Abstract
Infection of T cells with HIV-1 induces apoptosis and modulates apoptosis regulatory molecules. Similar effects occur following treatment of cells with individual HIV-1 encoded proteins. While HIV-1 protease is known to be cytotoxic, little is known of its effect on apoptosis and apoptosis regulatory molecules. The ability of HIV-1 protease to kill cells, coupled with the degenerate substrate specificity of HIV-1 protease, suggests that HIV-1 protease may activate cellular factor(s) which, in turn, induce apoptosis. We demonstrate that HIV-1 protease directly cleaves and activates procaspase 8 in T cells which is associated with cleavage of BID, mitochondrial release of cytochrome c, activation of the downstream caspases 9 and 3, cleavage of DFF and PARP and, eventually, to nuclear condensation and DNA fragmentation that are characteristic of apoptosis. The effect of HIV-1 protease is not seen in T cell extracts which have undetectable levels of procaspase 8, indicating a specificity and requirement for procaspase 8.
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Abbreviations
- AICD:
-
activation induced cell death
- ATCC:
-
American type cell culture
- ATP:
-
adenine trinucleotide phosphate
- BA:
-
bongkrekic acid
- BSA:
-
bovine serum albumin
- CHAPS:
-
cholamidopropyl dimethylammonio propane sulfonate
- DFF:
-
DNA fragmentation factor
- DMSO:
-
dimethyl sulfoxide
- DTT:
-
dithiothreitol
- EDTA:
-
ethylene diamine tetracetic acid
- EGTA:
-
ethylene glycol tetracetic acid
- FLIP:
-
FLICE-like inhibitory peptide
- HEPES:
-
hydroxyethyl piperazine ethane sulfonic acid
- HIV:
-
human immunodeficiency virus
- HIV-1 PI:
-
HIV-1 protease inhibitor
- HPLC:
-
high performance liquid chromatography
- HRP:
-
horseradish peroxidase
- PAGE:
-
poly crylamide gel electropheresis
- PARP:
-
poly (ADP Ribose) polymerase
- PBL:
-
peripheral blood lymphocyte
- PMSF:
-
phenylmethylsulfonyl fluoride
- SDS:
-
sodium dodecyl sulphate
- TRAIL:
-
TNF related apoptosis inducing ligand
References
Badley AD, McElhinny JA, Leibson PJ, Lynch DH, Alderson MR, Paya CV . 1996 Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes J. Virol. 70: 199–206
Badley AD, Dockrell D, Simpson M, Schut R, Lynch DH, Leibson P, Paya CV . 1997 Macrophage-dependent apoptosis of CD4+T lymphocytes from HIV-infected individuals is mediated by FasL and tumor necrosis factor J. Exp. Med. 185: 55–64
Banda NK, Bernier J, Kurahara DK, Kurrle R, Haigwood N, Sekaly RP, Finkel TH . 1992 Crosslinking CD4 by Human Immunodeficiency virus gp120 primes T cells for activation-induced apoptosis J. Exp. Med. 176: 1099–1106
Cottrez F, Manca F, Dalgleish AG, Arenzana-Seisdedos F, Capron A, Groux H . 1997 Priming of human CD4+antigen-specific T cells to undergo apoptosis by HIV-infected monocytes J. Clin. Invest. 99: 257–266
Herbein G, Van Lint C, Lovett JL, Verdin E . 1998 Distinct mechanisms trigger apoptosis in human immunodeficiency virus type-1 infected and in uninfected bystander T lymphocytes J. Virol. 72: 660–670
Kameoka M, Suzuki S, Kimura T, Fujinaga K, Auwanit W, Luftig RB, Ikuta K . 1997 Exposure of resting peripheral blood T cells to HIV-1 particles generates CD25+killer cells in a small subset, leading to induction of apoptosis in bystander cells Int. Immunol. 9: 1453–1462
Nardelli B, Gonzalez CJ, Schechter M, Valentine FT . 1995 CD4+blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells Proc. Natl. Acad. Sci. USA. 92: 7312–7316
Orlikowsky T, Wang Z-Q, Dudhane A, Horowitz H, Riethmuller G, Hoffman MK . 1997 Cytotoxic monocytes in the blood of HIV type-1 infected subjects destroy targeted T cells in a CD-95-dependent fashion AIDS Res. Hum. Retro. 13: 953–960
Mitra D, Steiner M, Lynch DH, Staiano-Coico L, Laurence J . 1996 HIV-1 upregulates Fas ligand expression in CD4+T cells in vitro and in vivo: association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid Immunol. 87: 581–585
Sloand EM, Young NS, Kumar P, Weichold FF, Sato T, Maciejewski JP . 1997 Role of Fas ligand and receptor in the mechanism of T-cell depletion in acquired immunodeficiency syndrome: effect on CD4+lymphocyte depletion and human immunodeficiency virus replication Blood 89: 1357–1363
Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley A, Goodwin RG, Smith CA, Ramsdell F, Lynch DH . 1995 Fas ligand mediates activation-induced cell death in human T lymphocytes J. Exp. Med. 181: 71–77
