Apoptosis mediated by HIV protease is preceded by cleavage ... - PNAS

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9571-9576, September 1996 Cell Biology

Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2 (AIDS/cell death/NFicB/proteolysis) PETER R. STRACK*, MICHELLE WEST FREY, CHRISTOPHER J. RIzzo, BEVERLY CORDOVA, HENRY J. GEORGE, RAYMOND MEADE, SIEW PENG Ho, JEANNE CORMAN, RADONNA TRITCH, AND BRUCE D. KORANTt Molecular Biology Department, DuPont Merck Pharmaceutical Company, Experimental Station, Building 336, Wilmington, DE 19880-0336

Communicated by James E. Damell, Jr., The Rockefeller University, New York, NY May 20, 1996 (received for review March 1, 1996)

genes were used for transient transfection of cells. Other strong promoters could be substituted with no difference in results. Plasmid DNA was purified using cesium chloride density gradient centrifugation. Cells (107) were transfected with purified plasmid DNA using a Bio-Rad Genepulser, and incubated at 37°C in 5% C02/95% air in Dulbecco's modified minimal essential medium or RPMI 1640 (GIBCO), containing 10% fetal calf serum. Assays. Measurement of cellular oxidative status. Cells were assayed for glutathione levels (9), intracellular lipid peroxidation (10), binding of nuclear extracts to a 32P end-labeled oligonucleotide specific for NFKB (11) or TNF antigen released into the extracellular medium using a monoclonal antibody (R & D Systems). Cleavage of 35S-labeled bcl-2. 35S-L-methionine-labeled bcl-2 was synthesized in a rabbit reticulocyte lysate coupled transcription/translation system (Promega), and reactions were terminated with cycloheximide (20 ,ug/ml). Canine pancreatic microsomal membranes (four equivalents) were added and incubated for an additional 30 min at 37°C. Membraneassociated bcl-2 was recovered by centrifugation in an Airfuge (Beckman) for 10 min at 25 psi. Pellets were washed (50 mM triethanolamine, pH 7.5/250 mM sucrose/2 mM DTT), centrifuged, and suspended in 40 ,l of assay buffer (12). HIV protease (0.18 mg/ml) was added to membrane-associated bcl-2, incubated at 37°C for 30 min, and the products separated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate (SDS/PAGE) and detected by autoradiography. Determination of HIVprotease cleavage sites in bcl-2. Bcl-2 was purified from Sf21 insect cells infected with a baculovirus expressing human bcl-2 cDNA (38). Cells (109) were lysed by sonication (3 x 20 sec) in 50 ml of lysis buffer [25 mM Tris-HCl, pH 8.0/5 mM EDTA/2 mM 2-mercaptoethanol/1 mM Pefabloc SC (Boehringer Mannheim)/5 ,uM leupeptin/ 1.6 mM benzamidine/0.5 ,uM aprotinin) and centrifuged for 1 h at 105,000 x g. The pellet was washed 2 times in lysis buffer containing 1% Triton X-100 and 1 M NaCl, solubilized in lysis buffer containing 6 M guanidine-HCI (GdnHCI) using 10 strokes of a Dounce homogenizer each time, gently stirred overnight at 4°C, and centrifuged (39). Soluble material was diluted to 2 M GdnHCl with lysis buffer, applied to a phenylsepharose high performance column (2.6 x 14 cm, Pharmacia) and the column washed with lysis buffer (100 ml) containing 2 M GdnHCl. Bound bcl-2 was eluted with 200 ml of lysis buffer containing 6 M GdnHCl. Eluant was concentrated to 7.0 ml (1.8 mg/ml) using an Amicon stirred cell (3 kDa Mr cutoff) and stored at -80°C. Concentrated material (100 ,lA) was adjusted to 20% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA), centrifuged for 2 min at 14,000 x g and the soluble material applied to an HPLC-C18 (Vydak

