- Email: [email protected]
Mitochondrial membrane permeabilization by HIV-1 Vpr
‘ Mitochondrion 4 (2004) 223–233 www.elsevier.com/locate/mito
Review
Mitochondrial membrane permeabilization by HIV-1 Vpr Aure´lien Deniauda, Catherine Brennera,*, Guido Kroemerb a
CNRS FRE 2445, Universite´ de Versailles/St Quentin, 45, avenue des Etats-Unis, 78035 Versailles, France Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif, France
b
Received 1 February 2004; received in revised form 17 May 2004; accepted 2 June 2004
Abstract The mitochondrion is a privileged target for apopotosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins. q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: Apoptosis; Adenine nucleotide translocase; Mitochondrion; Bcl-2; Viral target
1. Introduction HIV infection is characterized by a dramatic decrease in CD4C T cells, the progresive loss of immune function and the eventual evolution to AIDS in the absence of any therapeutic intervention
Abbreviations: ANT, adenine nucleotide translocator; BA, bongkrekic acid; CsA, cyclosporin A; DJm, mitochondrial transmembrane potential; HIV-1, human immunodeficiency virus-1; MMP, mitochondrial membrane permeabilization; MUP, 4-methylumbelliferyl phosphate; MU, 4-methylumbelliferone; IM, inner membrane; OM, outer membrane; PTPC, permeability transition pore complex; VDAC, voltage-dependent anion channel; Vpr, viral protein R. * Corresponding author. Tel.: C33-139-254-552; fax: C33-139254-572. E-mail address: [email protected] (C. Brenner).
such as highly active antiretroviral therapy (De Oliveira Pinto et al., 2002; Gougeon, 2003). Among various hypotheses, it has been postulated that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) protein may contribute to AIDS pathogenesis by inducing apoptotic death of CD4C T cells and depletion of bystander cells (Azad, 2000; Bukrinsky and Adzhubei, 1999). Indeed, in HIV-infected patients, Vpr is found associated with the virion, within infected cells, and as a soluble protein in the serum and the cerebrospinal fluid of patients (Levy et al., 1994; Muthumani et al., 2003). On theorical ground, free Vpr can account for the death of uninfected cells since the external addition of Vpr results in its penetration into cells, loss of the inner membrane potential (DJm), DNA fragmentation and cell death
1567-7249/$ - see front matter q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2004.06.012
224
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
(Muthumani et al., 2003). This is not restricted to CD4C T cells, because Vpr can kill a variety of cells including hippocampal neurons or astrocytes (Huang et al., 2000), cell lines of various species (Matsuda et al., 2003) as well as yeast cells (Macreadie et al., 1995). This suggests that Vpr activates a general step of the apoptotic cascade common to many eukaryotic cells. Thus, Vpr has been suggested to trigger the mitochondrial membrane permeabilization (MMP), namely via the opening of the permeability transition pore (PTPC) (Kroemer and Reed, 2000). The PTPC is a dynamic polyprotein complex formed in the contact site between the inner and the outer mitochondrial membranes (IM and OM, respectively) (Zoratti and Szabo, 1994, 1995) (Fig. 1). Despite tissue-, development- and differentiation state variations of protein expression (Belzacq et al., 2002; Doerner et al., 1997, 1999), it contains permanently the two most abundant IM and OM proteins, the adenine nucleotide translocator (ANT, in the IM), the voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well
as apoptosis-regulatory proteins from the Bax/Bcl-2 family (Beutner et al., 1996; Halestrap and Brenner, 2003; Marzo et al., 1998a,b). In physiological conditions, ANT is a vital, specific antiporter, which catalyzes the exchange of ATP and ADP on IM (Pfaff et al., 1969; Pfaff and Klingenberg, 1968). Upon calcium-induced conformational changes, ANT can form a non-specific pore (Brustovetsky and Klingenberg, 1996; Beutner et al., 1996). Moreover, pore formation by ANT elicited by a variety of different agents (e.g. Ca2C, atractyloside (Ru¨ck et al., 1998), thiol oxidation (Costantini et al., 2000), lipidic messengers (Jan et al., 2002), chemotherapeutic drugs (Belzacq et al., 2001a,b), etc.), is enhanced by Bax (Marzo et al., 1998b) and Bid (Zamzami et al., 2000) and inhibited by Bcl-2 (Marzo et al., 1998b), ADP as well as ATP (for review see Halestrap and Brenner, 2003). During MMP (for review Kroemer and Reed, 2000), pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, loss of inner membrane potential (Zamzami et al., 1995) and OM permeabilization, presumably due to physical rupture of OM (Vander Heiden et al., 1997). Although alternative mechanisms of MMP may exist (Martinou et al., 2000; Tsujimoto and Shimizu, 2000), ANT emerged as a major player in the regulation of cell death (for review Belzacq et al., 2002; Halestrap and Brenner, 2003; Vieira et al., 2000). This review discusses recent advances in the characterization of the pro-apoptotic mitochondrial damage induced by Vpr, the underlying molecular mechanisms of Vpr/PTPC cooperation to induce MMP and the place of Vpr in AIDS pathogenesis.
2. Pleitropic functions of Vpr
Fig. 1. Scheme of the PTPC organization. PTPC is a polyprotein complex built up at the contact site of mitochondrial inner and outer membranes (IM and OM, respectively). The opening of PTPC allows the diffusion solutes of MW!1500 Da between the cytosol and the mitochondrial matrix. HK, hexokinase; VDAC, voltagedependent anion channel; PBR, peripheral benzodiazepine receptor; CK, creatine kinase; ANT, adenine nucleotide translocator and CypD, cyclophilin D.