Dhein J, Walczak H, Baumler C, Debatin KM, Krammer PH . 1995 Autocrine T-cell suicide mediated by APO-1/(Fas/CD95) Nature 373: 438–443
Ju ST, Panka DJ, Cui H, Ettinger R, El-Khatib M, Sherr DH, Stanger BZ, Marshak-Rothstein A . 1995 Fas (CD95) FasL interactions required for programmed cell death after T-cell activation Nature 373: 444–448
Wesselborg S, Janssen O, Kabelitz D . 1993 Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells J. Immunol. 150: 4338–4345
Gandhi RT, Chen BK, Straus SE, Dale JK, Lenardo MJ, Baltimore D . 1998 HIV-1 directly kills CD4+T cells by a Fas-independent mechanism J. Exp. Med. 187: 1113–1122
Katsikis PD, Garcia-Ojeda ME, Wunderlich ES, Smith CA, Yagita H, Okumura K, Kayagaki N, Alderson M, Herzenberg LA, Herzenberg LA . 1996 Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals Int. Immunol. 8: 1311–1317
Katsikis PD, Garcia-Ojeda ME, Torres-Roca JF, Tijoe IM, Smith CA, Herzenberg LA, Herzenberg LA . 1997 Interleukin-1B converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection J. Exp. Med. 186: 1365–1372
Ledru E, Lecoeur H, Garcia S, Debord T, Gougeon ML . 1998 Differential susceptibility to activation-induced apoptosis among peripheral Th1 subsets: correlation with Bcl-2 expression and consequences for AIDS pathogenesis J. Immunol. 160: 3194–3206
Groux H, Torpier G, Monte D, Mouton Y, Capron A, Ameisen JC . 1992 Activation-induced death by apoptosis in CD4+T cells from human immunodeficiency virus-infected asymptomatic individuals J. Exp. Med. 175: 331–340
Algeciras A, Dockrell DH, Lynch DH, Paya CV . 1998 CD4 regulates susceptibility to Fas ligand - and tumor necrosis factor-mediated apoptosis J. Exp. Med. 187: 711–720
Laurent-Crawford AG, Krust B, Riviere Y, Desgranges C, Muller S, Kieney MP, Dauguet C, Hovanessian AG . 1993 Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells AIDS Res. Hum. Retro. 9: 761–773
Oyaizu N, McCloskey TW, Coronesi M, Chirmule N, Kalyanaraman VS, Pahwa S . 1993 Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals Blood 82: 3392–3400
Jeremias I, Herr I, Boehler T, Debatin KM . 1998 TRAIL/Apo-2-ligand-induced apoptosis in human T cells Eur. J. Immunol. 28: 143–152
Glynn JM, McElligott DL, Mosier D . 1996 Apoptosis induced by HIV infection in H9 T cells is blocked by ICE-Family protease inhibition but not by a Fas (CD95) antagonist J. Immunol. 157: 2754–2758
Noraz N, Gozlan J, Corbeil J, Brunner T, Spector SA . 1997 HIV-induced apoptosis of activated primary CD4+T lymphocytes is not mediated by Fas–Fas ligand AIDS 11: 1671–1680
Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB . 1995 Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein Science 268: 429–431
New DR, Maggirwar SB, Epstein LG, Dewhurst S, Gelbard HA . 1998 HIV-1 Tat induces neuronal death via tumor necrosis factor-α and activation of non-N-methyl-D-aspartate receptors by a NFkB-independent mechanism J. Biol. Chem. 273: 17852–17858
Patki AH, Lederman MM . 1996 HIV-1 Tat protein and its inhibitor Ro 24-7429 inhibit lymphocyte proliferation and induce apoptosis in peripheral blood mononuclear cells from healthy donors Cell Immunol. 169: 40–46
Fujii Y, Otake K, Tashiro M, Adachi Y . 1996 Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+T cells FEBS Letters 393: 105–108
Fujii Y, Otake K, Tashiro M, Adachi Y . 1996 Soluble Nef antigen of HIV-1 is cytotoxic for human CD3+T cells FEBS Letters 393: 93–96
Fujii Y, Otake K, Tashiro M, Adachi Y . 1996 In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+T cells without MHC restriction J. Gen. Virol. 77: 2943–2951
Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P . 1998 Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice Cell 95: 163–175
Poon B, Grovit-Ferbas K, Stewart SA, Chen ISY . 1998 Cell cycle arrest by vpr in HIV-1 virions and insensitivity to antiretroviral agents Science 281: 266–269
Stewart SA, Poon B, Jowett JBM, Chen ISY . 1997 Human immunodeficiency virus type 1 vpr induces apoptosis following cell cycle arrest J. Virol. 71: 5579–5592
Yao X-J, Mouland AJ, Subbramanian RA, Forget J, Rougeau N, Bergeron D, Cohen EA . 1998 Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing jurkat T cells J. Virol. 72: 4686–4693