ABSTRACT Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor a. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NFKB, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site. Loss of immunocompetent lymphocytes, as well as other cell types, is the hallmark of progressive infection by HIV. Lymphocytes infected in vitro soon die by the process of apoptosis and cells taken from HIV-infected people proceed to apoptotic death when placed in culture (1, 2). Examination of biopsy or autopsy material from HIV-infected people reveals apoptosis coincident with or adjacent to infected cells (3). There are reports of the HIV gene products tat and gpl20 having cytotoxic or apoptotic effects, but usually a secondary stimulus must be included, such as serum factor depletion or addition of cell surface antibodies (4). In comparison, Krausslich (5) reported direct toxic effects following expression of the HIV aspartic protease in cultured cells. We have examined HIV protease-expressing cells in greater detail, and observe structural changes characteristic of apoptosis, a typical ladder-like DNA fragmentation pattern and depression of the signal from cellular enzymatic reporters (6, 7). Preceding cell death, bcl-2, a key negative regulator of apoptosis (8) is cleaved by the viral protease. We describe the sites in bcl-2 hydrolyzed by HIV protease and show that a circuit exists between virus proteasemediated loss of bcl-2 and activation of NFKB, a redoxsensitive transcription factor required for HIV replication. In contrast, in lymphocytes over-producing bcl-2, we report down-regulation of NFKB-containing sequences, specifically the HIV long terminal repeat and tumor necrosis factor a (TNF-a), leading to a cytoprotective and antiviral effect against HIV.

MATERIALS AND METHODS Transfection of Cell Lines. Expression plasmids containing the human cytomegalovirus immediate early promoter and either the firefly luciferase, human bcl-2, or HIV protease

Abbreviations: TNF-a, tumor necrosis factor a; IL-10, interleukin 10. *Present address: Mitotix, Inc., One Kendall Square, Building 600, Cambridge, MA 02139. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9571

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FIG. 1. HIV protease produces an apoptotic response in cultured cells that can be prevented by DMP323, a specific HIV protease inhibitor (14) or by supplying IL-10. (a-c) Electron microscopy: (a) control COS-7 cells; (b) COS-7 cells producing HIV protease (note chromatin condensation and formation of cytoplasmic vesicles); (c) human conjunctiva cells expressing HIV protease. (d) Scanning microscopy of MT2 lymphocytes expressing HIV protease, showing membrane blebbing. Autologous plasmid DNA coding for an inactive (asp-25 gly) form of HIV protease (12) produced no visible changes to the cells (not shown). (e) DNA fragmentation in COS-7 cells: lane 1, 1-kb DNA ladder; lane 2, immediately after transfection with HIV protease DNA; lanes 3-5: 6, 24, and 48 h after transfection, respectively; lane 6, 48 h after transfection with the inactive

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4.5 x 250 mm, Model 218TPC18) reverse phase column. The HPLC-C18 column was washed with 20% acetonitrile in 0.1% TFA, and bound material eluted with the following linear gradients in 0.1% TFA: 20 to 65% acetonitrile (4 ml), 65 to 85% acetonitrile (20 ml), 85 to 100% acetonitrile (4 ml). Bound bcl-2 eluted at 75% acetonitrile, with recovery of approximately 700 ,g from 109 cells. The peak fractions containing purified bcl-2 were concentrated 40-fold in a Speed Vac using siliconized tubes and adjusted to 20 mM Pipes (pH 6.1), 10% glycerol, 1 mM DTT, and 125 mM NaCl. Purified bcl-2 was reacted with HIV protease for 16 h at 23°C, using a substrate:enzyme ratio ranging from 100:1 to 4:1. Amino terminal residues were determined by automated Edman degradation in a Porton PI 2090 protein sequenater (Beckman). Electrophoresis. Proteins were resolved by SDS/PAGE, and detected by silver stain, autoradiography, or Western blot analysis. Proteins for Western blot analysis were transferred to Immobilon membranes (Millipore), reacted with appropriate primary antibody, and detected using a chemiluminescent peroxidase substrate (DuPont/NEN) with peroxidase-coupled anti-primary IgG (Amersham). DNA analysis was carried out in 0.8% agarose gels and products were detected by staining with 0.2 ,mg/ml of ethidium bromide under UV illumination. In Vivo Activity of bcl-2 in Mouse Muscle. Autologous plasmids expressing the HIV protease, firefly luciferase, or bcl-2 genes were constructed and purified as described above. Plasmid DNA samples (10 ,ug in 100 ,l of phosphate buffered saline, pH 7.2) and DMP323 (10 ,ug) were injected with a 27-gauge needle into quadriceps muscles of Avertinanesthetized, 30-day-old BALB/c female mice. DMP323 was provided by P. Lam (DuPont Merck). Animals were sacrificed after 48 h, and the quadriceps muscles removed, weighed, and extracted using mechanical homogenization in luciferase assay buffer, as described (13). Luciferase activity was measured in a Dynatech ML 1000 plate luminometer.