Vpr is a 14 kDa-protein endowed with pleiotropic functions. Indeed, the Vpr protein behaves as a transcriptional activator of HIV and heterologous promoters (Cohen et al., 1990; Wang et al., 1995, and for review Luo et al., 1998). It also induces a cell cycle arrest in G2 (Poon et al., 1998), blocks cell proliferation (Yamaguchi et al., 1999) and induces apoptosis (Stewart et al., 1997). All these effects may be mediated by protein–protein
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
225
Table 1 Eucaryotic proteins interacting with Vpr and role of the interaction Protein
Domain or aminoacids of Vpr
Role of the interaction
Reference
Cyclophilin A
N-terminus Pro 35
Zander et al. (2003)
Human nucleoporin (hCG1)
N-terminal region
Decrease of Vpr synthesis and loss of cell cycle effects Le Rouzic et al. (2002)
VprBP ANT HHR23A
ND (67–82) Vpr ND
Lys-tRNA synthetase (LysRS)
N- and C-terminus
Uracyl DNA glycosylase Sp1
Trp 54 Leu/Ile-rich domain
Cytoplasmic retention of Vpr Apoptosis induction via MMP Prevention of Vpr-induced cell cycle arrest Initiation of HIV-1 reverse transcription HIV-1 in vivo mutation rate Activation of HIV-1 long terminal repeat (LTR)-directed transcription
Le Rouzic et al. (2002); Fouchier et al. (1998) Zhang et al. (2001) Jacotot et al. (2000, 2001) Gragerov et al. (1998) Stark and Hay (1998) Mansky et al. (2000) Wang et al. (1995)
ND, not determined; MMP, mitochondrial membrane permeabilization.
interactions of Vpr with intracellular proteins (Table 1). These various functions are separable (Elder et al., 2000; Nishizawa et al., 2000) and presumably linked to the intracellular localization of Vpr in response to export and import signals contained in the N- and C-terminus of the protein and to the existence of different domains within the protein (Waldhuber et al., 2003) (Fig. 2). The pleitotropic effects of Vpr could also be related to the fact that there are three different sources of Vpr available in HIV-1 infected patients (for review Tungaturthi et al., 2003). These include cell-associated, virion-associated (infectious, infectious nonproductive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). Even if quantities of intracellular or circulating Vpr are difficult to estimate, it is interesting to note that purified Vpr (synthetic or expressed through baculovirus) when added in culture is able to penetrate into cells (Huang et al., 2000; Levy et al., 1995). This suggests that Vpr may use receptors present on the cell surface or may contain signal sequences that enable its entry into cells independently of the receptor pathway, for instance by endocytosis (for review Tungaturthi et al., 2003). 2.1. Vpr, elements of structure/function relationship Recently, a novel 3D structure of (1–96) full length Vpr has been resolved by NMR (Morellet et al.,
2003). The Vpr protein was synthesized with 22 labeled amino acids in pure water and in twater containing 10, 20, 30% of acetonitrile. The folding of the protein is characterized by three well-defined alpha-helices (amino acids 17–33, 38–50 and 56–77) surrounded by flexible N- and C-terminal domains (Fig. 2). In contrast to a previous structure obtained in the presence of the solvent trifluoroethanol (Wecker et al., 2002), the three alpha-helices are folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues. Interestingly, this structure is highly compatible with the interaction of Vpr with different effector targets. For example, amino acids contained in the N-terminus (e.g. Trp18, Leu 22 and Leu 26, Ile 63, Gln 65, Leu 67) and C-terminus basic amino acids, are accessible for interaction with the structural Gag polyprotein, with the uracil DNA glycosylase, with the nucleocapsid protein NCp7, and for activity to induce G2 arrest, respectively (Morellet et al., 2003; Schuler et al., 1999). Moreover, arginine residues R73, R77, and R80 would be accessible for interaction with the first cytoplasmic loop of the mitochondrial protein ANT (Brenner and Kroemer, 2003) (Fig. 2). Several groups have proposed putative structure– function map of Vpr (Fig. 2). Mutations in the alphahelical region reportedly eliminate the incorporation of Vpr into virions (Di Marzio et al., 1995; Mahalingam et al., 1997; Singh et al., 2000; Yao et al., 1995), as do point deletions at the C terminus
226
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
Fig. 2. Structure/function map of Vpr. The structure of Vpr depicts a folding of the protein characterized by three well-defined alpha-helices I, II and III (amino acids 17–33, 38–50 and 56–77, respectively) surrounded by flexible N- and C-terminal domains. The (72–83) domain, has been identified as the mitochondriotoxic domain, capable of inducing mitochondrial membrane permeabilization. Mutations of aminoacids R73, 77, and R80 abolish the mitochondriotoxic activity of the domain. Key residues for nucleus import of Vpr are localized from aminoacids 17 to 60. Helix I and aminoacids 35–46 are implicated in virion incorporation and aminoacids 84–96, plus R73, R77, R80 and R85 are critical for the Vpr-induced cell cycle arrest in G2.
(Zhou et al., 1998). Amino acids in the core of the protein (amino acids 30–60) have been found to be important for nuclear localization of Vpr (Mahalingam et al., 1997), and the C-terminal basic region is essential for protein stability and induction of cell cycle arrest (Di Marzio et al., 1995; Mahalingam et al., 1997; Yao et al., 1995) (Fig. 2). In addition, a mitochondriotoxic domain has been located in the C-terminal moiety of the protein and key aminoacids identified within the 52–96 domain of Vpr (Jacotot et al., 2000, 2001; Muthumani et al., 2002; Roumier et al., 2002) (Fig. 2). A point mutation in this domain (aminoacid R77) found in long-term non-progressors (that is HIV-1 carriers who do not develop AIDS), generates a mutant protein (aminoacid Q77), which exhibits an impaired pro-apoptotic activity. This observation
established for the first time a link between the mitochondriotoxic properties of Vpr and the clinical outcome of HIV-infected patients (Brenner and Kroemer, 2003; Lum et al., 2003). 3. Vpr targets the mitochondrion to induce MMP After its entry into a cell, a fraction of Vpr localizes to the mitochondrial compartment as well as to the nucleus (Arunagiri et al., 1997; Jacotot et al., 2000). By association with the mitochondrial membrane, Vpr triggers a typical mitochondrial pathway of apoptosis characterized by an early loss of the inner transmembrane potential (DJm), the release of cytochrome c, the loss of respiratory activity and activation of various caspases including caspase 9 and 3 (Jacotot et al., 2000, 2001; Muthumani et al., 2002).
