Human T-Lymphotropic Virus Type 1 Mitochondrion-Lo
http://www.100md.com
病菌学杂志 2005年第15期
Center for Retrovirus Research and Department of Veterinary Biosciences, College of Veterinary Medicine
Comprehensive Cancer Center
Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, Ohio
Department of Oncology and Surgical Sciences, University of Padova, Padua 35128, Italy
ABSTRACT
Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13II. We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13II localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13II, using both transient and stable expression vectors. Our data indicate that p13II-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13II-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13II-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13II's influence on lymphocyte survival. Our data are the first to demonstrate that p13II alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.
INTRODUCTION
Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL), a highly aggressive T-cell malignancy characterized by circulating activated CD4+ CD25+ T cells (12). The virus is also associated with a variety of lymphocyte-mediated diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (12, 16, 28). There are approximately 15 to 25 million HTLV-1-infected persons worldwide, and 3 to 5% of these infected subjects will develop HTLV-1-associated diseases (17). The underlying mechanism of virus-mediated lymphocyte transformation has been extensively investigated but is incompletely understood. Based on the long period of latency and the small percentage of individuals who develop ATL, the transformation of infected lymphocytes is believed to be initiated through the induction of cellular genes and alterations in cellular activation and death pathways by the viral proteins (28).
HTLV-1 is a member of the genus Deltaretrovirus of the family Retroviridae. The viral genome consists of long terminal repeats (LTRs) flanking genes encoding the structural and enzymatic proteins Gag, Pol, and Env. In addition, a region located between env and the 3' LTR, pX, encodes the regulatory proteins Tax and Rex as well as several accessory proteins, namely, p12I, p27I, p13II, and p30II (1). The ability of HTLV-1 to produce these regulatory and accessory proteins through alternative splicing and selective codon usage classifies the virus among the complex retroviruses (4, 23). Recent studies have indicated a significant role for HTLV-1 accessory proteins in the life cycle of HTLV-1, particularly during the early phase of the viral infection of lymphocytes (1, 9, 16, 27, 30, 32, 40). Less is known, however, about the accessory protein p13II, a singly spliced product of the second open reading frame (ORF II) of the pX gene region.
This protein selectively localizes to the inner membranes of mitochondria (5, 8) and directly binds to cellular protein farnesyl pyrophosphate synthetase (25). p13II mRNA is expressed in various HTLV-1-infected cell lines isolated from clinical patients with ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis, and circulating cytotoxic lymphocytes specific to ORF II products (i.e., p13II and p30II) have been detected in both HTLV-1-infected ATL patients and asymptomatic persons (3, 11, 31). Furthermore, although initial studies reported that HTLV-1 ORF II was dispensable for viral infection in vitro (14, 33), the selective ablation of pX ORF II protein expression encoded by infectious HTLV-1 proviral clones dramatically reduced viral infectivity and host humoral responses in rabbits (2, 37), indicating the requirement of the pX ORF II-encoded proteins, p13II and p30II, for natural HTLV-1 infection. In addition, we reported the suppressive effect of p13II on both cell growth in vitro and tumorigenicity in a murine model (36). Collectively, these observations indicate a distinct role for p13II in HTLV-1 infection and a potential role in HTLV-1-mediated lymphocyte transformation.
For this study, we used both transient and stable expression methods to test the effect of mitochondrion-localizing HTLV-1 p13II in Jurkat T cells in response to apoptotic stimuli. Annexin V staining assays indicated that the Jurkat T cells expressing p13II were more sensitive to apoptosis in a dose-dependent manner when treated with synthetic ceramide and Fas ligand (FasL), known apoptosis inducers of T lymphocytes. Furthermore, preincubation of p13II-expressing Jurkat T cells with a farnesyl transferase inhibitor (FTI), which blocks the posttranslational modification of Ras to its active form, resulted in a marked, dose-dependent protection against apoptosis, indicating the involvement of Ras-mediated signaling in the p13II alteration of lymphocyte responses to apoptotic stimuli. Our study is the first to demonstrate a role of the mitochondrion-localizing protein p13II of HTLV-1 in the lymphocyte response to Ras-mediated apoptosis, and it reveals a potential new mechanism for the HTLV-1-induced alteration of lymphocyte proliferation.
MATERIALS AND METHODS
Cells. Jurkat T cells (clone E6-1; American Type Culture Collection) were maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum, 100 μg of streptomycin plus penicillin/ml, 2 mM L-glutamine, and 10 mM HEPES (cRPMI) (Invitrogen, Carlsbad, CA). 293T cells (American Type Culture Collection) and HeLa-Tat cells (AIDS Research and Reference Reagent Program, National Institutes of Health) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg of streptomycin plus penicillin/ml, and 2 mM L-glutamine (Invitrogen).
Plasmids. To generate the pMEp13II plasmid, the coding sequence of p13II was amplified from pACH (22), an HTLV-1 infectious molecular clone, using the primers 5' ATA CCT CGA GAT GCT TAT TAT CAG CCC A and 3' TAT AGC GGC CGC TCG ATG CAA GC. The amplified product was cloned into the pBR322 (Stratagene, La Jolla, CA) shuttle vector. Concurrently, a 39-bp sequence of the influenza virus hemagglutinin (HA) epitope domain was amplified by oligonucleotide annealing using the primer pair 5' AGC TTT ACC CAT ACG ATG TTC CAG ATT ACG CTA GCT TGC ATC GAT AAG 3' and 5' AAT TCT TAT CGA TGC AAG CTA GCG TAA TCT GGA ACA TCG TAT GGG TAA 3' and was cloned into the 3' end of the p13II sequence to tag the p13II protein with the HA epitope for immunodetection (pBR322p13IIHA). For the generation of the pMEp13II plasmid, the p13IIHA segment was obtained from pBR322p13IIHA by endonuclease digestion with XhoI and NotI and was then cloned into the pME18S plasmid (29; G. Franchini, National Cancer Institute, Bethesda, MD). The correct sequence was confirmed by Sanger DNA sequencing, and the expression of p13IIHA was confirmed by anti-HA immunoblotting following transient transfection by electroporation in Jurkat cells.
For the construction of a transfer vector expressing p13II (pWPTp13IIHA), the p13IIHA-encoding fragment was obtained by restriction digestion and inserted into the pWPT-eGFP lentiviral transfer vector (D. Trono, University of Geneva, Geneva, Switzerland). The encephalomyocarditis virus internal ribosomal entry site element from the pHRCMV-Tax-eGFP plasmid (39) was obtained by PCR amplification and cloned downstream of p13IIHA for the translation of a bicistronic enhanced green fluorescent protein (eGFP)- and p13II-expressing mRNA. The pWPT transfer vector with p13IIHA inserted in a reverse orientation (pWPT) was also made and used as a mock control vector.
Production of vesicular stomatitis virus glycoprotein-pseudotyped p13II lentiviral expression vector. To stably express p13II, a lentiviral vector was generated using a packaging plasmid (pCMV8.2) and a vesicular stomatitis virus glycoprotein plasmid (pHCMV-G) for packaging. The vector was produced by transient transfection of three plasmids (pCMV8.2, pHCMV-G, and pWPT transfer vectors) into 293T packaging. In brief, 1.2 x 107 early-passage 293T cells were seeded into 0.01% poly-L-lysine (Sigma, St. Louis, MO)-coated 15-cm culture dishes on the day before transfection. The cells were transfected with 5 μg of pCMV-G, 25 μg of pHCMVR8.2, and 25 μg of either pWPT- p13IIHA or pWPT by calcium phosphate-mediated transfection. The culture supernatants were collected at 24-h intervals for 96 h, cleared of cellular debris by centrifugation at 500 x g for 10 min, and filter sterilized (0.2 μm). The filtered supernatants were concentrated by centrifugation (6,000 x g for 16 h at 4°C). The pellet was suspended in 300 μl of complete Dulbecco's modified Eagle's medium overnight at 4°C, and the concentrated virus was stored at –80°C.
