HIV Nef-mediated Cellular Phenotypes Are Differentially Expressed as a Function of Intracellular NEF Concentrations
2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês
10.1074/jbc.m101025200
ISSN1083-351X
AutoresXunxian Liu, Jeffrey A. Schrager, G. David Lange, Jon W. Marsh,
Tópico(s)interferon and immune responses
ResumoNef is a regulatory protein encoded by the genome of both human and simian immunodeficiency virus. Its expression in T cells leads to CD4 and major histocompatibility complex class I modulation and either enhancement or suppression of T cell activation. How this viral protein achieves multiple and at times opposing activities has been unclear. Through direct measurements of Nef and the Nef-GFP fusion protein, we find that these events are mediated by different Nef concentrations. Relative to the intracellular concentration that down-modulates surface CD4, an order of magnitude increase in Nef-GFP expression is required for a comparable modulation of major histocompatibility complex class I, and a further 3-fold increase is necessary to suppress T cell activation. Nef is a regulatory protein encoded by the genome of both human and simian immunodeficiency virus. Its expression in T cells leads to CD4 and major histocompatibility complex class I modulation and either enhancement or suppression of T cell activation. How this viral protein achieves multiple and at times opposing activities has been unclear. Through direct measurements of Nef and the Nef-GFP fusion protein, we find that these events are mediated by different Nef concentrations. Relative to the intracellular concentration that down-modulates surface CD4, an order of magnitude increase in Nef-GFP expression is required for a comparable modulation of major histocompatibility complex class I, and a further 3-fold increase is necessary to suppress T cell activation. human immunodeficiency virus major histocompatibility complex class I human major histocompatibility complex class I region A green fluorescence protein (R)-phycoerythrin cytomegalovirus retroviral long terminal repeat, used here to define promoter molecular equivalents of soluble fluorochrome phorbol 12-myristate 13-acetate interleukin-2 The role of the viral nef gene in HIV1 and simian immunodeficiency virus infection has been defined by the Nef-mediated increase in in vivo viral titer and the development of pathogenesis (1Kestler H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1418) Google Scholar). Some reported Nef activities, such as enhancement of infectivity (2de Ronde A. Klaver B. Keulen W. Smit L. Goudsmit J. Virology. 1992; 188: 391-395Crossref PubMed Scopus (143) Google Scholar, 3Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Crossref PubMed Scopus (360) Google Scholar, 4Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Crossref PubMed Scopus (476) Google Scholar), could contribute to this in vivo picture. However, other findings, such as molecular associations, receptor modulations, and effects on cell activity (5Cullen B.R. Cell. 1998; 93: 685-692Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 6Piguet V. Schwartz O. Le Gall S. Trono D. Immunol. Rev. 1999; 168: 51-63Crossref PubMed Scopus (178) Google Scholar, 7Marsh J.W. Arch. Biochem. Biophys. 1999; 365: 192-198Crossref PubMed Scopus (34) Google Scholar), are less definitive and at times contradictory. For example, whereas some (8Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 9Alexander L. Du Z. Rosenzweig M. Jung J.U. Desrosiers R.C. J. Virol. 1997; 71: 6094-6099Crossref PubMed Google Scholar, 10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar, 11Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (249) Google Scholar) have concluded that Nef increases T cell activity, others (12Luria S. Chambers I. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5326-5330Crossref PubMed Scopus (162) Google Scholar, 13Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 14Collette Y. Chang H.L. Cerdan C. Chambost H. Algarte M. Mawas C. Imbert J. Burny A. Olive D. J. Immunol. 