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
This has been shown for primary human lymphoid cells, as well as for T cell lines, independently of the mode of delivery of Vpr, i.e. extracellular addition or adenovirus delivery of Vpr (Jacotot et al., 2000, 2001; Muthumani et al., 2002). However, the implication of caspases is questionable since the knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated cell killing (Roumier et al., 2002). The mitochondrial effect of Vpr is likely to be direct since all criteria of mitochondrial damage depicted above have been reproduced by the addition of synthetic (52–96) Vpr-derived peptide in cell-free systems, such as mice liver-isolated mitochondria or HeLa cell-isolated nuclei (Jacotot et al., 2000, 2001). As a negative control, the synthetic (1–51) Vpr peptide was devoid of any effect on human primary lymphoid cells, on culture cell lines (Jurkat, COS, etc.) or on isolated liver mitochondria (Jacotot et al., 2001). Altogether, this suggested that Vpr requires no extramitochondrial cofactor and/or cytoplasmic second messenger to exert its mitochondriotoxic activity via its C-terminal moiety. Whether the mitochondrial oncoprotein Bcl-2 is able to protect cells from Vpr-driven apoptosis is a matter of debate. Thus, by stable transfection of T cell lines, Bcl-2 prevented the loss of DJm, the exposure of phosphatidylserine residues, the nuclear chromatin condensation and the release of apoptogenic factors from the intermembrane mitochondrial compartment (Jacotot et al., 2000). These effects were comparable with the protective effects of two PTPC inhibitors, cyclosporin A (CsA), a cyclophilin D ligand that inhibits the binding of CypD to ANT, and bongkrekic acid (BA), an ANT ligand. Furthermore, Bcl-2 protection was confirmed in in vitro assays with recombinant Bcl-2. Thus, Bcl-2 blocked Vpr interaction with mitochondrial PTPC proteins, and the channel activity of Vpr in proteoliposomes 3 (Jacotot et al., 2001) (Fig. 3). In contrast, Muthumani, et al. (2002) found that Vpr reduces either the expression level of Bcl-2 or the level of Vpr-induced apoptosis, but does not suppress it, suggesting that experimental conditions (Bcl-2 and Vpr delivery mode, Bcl-2/Vpr ratios, etc.) can critically influence the capacity of Bcl-2 to inhibit Vpr-driven cell death.
227
3.1. Vpr and PTPC components, the lethal interaction The previous functional observations prompted the question of how Vpr can mediate its mitochondrial effects, and notably, the MMP. The first pharmacological clue came from experiments showing that PTPC inhibitors, CsA and BA, can prevent all Vpr-driven mitochondrial damages, in cellular experiments as well as in cell-free systems (Jacotot et al., 2000). This led to the search of an intracellular target of Vpr and resulted in the identification of ANT as a Vpr-cooperating protein critical for the induction of MMP (Jacotot et al., 2000, 2001). Indeed, Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as ANT (Fig. 3) (Jacotot et al., 2001). This effect is prevented by addition of recombinant Bcl-2, CsA and BA, but not a Bcl-2 mutant lacking their pore forming helix nor a non-dimerizing mutant of Bcl-2 (Jacotot et al., 2001). The Vpr COOH terminus binds to purified ANT, as well as a molecular complex containing ANT and VDAC. In addition, Bcl-2 reduces the ANT/Vpr interaction, as determined by affinity purification and plasmon resonance studies (Jacotot et al., 2001). Concomitantly, Bcl-2 suppresses channel formation by the ANT/Vpr complex in synthetic membranes. In contrast to Ko¨nig’s polyanion (PA10) (Jacotot et al., 2001) and to NADH (Brenner, unpublished result), specific pore blockers of VDAC, Bcl-2 failed to prevent (52–96) Vpr from crossing the mitochondrial OM. This finding strongly suggests that VDAC would allow the access of Vpr to the inner mitochondrial membrane (Jacotot et al., 2001). Critical aminoacids for interaction with ANT were identified by point mutation of synthetic peptides (R73, R77 and R80) and the minimal mitochondriotoxic domain was defined to be a dodecapeptide (aminoacids 72–83; FRIGCRHSRIGI) that is located within the C-terminal a-helix. This minimal toxic domain could be engaged in cation–p interactions with aminoacids F109, W111, Y113 and/or F114 within ANT (Brenner and Kroemer, 2003). Indeed, these aminoacids are exposed to the intermembrane space as indicated by the X-ray crystallographic structure of ANT, at least in its ‘c-state’ conformation (Pebay-Peyroula et al., 2003). The Vpr/ANT interaction is direct, since it is abolished by the addition of
228
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
Fig. 3. Experimental methods to measure the capacity of Vpr and derived peptides to functionally interact with PTPC members. ANT, purified from rat heart mitochondria and PTPC, purified from rat brain mitochondria, are reconstituted into phosphatidyl/cardiolipin and phosphatidyl/cholesterol liposomes, repectively, in which 4-methylumbelliferyl phosphate (4-MUP) has been encapsulated. The conversion into a non-specific pore is measured by the release of 4-MUP which reacts with alkaline phosphatase to generate a fluorescent signal. Moreover, ANT is incorporated into planar lipid bilayers to measure its electrophysiological properties, such as single channel activity or macroscopic conductance, opening frequency and ionic specificity. In the two set-ups, ANT and PTPC are in a closed conformation and upon stimulation by sub-micromolar doses of Vpr or Vpr-derived peptides, they can be converted in large non-specific pores. Human recombinant Bcl-2, the peptide (104–116) derived from ANT, cyclosporin A (CsA) and bongrekic acid (BA) are inhibitors of ANT and/or PTPC channel activity and are used as control of the specificity of interaction.