To determine the virus titer, virus stocks diluted 1:100, 1:1,000, or 1:10,000 were used to infect 293T cells, and eGFP expression was measured by flow cytometry at 48 h postinfection as described previously (15). Briefly, on the day before infection, 1 x 105 293T cells were seeded into a six-well plate. The medium was removed the following day, and the cells were then incubated with diluted virus-containing medium with Polybrene (5 μg/ml) (Sigma). The cells were spin infected by centrifugation at 1,400 x g for 1 h at 32°C and then left in the virus-containing medium for 16 h. After being fed with fresh medium and cultured for 48 h, the cells were collected and analyzed by fluorescence-activated cell sorting (FACS) using an ELITE ESP flow cytometer (Beckman Coulter, Fullerton, CA).
Lentiviral infection. Jurkat T cells (2.5 x 106 cells in 2 ml), 293T cells, or HeLa-Tat cells (both at 1 x 106 cells in 2 ml) were spin infected (1,400 x g at 32°C for 1 h) with the pWPTp13IIHA or pWPT mock lentiviral vector at a multiplicity of infection of 5 in the presence of Polybrene at 5 μg/ml (Sigma). The cells were left in the virus-containing medium for 16 h, fed with fresh medium, and cultured for at least 4 days to analyze p13II expression by Western immunoblotting.
DNA plasmid transfection. Jurkat T cells (1 x 107) were electroporated with 5, 10, or 20 μg of pMEp13II plasmid, using a Nucleofector kit (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions. The total amount of plasmid transfected into each sample was adjusted to 20 μg with pME18S (pME) plasmid. For Ras transfection, 1 x 107 Jurkat T cells were transfected with 20 μg of an Ha-Ras expression vector (pCMV-Ha-Ras) (BD Biosciences, San Jose, CA), using a Nucleofector kit (Amaxa Biosystems) according to the manufacturer's instructions. At 48 h posttransfection, the cells were collected and treated as described below.
Immunoprecipitation and Western blot assays. The expression of p13II was analyzed by Western blot assays. In brief, lentiviral vector-infected or transiently transfected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 10 mM EDTA, 10 mM NaF, 10 mM Na4P2O7 · H2O, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and Complete protease inhibitor [Roche Applied Science, Indianapolis, IN]), and the cell lysates were cleared by centrifugation (4°C at 16,000 x g for 15 min). Protein concentrations were determined with a BCA assay (micro-BCA protein assay; Pierce, IL), and a total of 50 μg of 293T cell lysates or 300 μg of infected Jurkat T-cell lysates was separated by SDS-15% polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween overnight, incubated with a mouse anti-HA monoclonal antibody (Ab; 1:1,000) (clone 16B-12; Covance Research Products, Princeton, NJ) overnight at 4°C, and developed by using a horseradish peroxidase-labeled secondary Ab (1:1,000) and an enhanced chemiluminescence reagent (Cell Signaling Technology, Beverly, MA).
For the detection of Ras, whole-cell lysates were prepared as described above. Five hundred micrograms of cell lysates were incubated with a 1:150 dilution of a mouse monoclonal anti-Ras Ab (Oncogene Research Products, San Diego, CA) overnight at 4°C. The immune complex mixture was then incubated with 50 μl of protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h. The beads were washed five times in RIPA buffer and boiled in SDS sample buffer, and supernatants were analyzed by Western immunoblotting using a mouse anti-Ras monoclonal Ab (1:1,000).
Cell growth and viability loss assays. For comparisons of cell growth curves, 1 x 105 to 5 x 105 viable Jurkat T cells were seeded into 24-well culture plate with 1 ml fresh medium. Viable cell numbers were counted by trypan blue dye exclusion every 24 h for 5 days. For additional cell growth and viability loss assays, an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] colorimetric cell viability assay kit (Promega, Madison, WI) was used according to the manufacturer's instructions. For stimulation protocols, Jurkat T cells were incubated with medium containing 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma)/ml, 2 μM ionomycin (Sigma), or a combination of 10 U/ml human interleukin-2 (Roche Applied Science) and 2 μg/ml phytohemagglutinin (Sigma). For stimulation with CD3/CD28 antibodies, 1 μg each of a mouse anti-human CD3 monoclonal antibody (clone HIT3a) and a mouse anti-human CD28 monoclonal antibody (clone Cd28.2; BD Pharmingen, San Diego, CA) was immobilized on an enzyme immunoassay-radioimmunoassay plate (Costar, Chicago, IL) in binding buffer (0.2 M sodium bicarbonate [pH 8.0]) overnight at 4°C. The wells were washed three times with PBS, followed by the addition of cells. At the end of the incubation, 20 μl of MTS reagent was added to each well and incubated for an additional 60 min. A photospectrometric microplate reader (Beckman Coulter, Fullerton, CA) was used to measure the optical density at 490 nm. For MTS cell viability loss assays, 40,000 viable cells were seeded in 96-well plates with various concentrations of FasL (0.2 to 0.8 μg/ml; Biomol, Plymouth Meeting, PA) or C2-ceramide (30 μM to 120 μM; Biomol) in a total volume of 100 μl per well for 6 h. All MTS experiments were performed with three or four replicates, and a minimum of two independent experiments were performed.
Annexin V apoptosis assay. Viable Jurkat T cells (2 x 105) were seeded into 24-well plates with fresh medium containing FasL (0.2 to 0.8 μg/ml; Biomol) or C2-ceramide (20 μM to 120 μM; Biomol) in a total volume of 1.0 ml per well for 1 to 4 h. zVAD.fmk and zIETD.fmk are inhibitors of pan-caspase and caspase 8, respectively (Biomol). Where indicated, the cells were preincubated with 20 μM of either zVAD.fmk or zIETD.fmk for 4 h prior to exposure to FasL (see Fig. 4A). The annexin V apoptosis assay was performed using an annexin V Alexa Fluor 647 conjugate (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. In brief, the cells were collected, washed once with PBS, and resuspended in 100 μl of annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4), followed by incubation with 5 μl of annexin V conjugate solution and 5 μl of 30 μM propidium iodide (PI) for 15 min at room temperature. After the incubation period, 400 μl of the annexin-binding buffer was added and mixed gently, and the samples were kept on ice. The samples were analyzed by flow cytometry (FACS Calibur; BD Coulter), and the data were analyzed using ProQuest software (BD Biosciences). For each sample, 10,000 gated cells were examined for their annexin V and PI staining, and the percentage of cells in early apoptosis was defined by measuring the high-annexin V- and low-PI-staining cell population, represented in the lower right quadrant of FACS analysis data (see Fig. 4A). All annexin V assays were performed in triplicate, and a minimum of two independent experiments were performed. Statistical analysis was performed by using Student's t tests. P values of <0.05 were considered significant.
Chemical blocking of Ras-mediated apoptosis. B581 (Biomol) is a farnesyl transferase inhibitor (FTI). Where indicated, Jurkat T cells were incubated with cRPMI containing either 50 or 200 nM of B581 for 16 h prior to exposure to FasL. To assess the potential toxicity of B581, the cells were also treated with 200 nM of B581 alone without a subsequent exposure to ceramide or FasL. Annexin V apoptosis assays were then performed as described above.