1996; 156: 360-370PubMed Google Scholar) have demonstrated that Nef expression leads to suppression of T cell activation. In part, these differences may be due to either the use of stable cellular clones, which may not be representative of the entire cell population, or the use of promoters with varied activity. One study that used non-clonal cells examined T cell receptor stimulation-induced expression of the activation marker CD69 and convincingly found that all Nef-expressing cells (defined by CD4 down-modulation) also suppressed activation-induced CD69 expression (15Iafrate A.J. Bronson S. Skowronski J. EMBO J. 1997; 16: 673-684Crossref PubMed Scopus (181) Google Scholar). CD69 is one of several surface markers that identify activation of the biochemical pathways leading from the T cell receptor (16Ziegler S.F. Ramsdell F. Alderson M.R. Stem Cells. 1994; 12: 456-465Crossref PubMed Scopus (390) Google Scholar). Other Nef-mediated cellular phenotypes include down-modulation of CD4 and MHC I from the cell surface (17Guy B. Kieny M.P. Riviere Y. Le Peuch C. Dott K. Girard M. Montagnier L. Lecocq J.P. Nature. 1987; 330: 266-269Crossref PubMed Scopus (402) Google Scholar, 18Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (647) Google Scholar, 19Schwartz O. Marechal V. Le Gall S. Lemonnier F. Heard J.M. Nat. Med. 1996; 2: 338-342Crossref PubMed Scopus (867) Google Scholar). Our own studies have made use of T cell populations, continuous lines as well as primary CD4 T cells, that have been transduced with a retroviral vector. Nef expressed in these non-clonal populations resulted in enhanced T cell activation, as defined by IL-2 secretion, and down-modulation of surface CD4 (8Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar, 20Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar). A Nef-mediated suppression (or enhancement) of CD69 induction or down-modulation of MHC I in transduced cells had not been noted in our experimentation with Nef. In this report we have chosen to study the effect of one variable, the concentration of intracellular Nef, on the biological activity of this HIV protein. The development of a quantitative chemiluminescent Western assay for Nef permitted an estimation of Nef concentrations in cell lysates, either transduced with thenef gene or infected with HIV. In order to define Nef concentrations at the cellular level, we have made use of a Nef-green fluorescent protein (GFP) fusion protein. By flow cytometry, we correlated the level of cellular Nef-GFP directly to measurements of Nef-modulated cell surface markers of cell function. We found that T cell activation enhancement and CD4 modulation, decreased MHC I surface expression, and suppression of CD69 induction were dependent, respectively, on increasing concentrations of Nef. These findings imply that the numerous Nef-mediated cellular phenotypes are possible within the same cell but are differentially expressed as a function of Nef concentrations. For flow cytometry, (R)-phycoerythrin (RPE)-conjugated antibodies to CD4 and CD69 were purchased from Caltag (South San Francisco, CA). The RPE conjugate antibody to HLA-ABC antigen (clone W6/32) was purchased from Dako A/S (Denmark). Standard beads with known molecular equivalents of soluble fluorochromes (MESF) of fluorescein were from Sigma. pQBI25 (green fluorescence protein (GFP) under CMV promoter) was purchased from Quantum (Canada), and pNA7GFP (NefGFP under CMV promoter) was kindly provided by Drs. Michael E. Greenberg and Jacek Skowronski (21Greenberg M.E. Bronson S. Lock M. Neumann M. Pavlakis G.N. Skowronski J. EMBO J. 1997; 16: 6964-6976Crossref PubMed Scopus (198) Google Scholar). The pNA7GFP plasmid encodes a fusion product of HIV Nef NA7 and genetically modified green fluorescent protein GFPsg25 (22Palm G.J. Zdanov A. Gaitanaris G.A. Stauber R. Pavlakis G.N. Wlodawer A. Nat. Struct. Biol. 1997; 4: 361-365Crossref PubMed Scopus (232) Google Scholar). To construct the retroviral expression plasmids pLGFPSN and pLNefGFPSN, primers were first synthesized (Midland Certified Reagent Co.) as follows: gf5, 5′-ATATGAATTCATGGCTAGCAAAGGAGAAGAACTCTTCACTGG-3′; gf3, 