National Institutes of Health. Red blood cells were disrupted with ammonium chloride in potassium bicarbonate lysis solution and removed by gentle centrifugation. The final pellet of d0 peripheral blood mononuclear cells was suspended in RPMI with 10% FCS. For T-cell activation, peripheral blood mononuclear cells purified by banding after Ficoll-Hypaque centrifugation were stimulated by CD3 mAb for 36 h in the presence of interleukin-2 (20 IU/ml) in RPMI with 10% FCS, following which they were propagated in medium containing interleukin-2 (24Venkatesan S. Rose J.J. Lodge R. Murphy P.M. Foley J.F. Mol. Biol. Cell. 2003; 14: 3305-3324Crossref PubMed Scopus (99) Google Scholar).All the recombinant vaccinia viruses used in this work had been constructed by homologous recombination at the thymidine kinase locus of the vaccinia virus genome. Plasmid pSC11 was used as the transfer vector. pSC11 contains the vaccinia virus 7.5K promoter for the expression of foreign genes and also had an Escherichia coli lac-Z gene linked to the vaccinia virus 11k promoter. The following recombinant vaccinia viruses were used: 1) for negative control, the recombinant vaccinia virus (vSC8) encoding the E. coli lac-Z (25Chakrabarti S. Brechling K. Moss B. Mol. Cell. Biol. 1985; 5: 3403-3409Crossref PubMed Scopus (619) Google Scholar); 2) vvNef that was isogenic with vSC8 except for the Nef gene linked to the 7.5K promoter (26Koenig S. Fuerst T.R. Wood L.V. Woods R.M. Suzich J.A. Jones G.M. de la Cruz V.F. Davey R.J. Venkatesan S. Moss B. Biddeson W.E. Fauci A.S. J. Immunol. 1990; 145: 127-135PubMed Google Scholar); 3) vvCD4 (vCB-7) expressing WT full-length human CD4 (27Broder C.C. Berger E.A. J. Virol. 1993; 67: 913-926Crossref PubMed Google Scholar); 4) vvtCD4 (vCB-2) expressing CD4 truncated at residue 402 (and modified by the addition at the C terminus of a Leu-Ileu-Asn sequence during DNA cloning) and thus lacking the cytoplasmic domain (28Golding H. Dimitrov D.S. Manischewitz J. Broder C.C. Robinson J. Fabian S. Littman D.R. Lapham C.K. J. Virol. 1995; 69: 6140-6148Crossref PubMed Google Scholar); 5) vvgp160 (vPE16) expressing the HIV-1 envelope glycoprotein, gp160 (29Earl P.L. Hugin A.W. Moss B. J. Virol. 1990; 64: 2448-2451Crossref PubMed Google Scholar, 30Earl P.L. Doms R.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 648-652Crossref PubMed Scopus (298) Google Scholar); and 6) vvCD46, expressing the human CD46 protein (31Nussbaum O. Broder C.C. Moss B. Stern L.B.-L. Rozenblatt S. Berger E.A. J. Virol. 1995; 69: 3341-3349Crossref PubMed Google Scholar). Cloned virus stocks were plaque-purified thrice on CV-1 cells, propagated in HeLa S3 suspension cells, and purified by banding on sucrose gradients by ultracentrifugation (29Earl P.L. Hugin A.W. Moss B. J. Virol. 1990; 64: 2448-2451Crossref PubMed Google Scholar, 30Earl P.L. Doms R.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 648-652Crossref PubMed Scopus (298) Google Scholar). For expression studies, cells were infected at a multiplicity of infection of 5 or 10 plaque-forming units/cell unless indicated otherwise.Recombinant DNA Constructs—The original Nef expression plasmid, pCMV-Nef, has been described (32Maitra R.K. Ahmad N. Holland S.M. Venkatesan S. Virology. 1991; 182: 522-533Crossref PubMed Scopus (31) Google Scholar). For expression in mammalian cells, wild type or mutant Nef cDNAs were PCR-amplified from the respective HIV and SIV proviruses or other recombinant plasmids and cloned into EcoRI-SalI sites of pCI-neo vector (Promega, Madison, WI). CMV promoter-linked CD4 and CD8 plasmids were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Rockville MD. CD4 was mutated by a two-step PCR mutagenesis protocol (33Venkatesan S. Gerstberger S.M. Park H. Holland S.M. Nam Y. J. Virol. 