Immunofluorescence staining and confocal microscopy. To verify the mitochondrial localization of p13II, HeLa-Tat cells that were spin infected with the lentiviral vector at a multiplicity of infection of 5 were seeded into LAB-TEK chamber slides (Nalgene Nunc International, Rochester, NY). The cells were then fixed for 15 min in 4% paraformaldehyde at 24 h after being seeded, followed by permeabilization with 0.2% Triton X-100 (Sigma) for 5 min. Nonspecific binding sites were blocked with PBS containing 1% bovine serum albumin for 1 h at 37°C. After three washes, cells were incubated with mouse anti-human cytochrome c oxidase complex IV immunoglobulin G2a (IgG2a; 1:100) (Molecular Probes) as a mitochondrion-specific marker and with a secondary goat anti-mouse IgG2a Alexa Fluor 647 conjugate (1:200) (Molecular Probes) for 1 h each at room temperature. The p13II protein was stained with mouse anti-HA IgG1 (clone 16B-12; Covance Research Products) after preincubation with anti-mouse IgG1-Fc F(ab)2 Zenon anti-HA (Molecular Probe) for 1 h at room temperature. HeLa-Tat cells that were spin infected with mock viral vector were used as a negative control.
For analyses of Ras expression and subcellular localization, HeLa-Tat cells stably infected with p13II-expressing or mock control lentiviral vector were prepared as described above. Cell nuclei were stained with bisbenzimide H33528 (Hoechst 33528; Calbiochem, San Diego, CA) for 30 min at 37°C. The cells were fixed and blocked as described above and were then incubated with a mouse anti-pan-Ras antibody (1:150; Oncogene Research) and a secondary rabbit anti-mouse IgG-Alexa Fluor 546 conjugate (1:200) (Molecular Probes) for 1 h each at room temperature. A mouse IgG1 antibody (Caltag Laboratories, Burlingame, CA) was used as an isotype control. Fluorescence confocal microscopy and image collection were performed using a Leica TCS SP2 fluorescence microscope system, and images were analyzed with LCS software (Leica Microsystems, Bannockburn, IL).
RESULTS
Expression of HTLV-1 p13II in Jurkat T lymphocytes. A stable expression system for p13II in Jurkat T cells using a lentiviral vector system was established to test the influence of the HTLV-1 mitochondrion-localizing protein p13II on T-lymphocyte responses to apoptotic stimuli (Fig. 1). Corresponding mock cell lines were generated by spin infecting Jurkat T cells with a mock lentiviral vector. The stable expression of p13II was verified by a Western immunoblot assay (Fig. 2A). Specific mitochondrial localization and a mitochondrial targeting motif for p13II have been reported (5). Our HA epitope-tagged p13II expressed using the lentiviral vector system also colocalized with the cytochrome c dehydrogenase complex protein, a mitochondrion-specific marker, in HeLa-Tat cells (Fig. 2B).
Both direct cell counting and MTS colorimetric assays were performed to examine Jurkat T cells for possible changes in cell viability and growth pattern under normal culture conditions associated with the stable expression of p13II. Cultures with a low seeding density (5 x 104 to 10 x 104 cells/ml) did not reveal a significant difference in cell proliferation between two pairs of independently established p13II-expressing cell lines and their corresponding mock lines (data not shown). We then examined cell growth starting with a higher seeding density (5 x 105 cells/ml) (Fig. 3A). At later time points, the p13II-expressing line showed reduced proliferation compared to the mock line. The data indicated that the lentiviral vector-mediated constitutive expression of p13II did not alter the growth pattern of Jurkat T cells under normal culture conditions but resulted in reduced proliferation associated with a high-cell-density environment.
Next, we examined the proliferation responses of Jurkat T cells, with or without stable p13II expression, in the presence of various known growth stimulants, using MTS colorimetric assays. The cells were incubated with media containing ionomycin, phorbol myristate acetate, combinations of human IL-2 and phytohemagglutinin, or anti-CD3 and anti-CD28 antibodies in order to elicit proliferation as previously described (1). Our data indicated no significant difference in response to any combination tested between the p13II-expressing and mock cell lines (Fig. 3B) compared by average increases in optical density over the values for unstimulated samples of the corresponding parental cell lines. A series of proliferation assays performed under these conditions and measured by using tritiated thymidine revealed the same results (data not shown).
To further investigate potential phenotypic changes in Jurkat T cells due to stable p13II expression, the cells were analyzed for the magnitude of viability loss upon treatment with ceramide and FasL, known inducers of apoptotic cell death in lymphocytes. Compared to the untreated cells, the p13II-expressing Jurkat T cells exhibited a significant loss of viability (P < 0.05) at concentrations above 0.4 μg/ml and 30 μM for FasL and ceramide, respectively (Fig. 3C), indicating a significant magnitude of apoptotic events in p13II-expressing Jurkat T cells induced by these agents.
p13II-expressing lymphocytes are sensitive to apoptosis induced by FasL and ceramide. Based on the observation that the p13II-expressing Jurkat cells resulted in a pronounced viability loss upon stimulation with known inducers of lymphocyte apoptosis, we then performed apoptosis-specific annexin V assays to further characterize the differences in apoptosis responses among these cells. FasL treatment of p13II-expressing Jurkat T cells resulted in a marked induction of an early apoptotic cell population (42.0% for the FasL-treated versus 4.6% for the untreated cell population) (Fig. 4A, top panels). The specificity of the assay was verified by preincubation of the cells with zVAD.fmk (a pan-caspase inhibitor) or zIETD.fmk (a caspase 8 inhibitor) prior to treatment with FasL. Both inhibitors resulted in a nearly complete block of annexin V staining (4.2% and 3.5%, respectively) upon exposure to an apoptosis-inducing concentration (0.4 μg/ml) of FasL (Fig. 4A, bottom panels).
A comparison of the degrees to which apoptosis was induced by FasL was made by annexin V assays performed on two independently established Jurkat T-cell lines (p13-1 and p13-2) stably expressing p13II and on their corresponding mock control lines (Mock-1 and Mock-2) (Fig. 4B). The results showed that the p13II-expressing Jurkat T cells underwent significantly more apoptosis at all doses of FasL tested (P < 0.001). For each experiment, the constitutive expression of p13II in p13-1 and -2 and comparable levels of surface Fas expression were verified by Western immunoblot assays (data not shown). To answer the question of whether the sensitization to apoptosis was dependent on the amount of p13II expressed in Jurkat T cells, we performed transient transfection assays with the p13II expression plasmid (pMEp13II), followed by exposure of the cells to fixed, apoptosis-inducing concentrations of FasL (0.4 μg/ml) and ceramide (60 μM) (Fig. 5A and B). Although the increasing level of p13II expression itself did not induce apoptosis, subsequent exposures to both FasL and ceramide were associated with increased sensitivities to apoptosis (18.3 ± 2.9% and 4.9 ± 0.19% early apoptotic cells in Jurkat T cells without p13II versus 38.19 ± 0.56% and 26.11 ± 0.4% in cells transfected with 20 μg of pMEp13II plasmid, upon FasL and ceramide exposure, respectively).
FasL-induced apoptosis was reduced by pretreatment of p13II-expressing cells with a chemical inhibitor of Ras. We then sought to explore possible molecular pathways that predisposed the p13II-expressing cells to apoptosis. Both FasL and ceramide play a critical role in the regulation of apoptosis in Jurkat T cells, and Fas-FasL engagement results in a caspase 8-mediated activation of endogenous acidic sphingomyelinase, a potent inducer of ceramide accumulation during lymphocyte apoptosis (19, 34). Ras-mediated signaling is a key regulator of both FasL- and ceramide-induced apoptosis in lymphocytes (18), which can be altered using farnesyl transferase inhibitors (FTIs), which affect the posttranslational prenylation of Ras (7, 20, 35, 38).
To verify that an increased amount of Ras can sensitize Jurkat T cells to FasL-induced apoptosis and to test the specificity of FTI as an inhibitor of Ras function, we performed a transfection experiment where the Jurkat T cells were transiently transfected with a plasmid expressing wild-type Ha-Ras and subsequently exposed to apoptosis-inducing concentrations of FasL (Fig. 6A). The results showed that a transient overexpression of wild-type Ha-Ras was associated with an increased magnitude of apoptosis upon exposure to a fixed concentration of FasL (0.4 μg/ml), whereas the expression of Ha-Ras itself did not induce apoptosis (Fig. 6A, fourth bar). Secondly, the cells were pretreated with commercially available FTI posttransfection for 16 h prior to exposure to FasL to test its effectiveness at inhibiting Ras-mediated function (e.g., apoptosis). Our results showed a significant reduction in the magnitude of apoptosis in the cells overexpressing Ha-Ras, even upon exposure to FasL (Fig. 6A, last bar).