a peptide corresponding to the Vpr binding site of ANT, (104–116) ANT2; DKRTQFWRYFAGN, but not by control peptides such as a scrambled (104–116) ANT2, a mutated (104–116) ANT2 for positively charged aminoacids (K and R), and a topologically equivalent peptide of the human phosphate carrier, a member of mitochondrial carrier family as ANT (Jacotot et al., 2001). Because ANT is expressed as three distinct isoforms, which would exert various roles in apoptosis (Bauer et al., 1999; Schubert and Grimm, 2004), Vpr affinity for each ANT isoform could be slightly different according to differences in their primary sequence. Thus, these isoforms differ in aminoacid composition and in the number of positive charges within the (104–116) domain, i.e. RHK for (106–108) ANT1, KRT for (106–108) ANT2,
and KHT for (106–108) ANT3. In conclusion, this region overlapping with the binding domain of Bax and Bcl-2 to ANT, i.e. aminoacids 105–155 (Marzo et al., 1998b), Vpr modulates MMP through a direct interaction with the apoptogenic region of ANT.
4. Vpr and the yeast model Vpr also harbors multiple activities in yeast cells (for review Zhao and Elder, 2000). This includes nuclear import, induction of cell cycle G2 arrest, mitochondrial damage, morphological changes and cell death. Therefore, Vpr can be expected to target phylogenetically conserved cellular processes and molecules. Thus, studies involving fission yeast
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
(Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) established that Vpr is able to induce a G2 arrest through inhibitory phosphorylation of the cyclin-dependent kinase by a pathway in which protein phosphatase 2A would play a critical role (Zhao et al., 1996). The protein is imported in the nuclear as a viral pre-integration complex by binding to importin-alpha subunit and nucleoporin (Vodicka et al., 1998), and triggers apoptosis by directly permeabilizing the mitochondrial membrane (Macreadie et al., 1997). It should be noted that the Vpr-induced G2 arrest in yeast is independent of cell death induction and of the DNA damage checkpoint and of nuclear localization of Vpr (Chen et al., 1999; Elder et al., 2000). Moreover, the aminoacid residues of Vpr at position 29, 33 and 71 were shown to be important sites for maintaining the overall structure of Vpr (Chen et al., 1999) and the mitochondriotoxic domain defined as the conserved amino acid sequence motif HFRIGCRHSRIG (Macreadie et al., 1997). Taking advantage of knock-out yeasts, invalidated for one or several isoforms of VDAC and ANT, Jacotot et al. (2000) showed that either VDAC or ANT are required for cell killing. This helps to explain how Vpr induces MMP,
Fig. 4. Mechanisms of MMP-induced by viral proteins targeting the PTPC compounds VDAC, ANT and PBR. HBx translocates to mitochondria to interact with VDAC3 and to induce MMP independently of Bcl-2. Vpr enters into mitochondria via VDAC, binds to ANT in a Bcl-2-dependent manner and induces MMP. vMIA interacts with ANT (but not with VDAC) and prevents MMP. M11L inhibits MMP through a physical association with PBR in the OM.
229
possibly via a VDAC-mediated import of Vpr for a direct interaction with ANT (Fig. 4). Because of the great versatility of yeast and the existence of wide mutant libraries, future studies of Vpr in yeast should lead to important advances in understanding the mechanisms of Vpr activities. Moreover, yeast may be instrumental for the screening of inhibitory molecules of Vpr inhibitors (Yao et al., 2002). 4.1. Open questions and perspectives Vpr represents probably one of the viral protein for which the lethal effects are the best characterized (Boya et al., 2001, 2003). However, it is interesting to note that Vpr is not the sole viral protein affecting mitochondrial function (Fig. 4). Recently at least, three proteins, vMIA from cytomagalovirus (Goldmacher, 2002; Goldmacher et al., 1999), M11L from myxoma virus (Everett et al., 2000, 2002; Everett and McFadden, 2001) and HBx from Hepatitis B (Rahmani et al., 2000; Shirakata and Koike, 2003), have been shown to modulate MMP by targeting PTPC members. vMIA reportedly interacts with ANT and M11L with the peripheral benzodiazepine receptor to inhibit cell death, whereas HBx would interact with VDAC to promote apoptosis. As to Vpr, numerous issues should be addressed to elucidate the precise molecular mechanisms of its pro-apoptotic activity. Uncertainties remain on the mechanisms allowing the targeting of Vpr to the mitochondrial compartment, the precise hierarchy of mitochondrial events, and the existence of one single or several submitochondrial targets. Moreover, it will be important to determine the exact place of Vpr in AIDS pathogenesis. In HIV-1 carriers, the envelope glycoprotein complex has recently been shown to stimulate a p53-dependent transcriptional program leading to the overexpression of the BH3-only protein Puma, a pro-apoptotic protein that can activate pro-opapototic Bcl-2 multidomain family members such as Bax and Bak (Castedo et al., 2001, 2002; Perfettini et al., 2004). This suggests that several pro-apoptotic proteins, be they virus encoded (such as Vpr) or from endogenous origin (such as Puma, Bax and Bak) all of which can act on the PTPC cooperate in the patient to induce apoptosis of a specific subset of vulnerable cells. Whether these
230
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
effects are simply additive or whether they interact synergistically to trigger AIDS remains on open conundrum.
Acknowledgements Our work is supported by grants from CNRS (to GK and CB), ANRS (to GK), EC (QLG1-CT-199900739, to GK), LNC (to GK), ARC (to GK and CB), FRM (to CB), and Ministry of Science (to GK and CB). Aure´lien Deniaud recieves a fellowship from the French ministry of Science.