1992; 66: 7469-7480Crossref PubMed Google Scholar) to exchange the codons for 2 leucines at 438 – 439 to alanines and cloned into the pCI-neo vector to generate LL/AA CD4. Dominant-negative Eps15 deletion mutant fused N-terminally to GFP (GFP-Eps15Δ95/295) was obtained from Alexandre Benmerah at INSERM, Paris, France (34Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). GFP-tagged WT dynamin and the K44A dominant-negative dynamin mutant (35Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) were from Julie Donaldson at NICHD, National Institutes of Health, Bethesda, MD. EYFP-tagged Rab5 and Rab5-S34N have been described (24Venkatesan S. Rose J.J. Lodge R. Murphy P.M. Foley J.F. Mol. Biol. Cell. 2003; 14: 3305-3324Crossref PubMed Scopus (99) Google Scholar).Antibodies—The following mouse monoclonal antibodies were obtained: 1) against α, γ, δ, and ϵ-adaptins, unconjugated or dye-conjugated CD71 (transferrin receptor, TfnR), and unconjugated CD46 (BD Transduction and Pharmingen Laboratories) and 2) unconjugated, Alexa Fluor 488-, PE-, or APC-conjugated CD4 and CD8 mAbs (Caltag Laboratories, Burlingame, CA). The following polyclonal antibodies were also used: 1) against the σ3 subunit of AP-3 adaptor (36Dell'Angelica E.C. Ooi C.E. Bonifacino J.S. J. Biol. Chem. 1997; 272: 15078-15084Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and 2) against TfnR (Zymed Laboratories Inc., South San Francisco, CA). Monoclonal antibody (TW 2.3) against the vaccinia virus E3L gene product (37Yuwen H. Cox J.H. Yewdell J. Bennick J.R. Moss B. Virology. 1993; 195: 732-744Crossref PubMed Scopus (118) Google Scholar) was a gift from Jonathan Yewdell, NIAID, National Institutes of Health. Rabbit antiserum against HIV-1 gp160 protein was a gift of Ronald Willey, NIAID, National Institutes of Health. Secondary antibodies to mouse, rabbit, goat, and sheep IgG conjugated to Alexa Fluor 488, 568, or 647 dyes were from Molecular Probes Inc. (Eugene, OR).Cell Culture and Transfections—HeLa cells, cultured in DMEM supplemented with 10% FCS (v/v), were transfected using the Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. PBLs were isolated from whole blood, buffy coat, or lymphocyte-rich leukopaks provided by the Department of Transfusion Medicine at the Clinical Center, National Institutes of Health, as described (24Venkatesan S. Rose J.J. Lodge R. Murphy P.M. Foley J.F. Mol. Biol. Cell. 2003; 14: 3305-3324Crossref PubMed Scopus (99) Google Scholar). CD4+ T cells were enriched by negative selection using magnetic bead technology (Miltenyi Biotec Inc., Auburn, CA). PBLs and T cell lines were transfected using an Amaxa Biosystems electroporator with the recommended reagents (AMAXA Inc., Gaithersburg, MD).Flow Cytometry—General protocols of flow cytometry using HeLa cell transfectants, primary T cells, and lines have been described before (24Venkatesan S. Rose J.J. Lodge R. Murphy P.M. Foley J.F. Mol. Biol. Cell. 2003; 14: 3305-3324Crossref PubMed Scopus (99) Google Scholar). In all transfections, plasmids encoding EGFP or CD8 were included to normalize for transfection efficiency. For detection of internal antigens, the cells were permeabilized with 0.1% saponin treatment and fixed in 4% paraformaldehyde prior to antibody staining in the presence of 0.1% saponin. Vaccinia virus infection was monitored by FACS analysis of cells permeabilized by saponin, using a monoclonal antibody (TW 2.3) against the vaccinia virus E3L gene product (37Yuwen H. Cox J.H. Yewdell J. Bennick J.R. Moss B. Virology. 1993; 195: 732-744Crossref PubMed Scopus (118) Google Scholar). At 4 h after vaccinia virus infection, 40 – 60% of cells scored positively with this antibody.CD4 Endocytosis and Recycling—CD4 endocytic kinetics was determined by flow cytometry assay essentially as described previously (38Greenberg M.E. Bronson S. Lock M. Neumann M. Pavlakis G.N. Skowronski J. EMBO J. 1997; 16: 6964-6976Crossref PubMed Scopus (198) Google Scholar, 39Pelchen-Matthews A. Armes J.E. Marsh M. EMBO J. 1989; 8: 3641-3649Crossref PubMed Scopus (75) Google Scholar). HeLa cells were co-transfected with WT or mutated CD4 along with WT Nef or the NefXho (NX) Nef deletion mutant. Alternatively, Jurkat cells were transfected with Nef or NX plasmid. In all transfections, CD8 plasmid was included to enable gating of transfected cells. At 30 h after transfection, cells were reacted with PE-conjugated Leu3A mAb for 30 min on ice in DMEM or RPMI 1640 (for Jurkat cells) containing 0.5% bovine serum albumin. An aliquot of cells was co-stained at 0 – 4 °C with CD8 APC to determine the transfection efficiency and CD4 PE to quantify the CD4 receptor density at the cell surface. The bulk of the cells that had been stained with CD4 PE only were rinsed thrice with RPMI containing 0.5% bovine serum albumin at 2– 4 °C to remove the excess unbound antibody and incubated at 37 °C. At the indicated times, dual 50-μl aliquots were diluted with 5–10 volumes of RPMI