Subsequently, the pretreatment of the p13II-expressing Jurkat T cells with FTI was followed by FasL exposure, and alterations in apoptotic responses were measured by an annexin V assay. Our results indicated significant reductions of apoptosis in FTI-pretreated Jurkat T cells (Fig. 6B). The percentage of cells undergoing early apoptosis upon FasL exposure was reduced from 51.54 ± 0.57% without FTI treatment to 32.52 ± 2.76% in cells pretreated for 16 h with 50 nM of FTI prior to exposure to FasL. The increased concentration of FTI resulted in a reduction in apoptosis, as the percentage of early apoptotic cells was further reduced to 8.75 ± 1.20% when pretreated with a higher concentration of FTI (200 nM). These results indicated that Ras is a principal modulator of apoptosis in p13II-expressing Jurkat T cells. Western blot assays were performed to verify that the expression of p13II in the FTI-treated cells was unaffected (data not shown).
Subcellular localization of Ras is not altered by p13II expression. We then examined Jurkat T cells with or without p13II to determine whether the increased sensitivity of p13II-expressing cells to FasL- and ceramide-induced apoptosis was due to increased amounts of or alterations in the subcellular localization of Ras. Immunoprecipitation of Ras in the cellular lysates under normal culture conditions followed by Western immunoblotting revealed no apparent difference in the total amount of Ras between p13II-expressing and corresponding mock cell lines (Fig. 7A). There were also no observable differences in the subcellular localization of Ras in p13II-expressing and mock-infected HeLa-Tat cells (Fig. 7B). Furthermore, Ras did not appear to be colocalized with p13II in mitochondria in HeLa-Tat cells (data not shown).
DISCUSSION
The HTLV-1 accessory protein p13II selectively localizes to the inner membranes of mitochondria and also interacts with farnesyl pyrophosphate synthetase, which catalyzes the synthesis of farnesyl pyrophosphate, an essential substrate of Ras posttranslational modifications (5, 8, 24, 25). In this study, we analyzed the ability of HTLV-1 p13II to alter the response of Jurkat T lymphocytes to apoptosis-inducing stimuli. Our data revealed that stable and transient p13II expression in Jurkat T cells was associated with an increased sensitivity to apoptosis induced by both ceramide and FasL, in a dose-responsive manner. We demonstrated that this activity was caspase dependent, using inhibitors of pan-caspases and caspase 8. Furthermore, sensitization of the cells to apoptosis by p13II was reduced by pretreatment of the cells with B581, a farnesyl transferase inhibitor (FTI) which blocks the posttranslational prenylation of Ras. We extended this observation by correlating the overexpression of Ras with an increased sensitivity to FasL-induced apoptosis in Jurkat T cells. Collectively, our data reveal that p13II, a mitochondrion-localizing protein of HTLV-1, alters the lymphocyte response to apoptotic stimuli in a Ras-mediated manner.
Ras-mediated signaling plays many roles in cell survival, proliferation, transformation, and the modulation of apoptosis (7, 26). The regulation of apoptosis by Ras depends on many factors, including the isoform, subcellular localization, and posttranslational modification of the signaling protein (7). The detailed mechanisms by which Ras mediates apoptosis remain unclear, but they appear to involve the dysregulation of proteins downstream of Ras signaling, including transcription factors (6). In addition, unique isoforms of Ras appear to traffic to mitochondria upon FasL stimulation and directly bind Bcl-2, counteracting Bcl-2's antiapoptotic function in Jurkat T lymphocytes (13). A number of viral proteins that localize to mitochondria play roles in the regulation of apoptosis (10). Our data are the first to suggest that p13II in HTLV-1-infected cells serves to modify the functional response of mitochondria to Ras-mediated signaling that controls cell survival. Interestingly, Bovine leukemia virus, another member of the Deltaretrovirus genus, encodes an analogous accessory protein, G4, which also localizes to mitochondria, and both p13II and G4 directly interact with farnesyl pyrophosphate synthetase (25). These observations suggest a role for these proteins in the function of farnesyl pyrophosphate, an essential substrate of Ras posttranslational modifications. In this regard, it is interesting that bovine leukemia virus G4 was reported to require the cotransfection of Ras to enhance the tumorigenicity of transplanted rat embryonal fibroblasts in mice (21). In addition, we have recently reported that the expression of p13II in HeLa cells was associated with a significant suppression of the tumorigenicity of c-Myc- and Ha-Ras-cotransfected rat embryonal fibroblasts in a nude mouse model, providing additional evidence for the involvement of Ras in p13II-associated alterations in cell functions (36). Consistent with our previous report (36), the p13II-expressing Jurkat T-cell lines used for the present study also exhibited reduced proliferation at a high seeding density compared to mock vector cell lines. It is likely that p13II-expressing cells are reactive to cellular stress (e.g., reduced growth factors) associated with high-density culture conditions in a similar manner to their reactivity to apoptotic signals. Our findings reported herein further support the role of this accessory protein in modifying mitochondrion-mediated responses of lymphocytes to signaling events involving cell survival.
Our data provide intriguing evidence of a link between the Ras pathway and the HTLV-1 viral protein p13II in human lymphocytes. It seems that p13II does not alter the constitutive level of cellular Ras in Jurkat T cells or its subcellular localization in HeLa-Tat cells under normal culture conditions. We found that Ras does not appear to be colocalized with p13II in HeLa-Tat cells by fluorescence confocal microscopy (data not shown). The biological significance of the potential association of p13II with the Ras pathway, particularly in the context of HTLV-1 infection in human lymphocytes, remains to be determined. Unfortunately, there are no published reports that directly address the expression of this unique viral accessory protein during infection in HTLV-1-infected subjects or patients with disease. We therefore are unable to compare the levels of p13II expression achieved in the present study with those in lymphocytes during natural HTLV-1 infections. Determining the amount of any HTLV-1 protein in vivo has been difficult to do, in part because of the highly cell-associated manner of transmission of the virus in vivo. For our study, we designed p13II expression to be under the influence of the hematopoietic EF1 cellular promoter. This expression system resulted in low and stable constitutive expression of p13II to mimic the low expression level in lymphocytes. The p13II-mediated modulation of apoptotic stimuli in HTLV-1-infected cells may represent a mechanism by which the virus modifies the survival of a subset of infected cells (e.g., high-virus-expressing cells) to avoid the host immune system. To test the influence of p13II in the context of HTLV-1 infection, our present studies have focused on an HTLV-1 infectious molecular clone that lacks the ability to express p13II. The data presented herein are the first to indicate that HTLV-1 p13II alters the balance in Ras-mediated lymphocyte survival, and they provide new insights into HTLV-1-induced lymphocyte proliferation. These findings illustrate that further studies are needed to compare the complementary or competing roles of HTLV-1 regulatory (Tax and Rex) and accessory (e.g., p13II) proteins in HTLV-1 replication and associated disease. Additionally, future analyses focusing on the Ras-mediated function in lymphocytes will be of great interest and will provide a further understanding of the mechanisms by which HTLV-1 regulates the survival of infected lymphocytes.
ACKNOWLEDGMENTS
We thank G. Franchini and D. Trono for providing the plasmids, M. Kotour for technical support with confocal microscopy, B. Regner for flow cytometric analysis, and S. Fernandez for statistical consultation.