RESULTS AND DISCUSSION HIV Protease Causes Apoptosis in Cultured Cells. Following transfection of a plasmid coding for the HIV protease, fibroblast, epithelial, and lymphocyte cell lines die (Fig. 1). The death pattern is apoptotic in appearance with cell rounding, chromatin condensation, formation of cytoplasmic vesicles, and blebbing, accompanied by characteristic DNA laddering. Inhibition of HIV protease-mediated apoptosis is accomplished by addition of the HIV protease inhibitor DMP323 (14), or by treating the cells with interleukin 10 (IL-10), stem cell factor, or pentoxifylline, a cytoprotectant that has metal chelating and anti-NFKB properties (15, 16). Similar treatments also suppress replication of HIV (16, 17). The level of HIV protease detected after transfection is similar to the quantity of protease found in HIV-infected cells, as estimated from Western blot analysis (not shown). bcl-2 Is Cleaved by HIV Protease. We observed no changes to most cellular proteins in the protease-expressing cells when we employed one and two-dimensional gel electrophoresis and Western blot analyses (Fig. 2), although there have been several reports of cellular cytoskeletal proteins serving as HIV protease substrates in cell-free experiments (reviewed in ref. 18). However, a cellular protein that is cleaved is bcl-2, a key negative regulator of apoptosis. When we examined the stability of endogenous bcl-2 in cell-free extracts or cells exposed to HIV protease, we saw a loss of full-size (26 kDa) bcl-2 and the appearance of proteolytic fragments; the predominant ones having apparent molecular sizes of approximately 12-14

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FIG. 2. Cleavage of bcl-2 by HIV protease. Lanes 1-4: Western blot detection of bcl-2 cleavage products. Lane 1, recombinant bcl-2 in extract of baculovirus-infected Sf21 insect cells; lane 2, Sf21 lysate incubated with 50 ng of HIV protease; lane 3, COS-7 cells transfected with bcl-2 for 24 h; lane 4, bcl-2 and HIV protease cotransfected in COS-7 cells (24 h). Lane 5, bcl-2 marker; silver stain detection of purified HIV protease (lane 6), purified bcl-2 (lane 7), bcl-2 and HIV protease (lane 8) incubated in assay buffer at 37°C for 4 h. Efficient recovery of recombinant bcl-2 from baculovirus-infected Sf21 cells was dependent on exposure of the protein to 6 M GdnHCl. As a control for possible misfolding of the bcl-2 used in lanes 7 and 8, microsome bound 35S-Met-bcl-2 was cleaved by HIV protease. Lane 9, 35S-Metbcl-2; lane 10, 35S-Met-bcl-2 plus HIV protease. Lanes 11-15, human MT-2 lymphocytes infected with HIV, strain Rf (0.5 infectious unit per cell). Bcl-2 levels were detected by Western blot analysis as described above. Lane 11, uninfected cells. Lanes 12-15: 6, 17, 24, 48 h post-infection, respectively. Lane 16, bcl-2 marker. Lanes 17 and 18 compare the total proteins in COS-7 cells without and with expression of HIV protease. No gross differences were observed using one- or two-dimensional gels, or by Western blotting with antibodies against a variety of proteins involved in DNA repair, the cytoskeleton, or cellular enzymes. Amino acids identified in sequence after hydrolysis of bcl-2 by HIV protease are: alanine, 85 pmol; glutamate, 100 pmol; methionine, 72 pmol; serine, 20.5 pmol; serine, 2 pmol; glutamine, 48.2 pmol; and leucine, 38.6 pmol, consistent with cleavage between phenylalanine 112 and alanine 113. Also detected were minor cleavages between amino acids 15 and 16, 27 and 28, and 97 and 98.