5′-TTAAGGATCCTCAATCGATGTTGTACAGTTCATCCATG-3′. By using gf5 and gf3 as primers and pQBI25 as the template, a polymerase chain reaction fragment was digested with EcoRI and BamHI and then inserted into the EcoRI-BamHI site of pLXSN. The recombinant is the retroviral expression vector pLGFPSN. Plasmid pNA7GFP was digested with XbaI and blunted with T4 DNA polymerase and then digested with BamHI. The small fragment was inserted into the HpaI and BamHI site of pLXSN, resulting in pLNefGFPSN. Recombinant retrovirus containing LGFPSN and LNefGFPSN were derived from the packaging line GP293 (CLONTECH) according to the protocol of the manufacturer. Briefly, pLGFPSN and pLNefGFPSN were cotransfected with pVSV (encoding the vesicular stomatitis virus envelope) into GP293 cells, and virus was collected 3 days later. Retroviral vectors LXSN, LNL43SN, and LG2ASN packaged by PA317 and used to transduce T cells were described previously (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar). VB (23Lifson J.D. Reyes G.R. McGrath M.S. Stein B.S. Engleman E.G. Science. 1986; 232: 1123-1127Crossref PubMed Scopus (375) Google Scholar) and Jurkat E6-1 (24Weiss A. Wiskocil R.L. Stobo J.D. J. Immunol. 1984; 133: 123-128PubMed Google Scholar) T cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. Cells were grown in complete growth medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 g/liter sodium bicarbonate, 1 mm non-essential amino acids, 10 mm sodium pyruvate, 4 µl/liter β-mercaptoethanol, and 50 µg/ml gentamicin, adjusted to pH 7.4). E6-1 T cells were transfected with pQBI25 or pNA7GFP using GenePORTER Transfection Reagent (Gene Therapy Systems) according to the manufacturer's protocol. VB T cells were transfected with pQBI25 or pNA7GFP by electroporation (T820; BTX Electronic Genetics, CA) set at 200 V, 65 ms, and 1 pulse. Cells (5 × 106/ml) were electroporated in RPMI 1640 (without fetal calf serum) with DNA at 30 µg/ml at room temperature. For transduction, cells were incubated with recombinant retroviruses carrying the expression vectors LXSN, LG2ASN, LNL43SN, LGFPSN, or LNefGFPSN and treated the next day with 2 mg/ml geneticin for E6-1 cells and 1 mg/ml for VB T cells. After 7 days in selection, cells were returned to complete medium, and whole populations of geneticin-resistant cells were used in subsequent studies. The peripheral lymphocyte fraction from healthy donors was obtained by leukapheresis and countercurrent centrifugal elutriation by the Department of Transfusion Medicine at the National Institutes of Health (25Czerniecki B.J. Carter C. Rivoltini L. Koski G.K. Kim H.I. Weng D.E. Roros J.G. Hijazi Y.M. Xu S.W. Rosenberg S.A. Cohen P.A. J. Immunol. 1997; 159: 3823-3837PubMed Google Scholar) and purified as described previously (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar). Proliferation of purified CD4 T cells was achieved by addition of CD3/CD28 beads (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar). CD4 T cells were infected with either wild type HIV-1 strain NL4-3 or a mutant NL4-3 virus containing a double single-point mutation in thenef gene (DS) (26Chowers M.Y. Spina C.A. Kwoh T.J. Fitch N.J. Richman D.D. Guatelli J.C. J. Virol. 1994; 68: 2906-2914Crossref PubMed Google Scholar) or mock-infected with complete media. After a 2-h incubation period at 37 °C, the cells were washed in 20 ml of RPMI 1640 to remove unadsorbed virions. Cell pellets were resuspended in complete media at 2 × 106 cells/ml and returned to 37 °C. Cells were then stimulated with anti-CD3/CD28 beads. Viral replication was followed by determination of secreted HIV core antigen p24 by enzyme-linked immunosorbent assay (Coulter, Miami, FL). At peak p24 secretion (1–2 weeks), cells were pelleted, lysed, and examined for Nef protein as described below. Analysis of Nef and green fluorescent protein on Western blots was performed essentially as reported previously (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar). Prior to lysis, cell count and volume were determined on a Coulter Z2 particle analyzer. Defined numbers of cells were lysed in 1% Triton X-100 in 20 mm Tris (pH 8.0), 150 mm NaCl, 2.0 mm EDTA and supplemented with 1.0 µg/ml leupeptin, 1.0 µg ml aprotinin, 1 µg/ml pepstatin A, and 250 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, followed by protein determination by BCA microassay (Pierce). Nef was then precipitated by rabbit polyclonal anti-Nef (27Shugars D.C. Smith M.S. Glueck D.H. Nantermet P.V. Seillier-Moiseiwitsch F. Swanstrom R. J. Virol. 