This work was supported by grants P01 CA100730 and R01 RR14324 (M.L.) from the National Cancer Institute and R03 TW05705 (M.L. and V.C.) from Fogarty Foundation, National Institutes of Health.
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Comprehensive Cancer Center
Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, Ohio
Department of Oncology and Surgical Sciences, University of Padova, Padua 35128, Italy
ABSTRACT
Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13II. We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13II localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13II, using both transient and stable expression vectors. Our data indicate that p13II-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13II-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13II-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13II's influence on lymphocyte survival. Our data are the first to demonstrate that p13II alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.
INTRODUCTION
Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL), a highly aggressive T-cell malignancy characterized by circulating activated CD4+ CD25+ T cells (12). The virus is also associated with a variety of lymphocyte-mediated diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (12, 16, 28). There are approximately 15 to 25 million HTLV-1-infected persons worldwide, and 3 to 5% of these infected subjects will develop HTLV-1-associated diseases (17). The underlying mechanism of virus-mediated lymphocyte transformation has been extensively investigated but is incompletely understood. Based on the long period of latency and the small percentage of individuals who develop ATL, the transformation of infected lymphocytes is believed to be initiated through the induction of cellular genes and alterations in cellular activation and death pathways by the viral proteins (28).
HTLV-1 is a member of the genus Deltaretrovirus of the family Retroviridae. The viral genome consists of long terminal repeats (LTRs) flanking genes encoding the structural and enzymatic proteins Gag, Pol, and Env. In addition, a region located between env and the 3' LTR, pX, encodes the regulatory proteins Tax and Rex as well as several accessory proteins, namely, p12I, p27I, p13II, and p30II (1). The ability of HTLV-1 to produce these regulatory and accessory proteins through alternative splicing and selective codon usage classifies the virus among the complex retroviruses (4, 23). Recent studies have indicated a significant role for HTLV-1 accessory proteins in the life cycle of HTLV-1, particularly during the early phase of the viral infection of lymphocytes (1, 9, 16, 27, 30, 32, 40). Less is known, however, about the accessory protein p13II, a singly spliced product of the second open reading frame (ORF II) of the pX gene region.
This protein selectively localizes to the inner membranes of mitochondria (5, 8) and directly binds to cellular protein farnesyl pyrophosphate synthetase (25). p13II mRNA is expressed in various HTLV-1-infected cell lines isolated from clinical patients with ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis, and circulating cytotoxic lymphocytes specific to ORF II products (i.e., p13II and p30II) have been detected in both HTLV-1-infected ATL patients and asymptomatic persons (3, 11, 31). Furthermore, although initial studies reported that HTLV-1 ORF II was dispensable for viral infection in vitro (14, 33), the selective ablation of pX ORF II protein expression encoded by infectious HTLV-1 proviral clones dramatically reduced viral infectivity and host humoral responses in rabbits (2, 37), indicating the requirement of the pX ORF II-encoded proteins, p13II and p30II, for natural HTLV-1 infection. In addition, we reported the suppressive effect of p13II on both cell growth in vitro and tumorigenicity in a murine model (36). Collectively, these observations indicate a distinct role for p13II in HTLV-1 infection and a potential role in HTLV-1-mediated lymphocyte transformation.
For this study, we used both transient and stable expression methods to test the effect of mitochondrion-localizing HTLV-1 p13II in Jurkat T cells in response to apoptotic stimuli. Annexin V staining assays indicated that the Jurkat T cells expressing p13II were more sensitive to apoptosis in a dose-dependent manner when treated with synthetic ceramide and Fas ligand (FasL), known apoptosis inducers of T lymphocytes. Furthermore, preincubation of p13II-expressing Jurkat T cells with a farnesyl transferase inhibitor (FTI), which blocks the posttranslational modification of Ras to its active form, resulted in a marked, dose-dependent protection against apoptosis, indicating the involvement of Ras-mediated signaling in the p13II alteration of lymphocyte responses to apoptotic stimuli. Our study is the first to demonstrate a role of the mitochondrion-localizing protein p13II of HTLV-1 in the lymphocyte response to Ras-mediated apoptosis, and it reveals a potential new mechanism for the HTLV-1-induced alteration of lymphocyte proliferation.
MATERIALS AND METHODS
Cells. Jurkat T cells (clone E6-1; American Type Culture Collection) were maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum, 100 μg of streptomycin plus penicillin/ml, 2 mM L-glutamine, and 10 mM HEPES (cRPMI) (Invitrogen, Carlsbad, CA). 293T cells (American Type Culture Collection) and HeLa-Tat cells (AIDS Research and Reference Reagent Program, National Institutes of Health) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg of streptomycin plus penicillin/ml, and 2 mM L-glutamine (Invitrogen).
Plasmids. To generate the pMEp13II plasmid, the coding sequence of p13II was amplified from pACH (22), an HTLV-1 infectious molecular clone, using the primers 5' ATA CCT CGA GAT GCT TAT TAT CAG CCC A and 3' TAT AGC GGC CGC TCG ATG CAA GC. The amplified product was cloned into the pBR322 (Stratagene, La Jolla, CA) shuttle vector. Concurrently, a 39-bp sequence of the influenza virus hemagglutinin (HA) epitope domain was amplified by oligonucleotide annealing using the primer pair 5' AGC TTT ACC CAT ACG ATG TTC CAG ATT ACG CTA GCT TGC ATC GAT AAG 3' and 5' AAT TCT TAT CGA TGC AAG CTA GCG TAA TCT GGA ACA TCG TAT GGG TAA 3' and was cloned into the 3' end of the p13II sequence to tag the p13II protein with the HA epitope for immunodetection (pBR322p13IIHA). For the generation of the pMEp13II plasmid, the p13IIHA segment was obtained from pBR322p13IIHA by endonuclease digestion with XhoI and NotI and was then cloned into the pME18S plasmid (29; G. Franchini, National Cancer Institute, Bethesda, MD). The correct sequence was confirmed by Sanger DNA sequencing, and the expression of p13IIHA was confirmed by anti-HA immunoblotting following transient transfection by electroporation in Jurkat cells.
For the construction of a transfer vector expressing p13II (pWPTp13IIHA), the p13IIHA-encoding fragment was obtained by restriction digestion and inserted into the pWPT-eGFP lentiviral transfer vector (D. Trono, University of Geneva, Geneva, Switzerland). The encephalomyocarditis virus internal ribosomal entry site element from the pHRCMV-Tax-eGFP plasmid (39) was obtained by PCR amplification and cloned downstream of p13IIHA for the translation of a bicistronic enhanced green fluorescent protein (eGFP)- and p13II-expressing mRNA. The pWPT transfer vector with p13IIHA inserted in a reverse orientation (pWPT) was also made and used as a mock control vector.
Production of vesicular stomatitis virus glycoprotein-pseudotyped p13II lentiviral expression vector. To stably express p13II, a lentiviral vector was generated using a packaging plasmid (pCMV8.2) and a vesicular stomatitis virus glycoprotein plasmid (pHCMV-G) for packaging. The vector was produced by transient transfection of three plasmids (pCMV8.2, pHCMV-G, and pWPT transfer vectors) into 293T packaging. In brief, 1.2 x 107 early-passage 293T cells were seeded into 0.01% poly-L-lysine (Sigma, St. Louis, MO)-coated 15-cm culture dishes on the day before transfection. The cells were transfected with 5 μg of pCMV-G, 25 μg of pHCMVR8.2, and 25 μg of either pWPT- p13IIHA or pWPT by calcium phosphate-mediated transfection. The culture supernatants were collected at 24-h intervals for 96 h, cleared of cellular debris by centrifugation at 500 x g for 10 min, and filter sterilized (0.2 μm). The filtered supernatants were concentrated by centrifugation (6,000 x g for 16 h at 4°C). The pellet was suspended in 300 μl of complete Dulbecco's modified Eagle's medium overnight at 4°C, and the concentrated virus was stored at –80°C.