at neutral or acid pH (pH 2 for HeLa cells, pH 3 for Jurkat cells) and incubated for 45 s at 2– 4 °C. After rinsing with neutral pH RPMI, cells were fixed in 4% paraformaldehyde in PBS for 10 min at 2– 4 °C, rinsed with PBS, and counterstained with CD8-APC and processed for flow cytometry. Mean fluorescence values (MFVs) for CD4 were calculated for cells gated for CD8 expression. To calculate the fraction of CD4 internalization at each time point, [F(int)ti], the CD4 MFV at zero time acid wash [MFV(pH 2)t0] was subtracted from the MFVs for each time point [MFV(pH 2)ti] times, and the resulting value for each time point was divided by the total MFV (neutral pH wash) [MFV(pH 7.4)ti] as indicated by the equation below.F(int)ti=[MFV(pH 2)ti−MFV(pH2)t0]MFV(pH 7.4)ti×100(Eq. 1) For microscopic visualization of CD4 endocytosis, fluorescent antibody was fed to living cells together with fluorescent transferrin as a monitor for clathrin-dependent endocytosis of TfnR. HeLa cells transiently expressing CD4, WT Nef, or Nef-Xho mutant and GFP- or YFP-tagged dominant-negative inhibitors of endocytosis were used. Cells on coverslips at 50% confluence were starved for 30 min at 37 °C in DMEM without serum. They were then incubated at 37 °C for 30 min in 200 μl of DMEM (with 0.5% bovine serum albumin) with Alexa Fluor 568-conjugated Tfn (50 μg/ml) and APC-labeled CD4 mAb (10 μl of mAbs or 1–2 μg of equivalent). Cells were rinsed thrice with PBS, fixed with paraformaldehyde, rinsed and mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). In some experiments (indicated in the relevant figure legends), cell surface bound antibody was stripped by treatment for 1–2 min with 0.5% acetic acid in 500 mm NaCl after the first rinse before fixation.CD4 recycling was calculated as described (39Pelchen-Matthews A. Armes J.E. Marsh M. EMBO J. 1989; 8: 3641-3649Crossref PubMed Scopus (75) Google Scholar) with slight modifications. HeLa cell transfectants were labeled with PE-conjugated CD4 mAb as for endocytosis studies, incubated at 37 °C for 30 min to maximize antibody internalization, rinsed, and suspended in 5 volumes of RPMI at pH 2 at 4 °C for 2 min. Cells were then transferred to neutral buffer, and a time 0 aliquot was collected, fixed, and counterstained with CD8-APC and analyzed by FACS. The remaining cells were warmed to 37 °C for the indicated times, placed in timed aliquots, washed in cold acid, fixed, and counterstained for CD8. CD4 MFVs in the CD8 gated population was determined by FACS analysis. The percentage of recycled CD4 was determined by dividing the MFVs during the rewarming period by the acid-resistant MFV before rewarming.Yeast Culture, Transformation, and Two- and Three-hybrid Assays— Two-hybrid genetic analysis between various Nefs and the adaptor subunits was carried out as described before (15Janvier K. Kato Y. Boehm M. Rose J.R. Martina J.A. Kim B.Y. Venkatesan S. Bonifacino J.S. J. Cell Biol. 2003; 163: 1281-1290Crossref PubMed Scopus (183) Google Scholar). The Saccharomyces cerevisiae strain HF7c (Clontech) was maintained on dropout agar plates lacking methionine. Transformation was performed by the lithium acetate procedure as described in the instructions for the MATCH-MAKER two-hybrid kit (Clontech). HF7c transformants were selected by spreading on plates lacking leucine, tryptophan, and methionine. For colony growth essays, HF7c transformants were dotted on plates lacking leucine, tryptophan, methionine, and histidine complemented with 3 mm 3-aminotriazole (Sigma) and allowed to grow at 30 °C for 3–5 days.Metabolic Labeling and Immunoprecipitation—For metabolic labeling experiments, transfected or virus-infected HeLa cells or T cells (106–107) were rinsed thrice with PBS and incubated in methionine and cysteine-free DMEM containing 1% dialyzed FCS (0.2 ml/sample) for 10 min. For steady-state labeling, cells were incubated for 1 h by the addition of 35S-labeled Trans-label (ICN Corp) to 1 mCi/ml. For measuring the kinetics of protein biosynthesis, 2 × 107 cells were labeled for 15 min in 500 μl of labeling medium (1 mCi/ml). At the end of the labeling, the cells were diluted with 10 volumes of complete RPMI medium. Aliquots were removed immediately after labeling and at the indicated periods during the chase (from 0 to 12 h). The cells were rinsed twice in PBS and processed for SDS-PAGE analysis. The cells