1993; 67: 4639-4650Crossref PubMed Google Scholar) (obtained through the AIDS Research and Reference Reagent Program, NIAID, National Institutes of Health) and run on SDS-gel electrophoresis, blotted onto a cellulose nitrate membrane. This was probed with an anti-C-terminal Nef monoclonal antibody (Advanced Biotechnologies, Inc., Columbia, MD, catalog number 13-160-100), followed by a secondary horseradish peroxidase-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and then developed with a chemiluminescence substrate (SuperSignal West, Pierce). Quantitation of chemiluminescence was achieved with a cooled charge coupled device camera system (ChemiImager, Alpha Innotech, San Leandro, CA). For detection of GFP and Nef-GFP, the blots were probed with rabbit anti-sera against GFP (Santa Cruz Biotechnology, Santa Cruz, CA). To make each experimental Western blot of unknowns quantitative, recombinant protein standards were included. T cells were activated and IL-2 was assayed, as described previously (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar). For CD69 induction, cells (106/ml) were incubated with 3 µg/ml anti-CD3 mAb (clone HIT3a, PharMingen) for 18 h. Data were acquired on a Becton Dickinson FACScan with CELLQuest software. Histogram subtraction was accomplished with the same software on two populations of cells of equivalent cell number and involved subtraction of cellular events of one histogram from events of another at each increment of fluorescence intensity. Only positive values are plotted in the resultant differential histogram. Because some of the analyses that we required were not available in the proprietary software, files were converted to Microsoft Excel format with FCS Assistant shareware (www.fscpress.com) and then to text suitable for input to programs written in the Mathematica programming language and executed under the Mathematica system (Wolfram Research, Inc., Champaign, IL). The data consisted of pairs of numbers for each of the cells (n = 300,000–700,000). The first of these was proportional to the log of the fluorescence due to GFP or Nef-GFP and the second was proportional to the log of the fluorescence of a surface marker for T cell activation CD69, CD4, or HLA-A. A two-dimensional 80 × 80 bin histogram was formed, smoothed with a 3 × 3 running average, and viewed as a contour plot with GFP or Nef-GFP displayed along thex axis and the T cell antigens along the y axis. Because the individual cell measurements were independent, each of the 80 columns in the unsmoothed histograms was considered to be an independent distribution. The means of these distributions were plotted against the x scale value of fluorescence of GFP or Nef-GFP. The 95% confidence bands were also calculated. These bands broadened at higher GFP levels because the distributions broaden and the numbers of cells diminish. For comparison purposes, the y values for all bins were adjusted so that the mean value of the left-hand no/low GFP region was identical for both plots. Jurkat E6-1 cells were transduced with either Nef from HIV NL4-3 or a mutant, non-myristoylated NL4-3 Nef, generated by a glycine to alanine switch at residue position 2 (G2A). This mutant Nef is known to lack the numerous bioactivities of the native Nef protein (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar, 15Iafrate A.J. Bronson S. Skowronski J. EMBO J. 1997; 16: 673-684Crossref PubMed Scopus (181) Google Scholar, 28Guy B. Riviere Y. Dott K. Regnault A. Kieny M.P. Virology. 1990; 176: 413-425Crossref PubMed Scopus (89) Google Scholar, 29Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 30Goldsmith M.A. Warmerdam M.T. Atchison R.E. Miller M.D. Greene W.C. J. Virol. 