To determine the virus titer, virus stocks diluted 1:100, 1:1,000, or 1:10,000 were used to infect 293T cells, and eGFP expression was measured by flow cytometry at 48 h postinfection as described previously (15). Briefly, on the day before infection, 1 x 105 293T cells were seeded into a six-well plate. The medium was removed the following day, and the cells were then incubated with diluted virus-containing medium with Polybrene (5 μg/ml) (Sigma). The cells were spin infected by centrifugation at 1,400 x g for 1 h at 32°C and then left in the virus-containing medium for 16 h. After being fed with fresh medium and cultured for 48 h, the cells were collected and analyzed by fluorescence-activated cell sorting (FACS) using an ELITE ESP flow cytometer (Beckman Coulter, Fullerton, CA).
Lentiviral infection. Jurkat T cells (2.5 x 106 cells in 2 ml), 293T cells, or HeLa-Tat cells (both at 1 x 106 cells in 2 ml) were spin infected (1,400 x g at 32°C for 1 h) with the pWPTp13IIHA or pWPT mock lentiviral vector at a multiplicity of infection of 5 in the presence of Polybrene at 5 μg/ml (Sigma). The cells were left in the virus-containing medium for 16 h, fed with fresh medium, and cultured for at least 4 days to analyze p13II expression by Western immunoblotting.
DNA plasmid transfection. Jurkat T cells (1 x 107) were electroporated with 5, 10, or 20 μg of pMEp13II plasmid, using a Nucleofector kit (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions. The total amount of plasmid transfected into each sample was adjusted to 20 μg with pME18S (pME) plasmid. For Ras transfection, 1 x 107 Jurkat T cells were transfected with 20 μg of an Ha-Ras expression vector (pCMV-Ha-Ras) (BD Biosciences, San Jose, CA), using a Nucleofector kit (Amaxa Biosystems) according to the manufacturer's instructions. At 48 h posttransfection, the cells were collected and treated as described below.
Immunoprecipitation and Western blot assays. The expression of p13II was analyzed by Western blot assays. In brief, lentiviral vector-infected or transiently transfected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 10 mM EDTA, 10 mM NaF, 10 mM Na4P2O7 · H2O, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and Complete protease inhibitor [Roche Applied Science, Indianapolis, IN]), and the cell lysates were cleared by centrifugation (4°C at 16,000 x g for 15 min). Protein concentrations were determined with a BCA assay (micro-BCA protein assay; Pierce, IL), and a total of 50 μg of 293T cell lysates or 300 μg of infected Jurkat T-cell lysates was separated by SDS-15% polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween overnight, incubated with a mouse anti-HA monoclonal antibody (Ab; 1:1,000) (clone 16B-12; Covance Research Products, Princeton, NJ) overnight at 4°C, and developed by using a horseradish peroxidase-labeled secondary Ab (1:1,000) and an enhanced chemiluminescence reagent (Cell Signaling Technology, Beverly, MA).
For the detection of Ras, whole-cell lysates were prepared as described above. Five hundred micrograms of cell lysates were incubated with a 1:150 dilution of a mouse monoclonal anti-Ras Ab (Oncogene Research Products, San Diego, CA) overnight at 4°C. The immune complex mixture was then incubated with 50 μl of protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h. The beads were washed five times in RIPA buffer and boiled in SDS sample buffer, and supernatants were analyzed by Western immunoblotting using a mouse anti-Ras monoclonal Ab (1:1,000).
Cell growth and viability loss assays. For comparisons of cell growth curves, 1 x 105 to 5 x 105 viable Jurkat T cells were seeded into 24-well culture plate with 1 ml fresh medium. Viable cell numbers were counted by trypan blue dye exclusion every 24 h for 5 days. For additional cell growth and viability loss assays, an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] colorimetric cell viability assay kit (Promega, Madison, WI) was used according to the manufacturer's instructions. For stimulation protocols, Jurkat T cells were incubated with medium containing 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma)/ml, 2 μM ionomycin (Sigma), or a combination of 10 U/ml human interleukin-2 (Roche Applied Science) and 2 μg/ml phytohemagglutinin (Sigma). For stimulation with CD3/CD28 antibodies, 1 μg each of a mouse anti-human CD3 monoclonal antibody (clone HIT3a) and a mouse anti-human CD28 monoclonal antibody (clone Cd28.2; BD Pharmingen, San Diego, CA) was immobilized on an enzyme immunoassay-radioimmunoassay plate (Costar, Chicago, IL) in binding buffer (0.2 M sodium bicarbonate [pH 8.0]) overnight at 4°C. The wells were washed three times with PBS, followed by the addition of cells. At the end of the incubation, 20 μl of MTS reagent was added to each well and incubated for an additional 60 min. A photospectrometric microplate reader (Beckman Coulter, Fullerton, CA) was used to measure the optical density at 490 nm. For MTS cell viability loss assays, 40,000 viable cells were seeded in 96-well plates with various concentrations of FasL (0.2 to 0.8 μg/ml; Biomol, Plymouth Meeting, PA) or C2-ceramide (30 μM to 120 μM; Biomol) in a total volume of 100 μl per well for 6 h. All MTS experiments were performed with three or four replicates, and a minimum of two independent experiments were performed.
Annexin V apoptosis assay. Viable Jurkat T cells (2 x 105) were seeded into 24-well plates with fresh medium containing FasL (0.2 to 0.8 μg/ml; Biomol) or C2-ceramide (20 μM to 120 μM; Biomol) in a total volume of 1.0 ml per well for 1 to 4 h. zVAD.fmk and zIETD.fmk are inhibitors of pan-caspase and caspase 8, respectively (Biomol). Where indicated, the cells were preincubated with 20 μM of either zVAD.fmk or zIETD.fmk for 4 h prior to exposure to FasL (see Fig. 4A). The annexin V apoptosis assay was performed using an annexin V Alexa Fluor 647 conjugate (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. In brief, the cells were collected, washed once with PBS, and resuspended in 100 μl of annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4), followed by incubation with 5 μl of annexin V conjugate solution and 5 μl of 30 μM propidium iodide (PI) for 15 min at room temperature. After the incubation period, 400 μl of the annexin-binding buffer was added and mixed gently, and the samples were kept on ice. The samples were analyzed by flow cytometry (FACS Calibur; BD Coulter), and the data were analyzed using ProQuest software (BD Biosciences). For each sample, 10,000 gated cells were examined for their annexin V and PI staining, and the percentage of cells in early apoptosis was defined by measuring the high-annexin V- and low-PI-staining cell population, represented in the lower right quadrant of FACS analysis data (see Fig. 4A). All annexin V assays were performed in triplicate, and a minimum of two independent experiments were performed. Statistical analysis was performed by using Student's t tests. P values of <0.05 were considered significant.
Chemical blocking of Ras-mediated apoptosis. B581 (Biomol) is a farnesyl transferase inhibitor (FTI). Where indicated, Jurkat T cells were incubated with cRPMI containing either 50 or 200 nM of B581 for 16 h prior to exposure to FasL. To assess the potential toxicity of B581, the cells were also treated with 200 nM of B581 alone without a subsequent exposure to ceramide or FasL. Annexin V apoptosis assays were then performed as described above.
Immunofluorescence staining and confocal microscopy. To verify the mitochondrial localization of p13II, HeLa-Tat cells that were spin infected with the lentiviral vector at a multiplicity of infection of 5 were seeded into LAB-TEK chamber slides (Nalgene Nunc International, Rochester, NY). The cells were then fixed for 15 min in 4% paraformaldehyde at 24 h after being seeded, followed by permeabilization with 0.2% Triton X-100 (Sigma) for 5 min. Nonspecific binding sites were blocked with PBS containing 1% bovine serum albumin for 1 h at 37°C. After three washes, cells were incubated with mouse anti-human cytochrome c oxidase complex IV immunoglobulin G2a (IgG2a; 1:100) (Molecular Probes) as a mitochondrion-specific marker and with a secondary goat anti-mouse IgG2a Alexa Fluor 647 conjugate (1:200) (Molecular Probes) for 1 h each at room temperature. The p13II protein was stained with mouse anti-HA IgG1 (clone 16B-12; Covance Research Products) after preincubation with anti-mouse IgG1-Fc F(ab)2 Zenon anti-HA (Molecular Probe) for 1 h at room temperature. HeLa-Tat cells that were spin infected with mock viral vector were used as a negative control.