kDa (Fig. 2, lanes 1-5). Purified bcl-2 and bcl-2 translated in reticulocyte lysates, after exposure to HIV protease, were cleaved into similar 12-14-kDa products (Fig. 2, lanes 6-10). Also shown in Fig. 2, lanes 11-15, is the time-dependent loss of bcl-2, which occurs in lymphocytes after infection with HIV (19), although discrete cleavage products were not consistently observed. In comparison, in cells overproducing HIV protease, there was no detectable change in the majority of cellular proteins (Fig. 2, lanes 17 and 18). Acute loss of bcl-2 by proteolysis has not been previously associated with apoptosis (20). Gradually reducing the intracellular level of bcl-2 message with antisense technology leads to onset of apoptosis, with a 24-48 h lag, as the endogenous bcl-2 protein level slowly declines (20). In transgenic mice in which the bcl-2 gene is interrupted, the surviving progeny progressively lose their lymphocytes within 4 weeks after birth (21). With all cell lines

(asp-25 gly) HIV protease mutant DNA. (f-) Phase microscopy of human 293 kidney cells 24 h after transfection with (f) no DNA, (g) HIV protease DNA, (h) HIV protease in the presence of the protease inhibitor DMP323 (100 ng/ml), (i) HIV protease in cells treated with human IL-10 (40 ng/ml, R & D Systems).

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in which bcl-2 cleavage mediated by HIV protease induced apoptosis, we found in parallel experiments that antisense to bcl-2 also caused death, although the initial apoptotic features were delayed by 12-24 h. Expression of HIV Protease and Loss of bcl-2 Leads to Oxidative Stress and NFKcB Activation. The mechanism of cytoprotection by bcl-2 is not clear, but may involve suppression of cellular production of reactive oxygen species or protection from such molecules (8, 22). In contrast, transcription of HIV is induced by reactive oxygen species (11, 23). We therefore tested if the expression of HIV protease affects intracellular oxidative status. Following protease expression, the intracellular concentration of glutathione is decreased, the level of lipid peroxidation increased, the DNA binding property of NFKB activated and TNF increased (Table 1). These effects, which can be partially mimicked in cells treated with bcl-2 antisense (Table 1), are each known to potentiate HIV transcription, and have been observed in clinical AIDS (11, 23). Although we cannot absolutely prove cause and effect, the cleavage and depletion of bcl-2 by HIV protease is consistent with loss of its claimed antioxidant function. Protective Effect of bcl-2 Against HIV Protease Toxicity. The anti-apoptosis protein of adenovirus, Elb-19k, protects Jurkat T cells against the cytopathic effect of HIV (24). We therefore examined the ability of bcl-2 to protect cells from HIV and the viral protease. As shown in Fig. 3, there is effective protection from the protease if a plasmid expressing bcl-2 is present in the cells. In lymphocytes, loss of a luciferase reporter signal caused by coexpression of HIV protease is prevented if bcl-2 is simultaneously overexpressed. Similarly, lymphocytes expressing bcl-2 do not become apoptotic when infected by HIV, and in contrast to earlier results with the adenovirus protein Elb-19k (24), HIV infectivity titers are reduced by up to 90%, as are the levels of viral p24 core and gp120 envelope proteins, the number of syncytia, and the quantity of TNF released into the medium (Fig. 3). These results provide additional evidence that above a threshold level of bcl-2, transcription of NFKB-activated promoters, such as the HIV long terminal repeat, TNF and p53 (11, 16, 23) are suppressed. Bcl-2 was reported to protect cultured mammalian cells against the cytopathic effects of influenza virus (25) and sindbis virus (26), possibly by inhibiting NFKB activation (27). The experiments so far describe results only in cultured cells. To test whether bcl-2 expression is protective in vivo, we Table 1. Effect of expression of the HIV protease or bcl-2 antisense on NFKB activation-related events in COS-7 cells

bcl-2 Control HIV protease antisense Assay Glutathione level ND 81.0 ± 2.0 5.0 ± 0.2 (nmol/107 cells) Lipid peroxidation 7.0 ± 1.0 17.0 ± 1.0 25.0 ± 2.0 (nmol/107 cells) Relative NFKB DNA 1.0 5.5 ± 0.6 3.5 ± 0.4 binding TNF released (ng/ml)