1995; 69: 4112-4121Crossref PubMed Google Scholar). Cells were transduced with these nef genes in the Moloney LTR-based LXSN vector (18Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (647) Google Scholar). The cellular concentration of Nef was determined by a chemiluminescence-based Western blot, where quantitation of emitted light was achieved with a cooled charge coupled device camera system (see "Experimental Procedures"). This analysis of cell lysates demonstrated the specific expression of the Nef proteins in cells transduced with either the wild type Nef or the G2A mutant Nef (Fig. 1A). Recombinant Nef at various levels is shown in lanes 1–4 of Fig. 1 A. The band intensities in this particular Western indicated that the NL4-3 (Fig. 1 A, lane 6) and the G2A (Fig.1 A, lane 7) Nef transduction of the Jurkat cell resulted in an expressed 2.0 ng of Nef per 7 × 106 cells for both transductants. Thus, each transductant contained ∼0.3 fg of Nef per cell. The VB T cell line used in this report is characterized in Fig.1 B, with recombinant Nef in lanes 1–4. The wild type Nef (Fig. 1 B, lane 5) is expressed at 0.3 fg per cell, and the G2A mutant Nef (Fig. 1 B, lane 6) is at 0.4 fg per cell. These results are representative of multiple T cell transductions using this retroviral expression system. Typically, geneticin-selected T cell populations expressed between 0.3 and 1.0 fg per cell. We also applied this Nef assay system to HIV-infected primary CD4 T cells. Purified and activated CD4 T cells were infected with either wild type NL4-3 HIV or a Nef-negative HIV mutant NL4-3 DS (26Chowers M.Y. Spina C.A. Kwoh T.J. Fitch N.J. Richman D.D. Guatelli J.C. J. Virol. 1994; 68: 2906-2914Crossref PubMed Google Scholar) as described under "Experimental Procedures." The HIV-infected cells were harvested following the peak of HIV core antigen p24 secretion for maximal Nef generation (31Page K.A. Liegler T. Feinberg M.B. AIDS Res. Hum. Retroviruses. 1997; 13: 1077-1081Crossref PubMed Scopus (40) Google Scholar). At this time a minimum of 50% of the cells displayed signs of infection by microscopic examination. The lysate was applied to Western analysis for Nef content. HIV infection of CD4 T cells yielded 1 ng per 106 cells or 1 fg Nef per cell (Fig. 1 C, lane 3). Although all cells stained positive for intracellular p24 (data not shown), this measured level of Nef has to be taken as a conservative estimate with a minimum of 50% of the cells displaying infection by microscopic examination. Lysate from cells infected with the Nef-negative DS mutant HIV lacked detectable protein (lane 4). These findings suggest that retroviral transduction as performed here results in cellular Nef concentrations that approximate those seen in HIV infection. Retroviral transduction of T cells with Nef has been demonstrated previously by this laboratory (10Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (159) Google Scholar, 20Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar) to enhance T cell activation and to modulate surface CD4. Accordingly, activation of the Nef-expressing Jurkat T cell resulted in an enhanced secretion of IL-2 (Fig. 2). Stimulation of the Jurkat cell can also be evaluated through measurement of CD69 induction, which occurs to a majority of stimulated cells and is easily measured by flow cytometry. In contrast, IL-2 synthesis in these cells following CD3/CD28 stimulation is confined to only a few percent of the cell population, and thus, a signal generated by intracellular staining of IL-2 is difficult to discriminate from the noise levels of flow cytometry. The CD69 induction pathway from the T cell receptor is essential for IL-2 production but is not sufficient for T cell activation and IL-2 secretion (32Testi R. D'Ambrosio D. De Maria R. Santoni A. Immunol. Today. 