Adams LD, Tomasselli AG, Robbins P, Moss P, Heinrikson RL . 1992 HIV-1 protease cleaves actin during acute infection human T-lymphocytes AIDS Res. Hum. Retro. 8: 291–295
Buttner J, Dornmair K, Schramm HJ . 1997 Screening of inhibitors of HIV-1 protease using an Escherichia coli cell assay Biochem. Biophy. Res. Commun. 233: 36–38
Konvalinka J, Litterst MA, Welker R, Kottler H, Rippman F, Heuser A-M, Krausslich H-G . 1995 An active site mutation in the HIV type 1 proteinase (PR) causes reduced PR activity and loss of PR mediated cytotoxicity without apparent effect on virus maturation and infectivity J. Virol. 69: 7180–7186
Rivi re Y, Blank V, Kourilsky P, Israel A . 1991 Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection Nature 350: 625–626
Shimizu T, Pommier Y . 1996 DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase 1 inhibitor campothecin: cell-free system studies Exper. Cell. Res. 226: 292–301
Kaplan AH, Swanstrom R . 1991 The HIV-1 gag precursor is processed via two pathways: implications for cytotoxicity Biomed. Biochim. Acta. 50: 647–653
Kaplan AH, Swanstrom R . 1991 Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments Proc. Natl. Acad. Sci. USA. 88: 4528–4532
Strack PR, West Frey M, Rizzo CJ, Cordova B, George HJ, Meade R, Ho W, Corman J, Tritch R, Korant BD . 1996 Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2 Proc. Natl. Acad. Sci. USA. 93: 9571–9576
Liu X, Kim CN, Yang J, Jemmerson R, Wang R . 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c Cell 86: 147–157
Cosulich SC, Green S, Clarke PR . 1996 Bcl-2 regulates activation of apoptotic proteases in a cell-free system Curr. Biology 6: 997–1005
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang H, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ . 1999 Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspase-2,-3,-6,-7,-8, and -10 in a caspase 9-dependent manner J. Cell. Biol. 144: 281–292
Muzio M, Salveson GS, Dixit VM . 1997 FLICE induced apoptosis in a cell-free system J. Biol. Chem. 272: 2952–2956
Kroemer G, Reed JC . 1998 Mitochondrial control of cell death Nat. Med. 6: 513–519
Green DR, Reed JC . 1998 Mitochondria and apotposis Science 281: 1309–1312
Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES . 1999 Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation J. Biol. Chem. 274: 17941–17945
Hu Y, Benedict MA, Ding L, Nunez G . 1999 Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis EMBO J. 18: 3586–3595
Li H, Zhu H, Xu CJ, Yuan J . 1998 Cleavage of BID by caspase 8 meidates the mitochondrial damage in the Fas pathway of apoptosis Cell 94: 491–501
Wei MC, Lindstein T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB, Korsmeyer SJ . 2000 tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c Genes Dev. 14: 2060–2071
Tomasselli AG, Hui JO, Chosay J, Lowery D, Greenberg B, Yem A, Deibel MR, Zurcher-Neely H, Heinrickson RL . 1991 Actin, troponin C, alzheimer amyloid precursor protein and pro-interleukin 1b as substrates of the protease from human immunodeficiency virus J. Biol. Chem. 266: 14548–14553
Krammer PH . 1999 CD95 (APO-1/Fas)-meidated apoptosis: live and let die Adv. Immunol. 71: 163–210
Medema JP, Scaffidi C, Kischek FC, Shevchenko A, Mann M, Krammer PH, Peter ME . 1997 FLICE is activated by association with the CD95 death-inducing signaling complex (DISC) EMBO J. 16: 2794–2804
Andrade F, Roy S, Nicholson D, Thornberry N, Rosen A, Casciola-Rosen L . 1998 Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis Immunity 8: 451–460
Chou KC, Zhang CT . 1993 Studies on the specificity of HIV protease: an application of Markov chain theory J. Protein Chem. 12: 709–724
Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM . 1995 Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase Cell 81: 801–809
Nicholson DW, Ali A, Thornberry N, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA . 1995 Identification and inhibition of the ICE/CED-2 protease necessary for mammalian apoptosis Nature 376: 37–43
Badley AD, Pilon AA, Landay A, Lynch DH . 2000 Mechanisms of HIV associated lymphocyte apoptosis Blood 96: 2951–2964
Barry MG, McFadden G . 1998 Apoptosis regulators from DNA viruses Curr. Opin. Immunol. 10: 422–430
Thome M, Schnelder P, Hofmann K, Foickenscher H, Neinl E, Neipel F, Mattmann C, Burns K, Bodmer JL, Schroter M, Scaffidi C, Krammer PH, Peter ME, Tschopp J . 1997 Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors Nature 386: 517–521