References Arunagiri, C., Macreadie, I., Hewish, D., Azad, A., 1997. A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4C lymphocytes. Apoptosis 2, 69–76. Azad, A., 2000. Could Nef and Vpr proteins contribute to disease progression by promoting depletion of bystander cells and prolonged survival of HIV-infected cells? Biochem. Biophys. Res. Commun. 267, 677–685. Bauer, M.K., Schubert, A., Rocks, O., Grimm, S., 1999. Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis. J. Cell Biol. 147 (7), 1493–1502. Belzacq, A.S., El Hamel, C., Vieira, H.L., Cohen, I., Haouzi, D., Metivier, D., et al., 2001a. Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20 (52), 7579–7587. Belzacq, A.S., Jacotot, E., Vieira, H.L., Mistro, D., Granville, D.J., Xie, Z., et al., 2001b. Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target. Cancer Res. 61 (4), 1260–1264. Belzacq, A.S., Vieira, H.L., Kroemer, G., Brenner, C., 2002. The adenine nucleotide translocator in apoptosis. Biochimie 84 (2/3), 167–176. Beutner, G., Ru¨ck, A., Riede, B., Welte, W., Brdiczka, D., 1996. Complexes between kinases, mitochondrial porin, and adenylate translocator in rat brain resemble the permeability transition pore. Fed. Eur. Biochem. Soc. Lett. 396, 189–195. Boya, P., Roques, B., Kroemer, G., 2001. Viral and bacterial proteins regulating apoptosis at the mitochondrial level. Eur. Mol. Biol. Org. J. 20 (16), 4325–4331. Boya, P., Roumier, T., Andreau, K., Gonzalez-Polo, R., Zamzami, N., Castedo, M., Kroemer, G., 2003. Mitochondrion-targeted apoptosis regulators of viral origin. Biochem. Biophys. Res. Commun. 2003;, 575–581.
Brenner, C., Kroemer, G., 2003. The mitochondriotoxic domain of Vpr determines HIV-1 virulence. J. Clin. Invest. 111, 1455– 1457. Brustovetsky, N., Klingenberg, M., 1996. Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2C. Biochemistry 35 (26), 8483–8488. Bukrinsky, M., Adzhubei, A., 1999. Viral protein R of HIV-1. Rev. Med. Virol. 9, 39–49. Castedo, M., Ferri, K.F., Blanco, J., Roumier, T., Larochette, N., Barretina, J., et al., 2001. Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J. Exp. Med. 194 (8), 1097–1110. Castedo, M., Roumier, T., Blanco, J., Ferri, K., Barretina, J., Tintignac, L., et al., 2002. Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope. Eur. Mol. Biol. Org. J. 21, 4070–4080. Chen, M., Elder, R., Yu, M., O’Gorman, M., Selig, L., Benarous, R., et al., 1999. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast. J. Virol. 73, 3236–3245. Cohen, E., Terwilliger, E., Jalinoos, Y., Proulx, J., Sodroski, J., Haseltine, W., 1990. Identification of HIV-1 vpr product and function. J. Acquir. Immune Defic. Syndr., 11–18. Costantini, P., Belzacq, A.S., Vieira, H.L., Larochette, N., de Pablo, M.A., Zamzami, N., et al., 2000. Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis. Oncogene 19 (2), 307–314. De Oliveira Pinto, L., Lecoeur, H., Ledru, E., Rapp, C., Patey, O., Gougeon, M., 2002. Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro. Acquir. Immune Defic. Syndr. 16, 329–339. Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R., Landau, N., 1995. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69, 7909–7916. Doerner, A., Pauschinger, M., Badorff, A., Noutsias, M., Giessen, S., Schulze, K., et al., 1997. Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans. Fed. Eur. Biochem. Soc. Lett. 414 (2), 258–262. Doerner, A., Olesch, M., Giessen, S., Pauschinger, M., Schultheiss, H.P., 1999. Transcription of the adenine nucleotide translocase isoforms in various types of tissues in the rat. Biochim. Biophys. Acta 1417 (1), 16–24. Elder, R., Yu, M., Chen, M., Edelson, S., Zhao, Y., 2000. Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint. Virus Res. 68, 161–173. Everett, H., McFadden, G., 2001. Viral proteins and the mitochondrial apoptotic checkpoint. Cytokine Growth Factor Rev. 12 (2/3), 181–188. Everett, H., Barry, M., Lee, S.F., Sun, X., Graham, K., Stone, J., et al., 2000. M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J. Exp. Med. 191 (9), 1487–1498.
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233 Everett, H., Barry, M., Sun, X., Lee, S.F., Frantz, C., Berthiaume, L.G., et al., 2002. The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J. Exp. Med. 196 (9), 1127–1139. Fouchier, R., Meyer, B., Simon, J., Fischer, U., Albright, A., Gonzalez-Scarano, F., Malim, M., 1998. Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J. Virol. 1998;, 6004–6013. Goldmacher, V.S., 2002. vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie 84 (2/3), 177–185. Goldmacher, V.S., Bartle, L.M., Skaletskaya, A., Dionne, C.A., Kedersha, N.L., Vater, C.A., et al., 1999. A cytomegalovirusencoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl Acad. Sci. USA 96 (22), 12536–12541. Gougeon, M., 2003. Apoptosis as an HIV strategy to escape immune attack. Nat. Rev. Immunol. 3, 392–404. Gragerov, A., Kino, T., Ilyina-Gragerova, G., Chrousos, G., Pavlakis, G., 1998. HHR23A, the human homologue of the yeast repair protein RAD23, interacts specifically with Vpr protein and prevents cell cycle arrest but not the transcriptional effects of Vpr. Virology 245, 323–330. Halestrap, A., Brenner, C., 2003. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10, 1507–1525. Huang, M., Weeks, O., Zhao, L., Saltarelli, M., Bond, V., 2000. Effects of extracellular human immunodeficiency virus type 1 vpr protein in primary rat cortical cell cultures. J. Neurovirol. 6, 202–220. Jacotot, E., Ravagnan, L., Loeffler, M., Ferri, K.F., Vieira, H.L., Zamzami, N., et al., 2000. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J. Exp. Med. 191 (1), 33–46. Jacotot, E., Ferri, K.F., El Hamel, C., Brenner, C., Druillennec, S.Q, Hoebeke, J., et al., 2001. Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein R and Bcl-2. J. Exp. Med. 193 (4), 509–520. Jan, G., Belzacq, A.S., Haouzi, D., Rouault, A., Metivier, D., Kroemer, G., Brenner, C., 2002. Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria. Cell Death Differ. 9 (2), 179–188. Kroemer, G., Reed, J., 2000. Mitochondrial control of cell death. Nat. Med. 6, 513–519. Le Rouzic, E., Mousnier, A., Rustum, C., Stutz, F., Hallberg, E., Dargemont, C., Benichou, S., 2002. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J. Biol. Chem. 277, 45091–45098. Levy, D., Refaeli, Y., MacGregor, R., Weiner, D., 1994. Serum Vpr regulates productive infection and latency of human immunodeficiency virus type 1. Proc. Natl Acad. Sci. USA 91, 10873– 10877. Levy, D., Refaeli, Y., Weiner, D., 1995. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency
231
virus replication and reactivates virus from latency. J. Virol. 69, 1243–1252. Lum, J., Cohen, J., Nie, Z., Weaver, J., Gomez, T., Yao, X., et al., 2003. Vpr R77Q is associated with long-term non progressive HIV infection and impaired apoptosis induction. J. Clin. Invest. 111, 1547–1554. Luo, Z., Butcher, D., Murali, R., Srinivasan, A., Huang, Z., 1998. Structural studies of synthetic peptide fragments derived from the HIV-1 Vpr protein. Biochem. Biophys. Res. Commun. 244, 732–736. Macreadie, I., Castelli, L., Hewish, D., Kirkpatrick, A., Ward, A., Azad, A., 1995. A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc. Natl Acad. Sci. USA 92, 2770–2774. Macreadie, I., Thorburn, D., Kirby, D., Castelli, L., de Rozario, N., Azad, A., 1997. HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae. Fed. Eur. Biochem. Soc. Lett. 410, 145–149. Mahalingam, S., Ayyavoo, V., Patel, M., Kieber-Emmons, T., Weiner, D., 1997. Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J. Virol. 71, 6339–6347. Mansky, L., Preveral, S., Selig, L., Benarous, R., Benichou, S., 2000. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate. J. Virol. 74, 7039–7047. Martinou, J.C., Desagher, S., Antonsson, B., 2000. Cytochrome c release from mitochondria: all or nothing. Nat. Cell Biol. 2 (3), E41–E43. Marzo, I., Brenner, C., Zamzami, N., Susin, S.A., Beutner, G., Brdiczka, D., et al., 1998a. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl2-related proteins. J. Exp. Med. 187 (8), 1261–1271. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., et al., 1998b. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281 (5385), 2027–2031. Matsuda, M., Matsuda, N., Watanabe, A., Fujisawa, R., Yamamoto, K., Masuda, M., 2003. Cell cycle arrest induction by an adenoviral vector expressing HIV-1 Vpr in bovine and feline cells. Biochem. Biophys. Res. Commun. 311, 748–753. Morellet, N., Bouaziz, S., Petitjean, P., Roques, B., 2003. NMR structure of the HIV-1 regulatory protein Vpr. J. Mol. Biol. 327, 215–227. Muthumani, K., Hwang, D., Desai, B., Zhang, D., Dayes, N., Green, D., et al., 2002. HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells. J. Biol. Chem. 277, 37820–37831. Muthumani, K., Choo, A., Hwang, D., Chattergoon, M., Dayes, N., Zhang, D., et al., 2003. Mechanism of HIV-1 viral protein Rinduced apoptosis. Biochem. Biophys. Res. Commun. 304, 583–592. Nishizawa, M., Kamata, M., Mojin, T., Nakai, Y., Aida, Y., 2000. Induction of apoptosis by the Vpr protein of human
232
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
immunodeficiency virus type 1 occurs independently of G(2) arrest of the cell cycle. Virology 276, 16–26. Pebay-Peyroula, E., Dahout-Gonzalez, C., Kahn, R., Trezeguet, V., Lauquin, G., Brandolin, G., 2003. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39–44. Perfettini, J., Roumier, T., Castedo, M., Larochette, N., Boya, P., Reynal, B., et al., 2004. NF-kB and p53 are the dominant apoptosis-inducing transcription factors elicited by the HIV-1 enveloppe. J. Exp. Med. 199, 629. Pfaff, E., Klingenberg, M., 1968. Adenine nucleotide translocation of mitochondria. 1. Specificity and control. Eur. J. Biochem. 6 (1), 66–79. Pfaff, E., Heldt, H.W., Klingenberg, M., 1969. Adenine nucleotide translocation of mitochondria. Kinetics of the adenine nucleotide exchange. Eur. J. Biochem. 10 (3), 484–493. Poon, B., Grovit-Ferbas, K., Stewart, S., Chen, I., 1998. Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents. Science 281, 266–269. Rahmani, Z., Huh, K.W., Lasher, R., Siddiqui, A., 2000. Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J. Virol. 74 (6), 2840–2846. Roumier, T., Vieira, H., Castedo, M., Ferri, K., Boya, P., Andreau, K., et al., 2002. The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway. Cell Death Differ. 9, 1212–1219. Ru¨ck, A., Dolder, M., Wallimann, T., Brdiczka, D., 1998. Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore. Fed. Eur. Biochem. Soc. Lett. 426 (1), 97–101. Schubert, A., Grimm, S., 2004. Cyclophilin d, a component of the permeability transition-pore, is an apoptosis repressor. Cancer Res. 64, 85–93. Schuler, W., Wecker, K., de Rocquigny, H., Baudat, Y., Sire, J., Roques, B.P., 1999. NMR structure of the (52–96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions. J. Mol. Biol. 285 (5), 2105–2117. Shirakata, Y., Koike, K., 2003. Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J. Biol. Chem. Singh, S., Tomkowicz, B., Lai, D., Cartas, M., Mahalingam, S., Kalyanaraman, V., et al., 2000. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr. J. Virol. 74, 10650–10657. Stark, L., Hay, R., 1998. Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription. J. Virol. 72, 3037–3044. Stewart, S., Poon, B., Jowett, J., Chen, I., 1997. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J. Virol. 71, 5579–5592. Tsujimoto, Y., Shimizu, S., 2000. Bcl-2 family: life-or-death switch. Fed. Eur. Biochem. Soc. Lett. 466 (1), 6–10.