For analyses of Ras expression and subcellular localization, HeLa-Tat cells stably infected with p13II-expressing or mock control lentiviral vector were prepared as described above. Cell nuclei were stained with bisbenzimide H33528 (Hoechst 33528; Calbiochem, San Diego, CA) for 30 min at 37°C. The cells were fixed and blocked as described above and were then incubated with a mouse anti-pan-Ras antibody (1:150; Oncogene Research) and a secondary rabbit anti-mouse IgG-Alexa Fluor 546 conjugate (1:200) (Molecular Probes) for 1 h each at room temperature. A mouse IgG1 antibody (Caltag Laboratories, Burlingame, CA) was used as an isotype control. Fluorescence confocal microscopy and image collection were performed using a Leica TCS SP2 fluorescence microscope system, and images were analyzed with LCS software (Leica Microsystems, Bannockburn, IL).
RESULTS
Expression of HTLV-1 p13II in Jurkat T lymphocytes. A stable expression system for p13II in Jurkat T cells using a lentiviral vector system was established to test the influence of the HTLV-1 mitochondrion-localizing protein p13II on T-lymphocyte responses to apoptotic stimuli (Fig. 1). Corresponding mock cell lines were generated by spin infecting Jurkat T cells with a mock lentiviral vector. The stable expression of p13II was verified by a Western immunoblot assay (Fig. 2A). Specific mitochondrial localization and a mitochondrial targeting motif for p13II have been reported (5). Our HA epitope-tagged p13II expressed using the lentiviral vector system also colocalized with the cytochrome c dehydrogenase complex protein, a mitochondrion-specific marker, in HeLa-Tat cells (Fig. 2B).
Both direct cell counting and MTS colorimetric assays were performed to examine Jurkat T cells for possible changes in cell viability and growth pattern under normal culture conditions associated with the stable expression of p13II. Cultures with a low seeding density (5 x 104 to 10 x 104 cells/ml) did not reveal a significant difference in cell proliferation between two pairs of independently established p13II-expressing cell lines and their corresponding mock lines (data not shown). We then examined cell growth starting with a higher seeding density (5 x 105 cells/ml) (Fig. 3A). At later time points, the p13II-expressing line showed reduced proliferation compared to the mock line. The data indicated that the lentiviral vector-mediated constitutive expression of p13II did not alter the growth pattern of Jurkat T cells under normal culture conditions but resulted in reduced proliferation associated with a high-cell-density environment.
Next, we examined the proliferation responses of Jurkat T cells, with or without stable p13II expression, in the presence of various known growth stimulants, using MTS colorimetric assays. The cells were incubated with media containing ionomycin, phorbol myristate acetate, combinations of human IL-2 and phytohemagglutinin, or anti-CD3 and anti-CD28 antibodies in order to elicit proliferation as previously described (1). Our data indicated no significant difference in response to any combination tested between the p13II-expressing and mock cell lines (Fig. 3B) compared by average increases in optical density over the values for unstimulated samples of the corresponding parental cell lines. A series of proliferation assays performed under these conditions and measured by using tritiated thymidine revealed the same results (data not shown).
To further investigate potential phenotypic changes in Jurkat T cells due to stable p13II expression, the cells were analyzed for the magnitude of viability loss upon treatment with ceramide and FasL, known inducers of apoptotic cell death in lymphocytes. Compared to the untreated cells, the p13II-expressing Jurkat T cells exhibited a significant loss of viability (P < 0.05) at concentrations above 0.4 μg/ml and 30 μM for FasL and ceramide, respectively (Fig. 3C), indicating a significant magnitude of apoptotic events in p13II-expressing Jurkat T cells induced by these agents.
p13II-expressing lymphocytes are sensitive to apoptosis induced by FasL and ceramide. Based on the observation that the p13II-expressing Jurkat cells resulted in a pronounced viability loss upon stimulation with known inducers of lymphocyte apoptosis, we then performed apoptosis-specific annexin V assays to further characterize the differences in apoptosis responses among these cells. FasL treatment of p13II-expressing Jurkat T cells resulted in a marked induction of an early apoptotic cell population (42.0% for the FasL-treated versus 4.6% for the untreated cell population) (Fig. 4A, top panels). The specificity of the assay was verified by preincubation of the cells with zVAD.fmk (a pan-caspase inhibitor) or zIETD.fmk (a caspase 8 inhibitor) prior to treatment with FasL. Both inhibitors resulted in a nearly complete block of annexin V staining (4.2% and 3.5%, respectively) upon exposure to an apoptosis-inducing concentration (0.4 μg/ml) of FasL (Fig. 4A, bottom panels).
A comparison of the degrees to which apoptosis was induced by FasL was made by annexin V assays performed on two independently established Jurkat T-cell lines (p13-1 and p13-2) stably expressing p13II and on their corresponding mock control lines (Mock-1 and Mock-2) (Fig. 4B). The results showed that the p13II-expressing Jurkat T cells underwent significantly more apoptosis at all doses of FasL tested (P < 0.001). For each experiment, the constitutive expression of p13II in p13-1 and -2 and comparable levels of surface Fas expression were verified by Western immunoblot assays (data not shown). To answer the question of whether the sensitization to apoptosis was dependent on the amount of p13II expressed in Jurkat T cells, we performed transient transfection assays with the p13II expression plasmid (pMEp13II), followed by exposure of the cells to fixed, apoptosis-inducing concentrations of FasL (0.4 μg/ml) and ceramide (60 μM) (Fig. 5A and B). Although the increasing level of p13II expression itself did not induce apoptosis, subsequent exposures to both FasL and ceramide were associated with increased sensitivities to apoptosis (18.3 ± 2.9% and 4.9 ± 0.19% early apoptotic cells in Jurkat T cells without p13II versus 38.19 ± 0.56% and 26.11 ± 0.4% in cells transfected with 20 μg of pMEp13II plasmid, upon FasL and ceramide exposure, respectively).
FasL-induced apoptosis was reduced by pretreatment of p13II-expressing cells with a chemical inhibitor of Ras. We then sought to explore possible molecular pathways that predisposed the p13II-expressing cells to apoptosis. Both FasL and ceramide play a critical role in the regulation of apoptosis in Jurkat T cells, and Fas-FasL engagement results in a caspase 8-mediated activation of endogenous acidic sphingomyelinase, a potent inducer of ceramide accumulation during lymphocyte apoptosis (19, 34). Ras-mediated signaling is a key regulator of both FasL- and ceramide-induced apoptosis in lymphocytes (18), which can be altered using farnesyl transferase inhibitors (FTIs), which affect the posttranslational prenylation of Ras (7, 20, 35, 38).
To verify that an increased amount of Ras can sensitize Jurkat T cells to FasL-induced apoptosis and to test the specificity of FTI as an inhibitor of Ras function, we performed a transfection experiment where the Jurkat T cells were transiently transfected with a plasmid expressing wild-type Ha-Ras and subsequently exposed to apoptosis-inducing concentrations of FasL (Fig. 6A). The results showed that a transient overexpression of wild-type Ha-Ras was associated with an increased magnitude of apoptosis upon exposure to a fixed concentration of FasL (0.4 μg/ml), whereas the expression of Ha-Ras itself did not induce apoptosis (Fig. 6A, fourth bar). Secondly, the cells were pretreated with commercially available FTI posttransfection for 16 h prior to exposure to FasL to test its effectiveness at inhibiting Ras-mediated function (e.g., apoptosis). Our results showed a significant reduction in the magnitude of apoptosis in the cells overexpressing Ha-Ras, even upon exposure to FasL (Fig. 6A, last bar).