Honer B, Shoeman RL, Traub P . 1991 Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture J. Cell. Sci. 100: 799–807
Shoeman RL, Honer B, Stoller TJ, Kesselmeier C, Miedel MC, Traub P, Graves MC . 1990 Human immunodeficiency virus type 1 protease cleaves the inermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein Natl. Acad. Sci. USA. 87: 6336–6340
Korant B, Strack P, Frey MW, Rizzo CJ . 1998 A cellular anti-apoptosis protein is cleaved by the HIV-1 protease Adv. Exp. Med. Biol. 436: 27–29
Martin SJ, Newmeyer DD, Mathias S, Farschon D, Wang H, Reed JC, Kolesnick RN, Green DR . 1995 Cell-free reconstitution of Fas-, UV radiation and ceramide-induced apoptosis EMBO J. 14: 5191–5200
Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE, Tschopp J . 1997 Inhibition of death receptor signals by cellular FLIP Nature 388: 190–195
Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, Peter ME . 1999 Differential modulation of apoptosis sensitivity in CD95 type I and type II cells J. Biol. Chem. 274: 22532–22538
Flexner C . 1998 HIV-protease inhibitors N. Engl. J. Med. 338: 1281–1292
Phenix BN, Lum JJ, Sanchez-Dardon J, Badley AD . 2001 Antiapoptotic mechanism of HIV protease inhibitors: preventing mitochondrial potential loss Blood 98: 1078–1085
Stennicke HR, Salvesen GS . 1997 Biochemical characteristics of caspase-3,-6,-7, and -8 J. Biol. Chem. 272: 25719–25723
Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S . 1998 Caspase-independent cell killing by Fas-associated protein with death domain J. Cell. Biol. 143: 1353–1360
Juo P, Kuo CJ, Yuan J, Blenis J . 1998 Essential requirement for caspase-8/FLICE in the initiation of the Fas- induced apoptotic cascade Curr. Biol. 8: 1001–1008
Wood ER, Earnshaw WC . 1990 Mitototic chromatin condensation in vitro using somatic cell extracts as nuclei with variable levels of endogenous topoisomerese II Cell. Biol. 111: 2389–2850
Zhu N, Wang Z . 1997 An assay for DNA fragmentation in apoptosis without phenol/chloroform extraction and ethanol precipitation Analytical Biochemistry 246: 155–158
Alam A, Braun MY, Hartgers F, Lesage G, Cohen L, Hugo P, Denis F, Sekaly R-P . 1997 Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus J. Exp. Med. 186: 1503–1512
Evans EK, Kuwana T, Strum SL, Smith JJ, Newmeyer DD, Kornbluth S . 1997 Reaper-induced apoptosis in a vertebrate system EMBO J. 16: 7372–7381
Schmitz I, Walczak H, Krammer PH, Peter ME . 2000 The two CD95 apoptosis signaling pathways may be a way of cells to respond to different amounts and/or forms of CD95 ligand produced in different tissues Cell Death and Diff. 7: 756–758
Stennicke HR, Salvesen GS . 1999 Catalytic properties of the caspases Cell Death Differ. 6: 1054–1059
Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhand M, Gentz R, Mann M, Krammer PH, Peter ME, Dixit VM . 1996 FLICE, a novel FADD/homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex Cell. 85: 817–827
Griffiths JT, Phylip LH, Konvalinka J, Strop P, Gustchina A, Wlodawer A, Davenport RJ, Briggs R, Dunn BM, Kay J . 1992 Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing - hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites Biochemistry 31: 5193–5200
Acknowledgements
Informed consent was obtained from all subjects prior to blood collection, and the study was reviewed and approved by the ethics committee of the Ottawa Hospital/University of Ottawa. The authors gratefully acknowledge the helpful discussions and advice of Dr. BWD Badley, as well as the administrative expertise of A Carisse. BN Phenix and JJ Lum are supported by a Studentship award from the Ontario HIV Treatment Network (OHTN). AD Badley is supported by an OHTN Career Scientist Award. This work is supported by grants from the Doris Duke Foundation (#T98026), the Canadian Institute of Health Research (#HOP-36047), the Canadian Foundation for AIDS Research and a Premier's Research Excellence Award.
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Nie, Z., Phenix, B., Lum, J. et al. HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation. Cell Death Differ 9, 1172–1184 (2002). https://doi.org/10.1038/sj.cdd.4401094
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