Tungaturthi, P., Sawaya, B., Singh, S., Tomkowicz, B., Ayyavoo, V., Khalili, K., et al., 2003. Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr. Biomed. Pharmacother. 20–24. Vander Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T., Thompson, C.B., 1997. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91 (5), 627–637. Vieira, H.L., Haouzi, D., Hamel, C.E., Jacotot, E., Belzacq, A.S., Brenner, C., Kroemer, G., 2000. Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator. Cell Death Differ. 7 (12), 1146–1154. Vodicka, M., Koepp, D., Silver, P., Emerman, M., 1998. HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 12, 175–185. Waldhuber, M., Bateson, M., Tan, J., Greenway, A., McPhee, D., 2003. Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization. Virology 313, 91–104. Wang, L., Mukherjee, S., Jia, F., Narayan, O., Zhao, L., 1995. Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and transactivation of viral long terminal repeat. J. Biol. Chem. 270, 25564–25569. Wecker, K., Morellet, N., Bouaziz, S., Roques, B., 2002. NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol. Comparison with the Vpr N-terminal (1–51) and Cterminal (52–96) domains. Eur. J. Biochem. 269, 3779–3788. Yamaguchi, T., Watanabe, N., Nakauchi, H., Koito, A., 1999. Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways. Microbiol. Immunol. 437–447. Yao, X., Subbramanian, R., Rougeau, N., Boisvert, F., Bergeron, D., Cohen, E., 1995. Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alphahelical structure in Vpr nuclear localization and virion incorporation. J. Virol. 69, 7032–7044. Yao, X., Lemay, J., Rougeau, N., Clement, M., Kurtz, S., Belhumeur, P., Cohen, E., 2002. Genetic selection of peptide inhibitors of human immunodeficiency virus type 1 Vpr. J. Biol. Chem. 277, 48816–48826. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssie`re, J.L., Petit, P.X., Kroemer, G., 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661–1672. Zamzami, N., Hamel, C.E., Maisse, C., Brenner, C., MunozPinedo, C., Belzacq, A.S., et al., 2000. Bid acts on the permeability transition pore complex to induce apoptosis. Oncogene 19 (54), 6342–6350. Zander, K., Sherman, M., Tessmer, U., Bruns, K., Wray, V., Prechtel, A., et al., 2003. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J. Biol. Chem. 278, 43202–43213. Zhang, S., Feng, Y., Narayan, O., Zhao, L., 2001. Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein–protein
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233 interaction with a novel human cytoplasmic protein VprBP. Gene 263, 131–140. Zhao, Y., Elder, R., 2000. Yeast perspectives on HIV-1 Vpr. Front. Biosci. 5, 905–916. Zhao, Y., Cao, J., O’Gorman, M.R., Yu, M., Yogev, R., 1996. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J. Virol. 70, 5821–5826.
233
Zhou, Y., Lu, Y., Ratner, L., 1998. Arginine residues in the Cterminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest. Virology 242, 414–424. Zoratti, M., Szabo, I., 1994. Electrophysiology of the inner mitochondrial membrane. J. Bioenerg. Biomembr. 26 (5), 543–553. Zoratti, M., Szabo, I., 1995. The mitochondrial permeability transition. Biochim. Biophys. Acta 1241 (2), 139–176.
Review
Mitochondrial membrane permeabilization by HIV-1 Vpr Aure´lien Deniauda, Catherine Brennera,*, Guido Kroemerb a
CNRS FRE 2445, Universite´ de Versailles/St Quentin, 45, avenue des Etats-Unis, 78035 Versailles, France Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif, France
b
Received 1 February 2004; received in revised form 17 May 2004; accepted 2 June 2004
Abstract The mitochondrion is a privileged target for apopotosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins. q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: Apoptosis; Adenine nucleotide translocase; Mitochondrion; Bcl-2; Viral target
1. Introduction HIV infection is characterized by a dramatic decrease in CD4C T cells, the progresive loss of immune function and the eventual evolution to AIDS in the absence of any therapeutic intervention
Abbreviations: ANT, adenine nucleotide translocator; BA, bongkrekic acid; CsA, cyclosporin A; DJm, mitochondrial transmembrane potential; HIV-1, human immunodeficiency virus-1; MMP, mitochondrial membrane permeabilization; MUP, 4-methylumbelliferyl phosphate; MU, 4-methylumbelliferone; IM, inner membrane; OM, outer membrane; PTPC, permeability transition pore complex; VDAC, voltage-dependent anion channel; Vpr, viral protein R. * Corresponding author. Tel.: C33-139-254-552; fax: C33-139254-572. E-mail address: [email protected] (C. Brenner).