Subsequently, the pretreatment of the p13II-expressing Jurkat T cells with FTI was followed by FasL exposure, and alterations in apoptotic responses were measured by an annexin V assay. Our results indicated significant reductions of apoptosis in FTI-pretreated Jurkat T cells (Fig. 6B). The percentage of cells undergoing early apoptosis upon FasL exposure was reduced from 51.54 ± 0.57% without FTI treatment to 32.52 ± 2.76% in cells pretreated for 16 h with 50 nM of FTI prior to exposure to FasL. The increased concentration of FTI resulted in a reduction in apoptosis, as the percentage of early apoptotic cells was further reduced to 8.75 ± 1.20% when pretreated with a higher concentration of FTI (200 nM). These results indicated that Ras is a principal modulator of apoptosis in p13II-expressing Jurkat T cells. Western blot assays were performed to verify that the expression of p13II in the FTI-treated cells was unaffected (data not shown).
Subcellular localization of Ras is not altered by p13II expression. We then examined Jurkat T cells with or without p13II to determine whether the increased sensitivity of p13II-expressing cells to FasL- and ceramide-induced apoptosis was due to increased amounts of or alterations in the subcellular localization of Ras. Immunoprecipitation of Ras in the cellular lysates under normal culture conditions followed by Western immunoblotting revealed no apparent difference in the total amount of Ras between p13II-expressing and corresponding mock cell lines (Fig. 7A). There were also no observable differences in the subcellular localization of Ras in p13II-expressing and mock-infected HeLa-Tat cells (Fig. 7B). Furthermore, Ras did not appear to be colocalized with p13II in mitochondria in HeLa-Tat cells (data not shown).
DISCUSSION
The HTLV-1 accessory protein p13II selectively localizes to the inner membranes of mitochondria and also interacts with farnesyl pyrophosphate synthetase, which catalyzes the synthesis of farnesyl pyrophosphate, an essential substrate of Ras posttranslational modifications (5, 8, 24, 25). In this study, we analyzed the ability of HTLV-1 p13II to alter the response of Jurkat T lymphocytes to apoptosis-inducing stimuli. Our data revealed that stable and transient p13II expression in Jurkat T cells was associated with an increased sensitivity to apoptosis induced by both ceramide and FasL, in a dose-responsive manner. We demonstrated that this activity was caspase dependent, using inhibitors of pan-caspases and caspase 8. Furthermore, sensitization of the cells to apoptosis by p13II was reduced by pretreatment of the cells with B581, a farnesyl transferase inhibitor (FTI) which blocks the posttranslational prenylation of Ras. We extended this observation by correlating the overexpression of Ras with an increased sensitivity to FasL-induced apoptosis in Jurkat T cells. Collectively, our data reveal that p13II, a mitochondrion-localizing protein of HTLV-1, alters the lymphocyte response to apoptotic stimuli in a Ras-mediated manner.
Ras-mediated signaling plays many roles in cell survival, proliferation, transformation, and the modulation of apoptosis (7, 26). The regulation of apoptosis by Ras depends on many factors, including the isoform, subcellular localization, and posttranslational modification of the signaling protein (7). The detailed mechanisms by which Ras mediates apoptosis remain unclear, but they appear to involve the dysregulation of proteins downstream of Ras signaling, including transcription factors (6). In addition, unique isoforms of Ras appear to traffic to mitochondria upon FasL stimulation and directly bind Bcl-2, counteracting Bcl-2's antiapoptotic function in Jurkat T lymphocytes (13). A number of viral proteins that localize to mitochondria play roles in the regulation of apoptosis (10). Our data are the first to suggest that p13II in HTLV-1-infected cells serves to modify the functional response of mitochondria to Ras-mediated signaling that controls cell survival. Interestingly, Bovine leukemia virus, another member of the Deltaretrovirus genus, encodes an analogous accessory protein, G4, which also localizes to mitochondria, and both p13II and G4 directly interact with farnesyl pyrophosphate synthetase (25). These observations suggest a role for these proteins in the function of farnesyl pyrophosphate, an essential substrate of Ras posttranslational modifications. In this regard, it is interesting that bovine leukemia virus G4 was reported to require the cotransfection of Ras to enhance the tumorigenicity of transplanted rat embryonal fibroblasts in mice (21). In addition, we have recently reported that the expression of p13II in HeLa cells was associated with a significant suppression of the tumorigenicity of c-Myc- and Ha-Ras-cotransfected rat embryonal fibroblasts in a nude mouse model, providing additional evidence for the involvement of Ras in p13II-associated alterations in cell functions (36). Consistent with our previous report (36), the p13II-expressing Jurkat T-cell lines used for the present study also exhibited reduced proliferation at a high seeding density compared to mock vector cell lines. It is likely that p13II-expressing cells are reactive to cellular stress (e.g., reduced growth factors) associated with high-density culture conditions in a similar manner to their reactivity to apoptotic signals. Our findings reported herein further support the role of this accessory protein in modifying mitochondrion-mediated responses of lymphocytes to signaling events involving cell survival.
Our data provide intriguing evidence of a link between the Ras pathway and the HTLV-1 viral protein p13II in human lymphocytes. It seems that p13II does not alter the constitutive level of cellular Ras in Jurkat T cells or its subcellular localization in HeLa-Tat cells under normal culture conditions. We found that Ras does not appear to be colocalized with p13II in HeLa-Tat cells by fluorescence confocal microscopy (data not shown). The biological significance of the potential association of p13II with the Ras pathway, particularly in the context of HTLV-1 infection in human lymphocytes, remains to be determined. Unfortunately, there are no published reports that directly address the expression of this unique viral accessory protein during infection in HTLV-1-infected subjects or patients with disease. We therefore are unable to compare the levels of p13II expression achieved in the present study with those in lymphocytes during natural HTLV-1 infections. Determining the amount of any HTLV-1 protein in vivo has been difficult to do, in part because of the highly cell-associated manner of transmission of the virus in vivo. For our study, we designed p13II expression to be under the influence of the hematopoietic EF1 cellular promoter. This expression system resulted in low and stable constitutive expression of p13II to mimic the low expression level in lymphocytes. The p13II-mediated modulation of apoptotic stimuli in HTLV-1-infected cells may represent a mechanism by which the virus modifies the survival of a subset of infected cells (e.g., high-virus-expressing cells) to avoid the host immune system. To test the influence of p13II in the context of HTLV-1 infection, our present studies have focused on an HTLV-1 infectious molecular clone that lacks the ability to express p13II. The data presented herein are the first to indicate that HTLV-1 p13II alters the balance in Ras-mediated lymphocyte survival, and they provide new insights into HTLV-1-induced lymphocyte proliferation. These findings illustrate that further studies are needed to compare the complementary or competing roles of HTLV-1 regulatory (Tax and Rex) and accessory (e.g., p13II) proteins in HTLV-1 replication and associated disease. Additionally, future analyses focusing on the Ras-mediated function in lymphocytes will be of great interest and will provide a further understanding of the mechanisms by which HTLV-1 regulates the survival of infected lymphocytes.
ACKNOWLEDGMENTS
We thank G. Franchini and D. Trono for providing the plasmids, M. Kotour for technical support with confocal microscopy, B. Regner for flow cytometric analysis, and S. Fernandez for statistical consultation.
This work was supported by grants P01 CA100730 and R01 RR14324 (M.L.) from the National Cancer Institute and R03 TW05705 (M.L. and V.C.) from Fogarty Foundation, National Institutes of Health.
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