were disrupted by three cycles of freeze-thawing (thawing to 37 °C for 1–2 min to disrupt rafts) in 500 μl of extraction buffer containing 0.05 m Tris-HCl, pH 7.4, 100 mm NaCl, 0.25% Nonidet P-40 (or CHAPS), 0.25% Triton X-100, and one tablet of protease inhibitor mix (Roche Applied Science) followed by extraction at 4 °C for 1 h. The extracts were centrifuged at 10,000 × g for 10 min, and supernatants were used for immunoprecipitation. The supernatants were precleared by incubation for 1 h at 4 °C with 30 μl of immobilized protein G agarose beads (Pierce) coated with preimmune rabbit or mouse sera. Labeled proteins were immunoprecipitated for 3 h at 4 °C with protein G agarose beads prebound to the corresponding anti-rabbit polyclonal or anti-mouse monoclonal antibodies. Following specific antibody binding, the beads were collected by centrifugation and washed five times with 10 –20 volumes of extraction buffer lacking protease inhibitors, and the labeled proteins were eluted by boiling in 50 μl of a buffer containing Tris-HCl, pH 7.4, 100 mm NaCl, 50 mm dithiothreitol, 2% SDS, glycerol (10% v/v), and bromphenol blue (0.1% w/v). The radiolabeled proteins were resolved by SDS-PAGE and visualized by phosphorimaging and quantified.Immunofluorescence Microscopy—Twenty-four hours after transfection, HeLa cells grown on glass coverslips were stained for immunofluorescence microscopy. Cells were fixed in 4% paraformaldehyde in PBS and permeabilized for 10 min with 0.1% (w/v) Triton X-100 in PBS. After permeabilization, the cells were blocked for 30 min with 1% normal goat serum in PBS and incubated for 30 min at room temperature with the primary antibody, washed with PBS, and then incubated for 30 min with the secondary antibody. The different combinations of 1° and 2° antibodies and GFP or GFP fusion proteins are indicated in the respective figure legends. The coverslips were washed and mounted on slides. Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exton, PA) using a ×63 or ×100 oil immersion objective NA 1.32 and digital zoom up to ×5. Fluorochromes were excited using an argon laser at 488 nm for Alexa Fluor 488 or fluorescein isothiocyanate, a krypton laser at 568 nm for Alexa Fluor 568 or Texas Red, and a helium/neon laser at 633 nm for APC. Fluorescent emission from Alexa Fluor 350 dye was visualized by excitation with a UV laser. Detector slits were configured to minimize any cross-talk between the channels, or the channels were collected separately and later superimposed. Differential interference contrast images were collected simultaneously with the fluorescence images using the transmitted light detector. Eight or more fields were examined per coverslip, and each experimental condition was repeated as indicated in the respective figure legends.RNA-mediated Interference—RNA-mediated interference of μ2 adaptor subunit or vps35 was performed using an siRNA duplex with the following sequence: μ2, 5′AAGUGGAUGCCUUUCGGGUCA-3′ starting at the 30th codon (86th nucleotide in the open reading frame); μ3, 5′AAGGAGAACAGUUCUUGCGGC-3′; and vps35, 5′AAGGUCCAGU-CAUUCCAAAUG-3′ starting at the 24th codon (70th nucleotide in the open reading frame). HeLa cells were transfected twice at 72-h intervals with 200 nm of the siRNAs, using Oligofectamine (Invitrogen). After 144 h of siRNA treatment, the cells were transfected as described above for expression of Nef, CD4, and CD8. Human T cell lines and PBLs were electroporated once with 100 nm siRNA using the Amaxa system. The cells were rested for 24 –36 h before electroporating with an additional 100 nm siRNA and expression plasmids as indicated in the relevant figure legends.RESULTSHIV-1 Nef Delayed the Cell Surface Expression and Reduced the Cell Surface Density of de novo Synthesized CD4 —Initially, we examined the kinetics of transport to the plasma membrane of de novo synthesized CD4 in the context of Nef expression. A2.01 (CD4-negative) T lymphocytes were co-infected with recombinant vaccinia viruses encoding WT CD4 (vCB-7) and NL4-3 HIV-1 Nef allele (vvNef) or a control virus (vSC8). Following 30 min of virus adsorption, cells were sampled at periodic intervals and examined by flow cytometry. Both sets of infected cells displayed similar levels of endogenous CD7 expression (not shown) that was used to monitor pot
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