1994; 15: 479-483Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 33Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (423) Google Scholar). As shown in Fig.3A, T cell receptor-mediated CD69 induction was unaffected by Nef expression from the retroviral vector. This suggests that the T cell receptor-mediated activation pathway involved in the Nef-mediated enhancement of IL-2 may be distinguishable from the pathway leading to CD69 expression. This is reasonable, since there are numerous pathways leading to IL-2 expression (33Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (423) Google Scholar). In all panels of Fig. 3 antigen measurements (CD4 or MHC I) in Nef-expressing cells are depicted by a solid line, whereas staining of non-transduced control cells is represented by thedashed line. The E6-1 Jurkat cells express high levels of HLA-A (MHC I); down-modulation of MHC I in Nef-transduced Jurkat cells (Fig. 3 B) was absent. Thus, Nef expression from the retroviral vector did not significantly alter either surface MHC I or CD69.Figure 3Surface expression of CD69, MHC I, and CD4 in transduced Nef-expressing cells. Flow cytometry of Jurkat E6-1 cells (A and B) and VB cells (Cand D) transduced with NL4-3 Nef retroviral expression vector. A, Nef-expressing Jurkat cells were stained with anti-CD69 antibody following T cell receptor stimulation with anti-CD3 antibody (solid line). Activated non-Nef control cells are represented by the dashed line, whereas CD69 levels for the non-activated cells are represented by the dotted line. B, Nef-expressing Jurkat cells were stained with anti-MHC I antibody (solid line) and are compared with non-Nef controls (dashed line). Dotted line represents the isotype control for Nef-expressing cells. C, flow cytometry of VB cells transduced with NL4-3 Nef and stained for either CD4 (solid line) or isotype 2A (dotted line). CD4 staining for the non-Nef VB control cell is represented by the dashed line. D, Nef-expressing VB cells were stained with either the anti-MHC I antibody (solid line) or the isotype 2A control antibody (dotted line). Control cell staining for MHC I is represented by the dashed line.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also examined Nef transduction of VB T cells which, like the E6-1 line, possess high surface expression of MHC I (in the form of HLA-A). Additionally, VB cells express a high level of CD4. The cells were stained for either CD4 (Fig. 3 C) or MHC I (Fig.3 D). Nef expression from the retroviral vector resulted in CD4 down-modulation (Fig. 3 C), but in the identical population there was no modulation of MHC I (Fig. 3 D). CD4 modulation does not occur to all cells in the transduced population (see Fig. 3 C and discussion below), but there is a 70% reduction in the mean fluorescence intensity for CD4 by an estimated 0.3 fg of native Nef per VB cell (Fig. 1 B). To permit a simultaneous measurement of surface antigen modulation with Nef concentration, we then constructed a Moloney LTR-based (LNefGFPSN) retrovirus for transduction of Nef-GFP. The Nef-GFP fusion protein had been shown previously (21Greenberg M.E. Bronson S. Lock M. Neumann M. Pavlakis G.N. Skowronski J. EMBO J. 1997; 16: 6964-6976Crossref PubMed Scopus (198) Google Scholar) to possess a biologically active Nef, the expressed levels of which correlated proportionally with CD4 modulation. Transduction of VB cells with either the Nef-GFP or a control (non-Nef) GFP vector yielded a bimodal distribution of cells either negative or positive for GFP (or for Nef-GFP) expression (Fig.4). Each of the positive populations represented ∼40% of the total population (see Fig. 4 legend). Instrumental measurements of fluorescence are in relative units and can fluctuate from day to day. To quantitate the generated fluorescence and permit comparisons of data, all flow cytometry experiments that measured GFP and the Nef-GFP fusion protein were co-run with standard beads with known MESF of fluorescein. As shown in Fig. 4, Aand B, the expression of GFP did not affect expressed levels either of CD4 or MHC I (defined by fluorescence on y axis). Cells positive for Nef-GFP expression displayed a 77% down-modulation of surface CD4 (Fig. 4 C), whereas with the identical cells stained for MHC I, there was no measurable modulation (Fig.4 D). The mode fluorescence of the Nef-GFP-positive population in Fig. 4 was 67 relative fluorescent units (xaxis), which in this run was equivalent to 4.4 × 104MESF (fluorescein equivalents). With the VB cell we have seen that transduction with either native Nef (Fig. 3 C) or Nef-GFP (Fig. 4 C) has resulted in CD4 modulation. However, in order to permit a direct comparison of the two transductants, we need to compare only Nef-expressing cells. Although a mechanism has not been defined, the lack of full expression of Nef from an integrated LnefSN (LTR-Nef-SV40-Neo) retroviral vector has been documented previously (18Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (647) Google Scholar). That is, cells that have been selected for geneticin resistance (Neo; neomycin phosphotransferase) do not always coexpress Nef. The Nef-GFP-positive cells in the transduced population displayed in Fig.4 C are 38% of the total geneticin-resistant population, and our measurement of CD4 modulation only included the Nef-GFP-expressing cells. Estimation of CD4 modulation in cells transduced by the native Nef, for example in Fig. 3, is not so straightforward. Indeed, our calculation for percent modulation, which comes from mean fluorescence, included all geneticin-resistant cells and thus included cells that could be negative for Nef expression. Corrections can be achieved, however, by only including cells that functionally display the presence of Nef. The modulation of CD4 in the Nef-transduced VB population of Fig. 3 C occurred to 65% of the selected cells (histogram subtraction, see "Experimental Procedures"). A comparison of the mode fluorescence between the CD4-modulated population (peak ofsolid line plot of Fig. 3 C; 179 relative fluorescent units on x axis) and the mode of the control CD4 levels (dashed line; 991 relative fluorescent units) yielded an 82% decrease in surface CD4. Additionally, if Nef expression is limited to 65% of the cells, one can deduce that the native Nef-transduced cells possessed a Nef concentration of 0.5 fg per cell (up from 0.3 fg in the total population). Thus, transduction of VB cells with either native Nef or Nef-GFP resulted in ∼80% modulation of surface CD4 but with no measurable change in MHC I. Also, transduction of the Jurkat E6-1 cell did not lead to MHC I modulation or loss of CD69 induction. Previous works have demonstrated that Nef can affect these latter functions, but there remains the need to define the mechanism by which the varied outcomes are possible. With the ability to measure Nef-GFP at the cellular level with flow cytometry, we then characterized T cell surface CD4, MHC I, and CD69 as a function of Nef-GFP concentration. In order to correlate MESH values from flow cytometry with protein concentration of cellular GFP, we first established a stable Jurkat clone expressing a high uniform level of GFP (Fig. 5A). Attempts to generate a stable highly fluorescent Nef-GFP clone were non-productive. The GFP clone yielded a mean fluorescence intensity of 95.1 relative units, which from standard beads yielded 1.22 × 105MESF. To correlate these values with cellular GFP, the lysate equivalent of 105 cells was applied to an SDS gel along with varied levels of recombinant GFP (Fig. 5 B). This permitted an estimate of 1.7 ng of GFP from the cell aliquot or 17 fg per cell. An additional two independent determinations yielded similar estimates (data not shown). We can use the relationship of 17 fg of GFP per cell yielding a fluorescence intensity equal to 1.22 × 105 MESF units per cell to estimate expressed GFP levels in cells from the cytometry-derived MESF values. The GFP VB population of Fig.4 B, the cells of which possess a fluorescence intensity of 1.45 × 105 MESF units, would thus be predicted to contain 20 fg of GFP per cell. If the GFP moiety of the Nef-GFP fusion product possessed fluorescence equivalent to the non-fusion GFP molecule, then the 4.4 × 104 MESF units of Nef-GFP population of VB cells (Fig. 4) multiplied by the same factor (17 fg of GFP
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