such as highly active antiretroviral therapy (De Oliveira Pinto et al., 2002; Gougeon, 2003). Among various hypotheses, it has been postulated that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) protein may contribute to AIDS pathogenesis by inducing apoptotic death of CD4C T cells and depletion of bystander cells (Azad, 2000; Bukrinsky and Adzhubei, 1999). Indeed, in HIV-infected patients, Vpr is found associated with the virion, within infected cells, and as a soluble protein in the serum and the cerebrospinal fluid of patients (Levy et al., 1994; Muthumani et al., 2003). On theorical ground, free Vpr can account for the death of uninfected cells since the external addition of Vpr results in its penetration into cells, loss of the inner membrane potential (DJm), DNA fragmentation and cell death
1567-7249/$ - see front matter q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2004.06.012
224
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
(Muthumani et al., 2003). This is not restricted to CD4C T cells, because Vpr can kill a variety of cells including hippocampal neurons or astrocytes (Huang et al., 2000), cell lines of various species (Matsuda et al., 2003) as well as yeast cells (Macreadie et al., 1995). This suggests that Vpr activates a general step of the apoptotic cascade common to many eukaryotic cells. Thus, Vpr has been suggested to trigger the mitochondrial membrane permeabilization (MMP), namely via the opening of the permeability transition pore (PTPC) (Kroemer and Reed, 2000). The PTPC is a dynamic polyprotein complex formed in the contact site between the inner and the outer mitochondrial membranes (IM and OM, respectively) (Zoratti and Szabo, 1994, 1995) (Fig. 1). Despite tissue-, development- and differentiation state variations of protein expression (Belzacq et al., 2002; Doerner et al., 1997, 1999), it contains permanently the two most abundant IM and OM proteins, the adenine nucleotide translocator (ANT, in the IM), the voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well
as apoptosis-regulatory proteins from the Bax/Bcl-2 family (Beutner et al., 1996; Halestrap and Brenner, 2003; Marzo et al., 1998a,b). In physiological conditions, ANT is a vital, specific antiporter, which catalyzes the exchange of ATP and ADP on IM (Pfaff et al., 1969; Pfaff and Klingenberg, 1968). Upon calcium-induced conformational changes, ANT can form a non-specific pore (Brustovetsky and Klingenberg, 1996; Beutner et al., 1996). Moreover, pore formation by ANT elicited by a variety of different agents (e.g. Ca2C, atractyloside (Ru¨ck et al., 1998), thiol oxidation (Costantini et al., 2000), lipidic messengers (Jan et al., 2002), chemotherapeutic drugs (Belzacq et al., 2001a,b), etc.), is enhanced by Bax (Marzo et al., 1998b) and Bid (Zamzami et al., 2000) and inhibited by Bcl-2 (Marzo et al., 1998b), ADP as well as ATP (for review see Halestrap and Brenner, 2003). During MMP (for review Kroemer and Reed, 2000), pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, loss of inner membrane potential (Zamzami et al., 1995) and OM permeabilization, presumably due to physical rupture of OM (Vander Heiden et al., 1997). Although alternative mechanisms of MMP may exist (Martinou et al., 2000; Tsujimoto and Shimizu, 2000), ANT emerged as a major player in the regulation of cell death (for review Belzacq et al., 2002; Halestrap and Brenner, 2003; Vieira et al., 2000). This review discusses recent advances in the characterization of the pro-apoptotic mitochondrial damage induced by Vpr, the underlying molecular mechanisms of Vpr/PTPC cooperation to induce MMP and the place of Vpr in AIDS pathogenesis.
2. Pleitropic functions of Vpr
Fig. 1. Scheme of the PTPC organization. PTPC is a polyprotein complex built up at the contact site of mitochondrial inner and outer membranes (IM and OM, respectively). The opening of PTPC allows the diffusion solutes of MW!1500 Da between the cytosol and the mitochondrial matrix. HK, hexokinase; VDAC, voltagedependent anion channel; PBR, peripheral benzodiazepine receptor; CK, creatine kinase; ANT, adenine nucleotide translocator and CypD, cyclophilin D.
Vpr is a 14 kDa-protein endowed with pleiotropic functions. Indeed, the Vpr protein behaves as a transcriptional activator of HIV and heterologous promoters (Cohen et al., 1990; Wang et al., 1995, and for review Luo et al., 1998). It also induces a cell cycle arrest in G2 (Poon et al., 1998), blocks cell proliferation (Yamaguchi et al., 1999) and induces apoptosis (Stewart et al., 1997). All these effects may be mediated by protein–protein
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
225
Table 1 Eucaryotic proteins interacting with Vpr and role of the interaction Protein
Domain or aminoacids of Vpr
Role of the interaction
Reference
Cyclophilin A
N-terminus Pro 35
Zander et al. (2003)
Human nucleoporin (hCG1)
N-terminal region
Decrease of Vpr synthesis and loss of cell cycle effects Le Rouzic et al. (2002)
VprBP ANT HHR23A
ND (67–82) Vpr ND
Lys-tRNA synthetase (LysRS)
N- and C-terminus
Uracyl DNA glycosylase Sp1
Trp 54 Leu/Ile-rich domain
Cytoplasmic retention of Vpr Apoptosis induction via MMP Prevention of Vpr-induced cell cycle arrest Initiation of HIV-1 reverse transcription HIV-1 in vivo mutation rate Activation of HIV-1 long terminal repeat (LTR)-directed transcription
Le Rouzic et al. (2002); Fouchier et al. (1998) Zhang et al. (2001) Jacotot et al. (2000, 2001) Gragerov et al. (1998) Stark and Hay (1998) Mansky et al. (2000) Wang et al. (1995)
ND, not determined; MMP, mitochondrial membrane permeabilization.
interactions of Vpr with intracellular proteins (Table 1). These various functions are separable (Elder et al., 2000; Nishizawa et al., 2000) and presumably linked to the intracellular localization of Vpr in response to export and import signals contained in the N- and C-terminus of the protein and to the existence of different domains within the protein (Waldhuber et al., 2003) (Fig. 2). The pleitotropic effects of Vpr could also be related to the fact that there are three different sources of Vpr available in HIV-1 infected patients (for review Tungaturthi et al., 2003). These include cell-associated, virion-associated (infectious, infectious nonproductive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). Even if quantities of intracellular or circulating Vpr are difficult to estimate, it is interesting to note that purified Vpr (synthetic or expressed through baculovirus) when added in culture is able to penetrate into cells (Huang et al., 2000; Levy et al., 1995). This suggests that Vpr may use receptors present on the cell surface or may contain signal sequences that enable its entry into cells independently of the receptor pathway, for instance by endocytosis (for review Tungaturthi et al., 2003). 2.1. Vpr, elements of structure/function relationship Recently, a novel 3D structure of (1–96) full length Vpr has been resolved by NMR (Morellet et al.,
2003). The Vpr protein was synthesized with 22 labeled amino acids in pure water and in twater containing 10, 20, 30% of acetonitrile. The folding of the protein is characterized by three well-defined alpha-helices (amino acids 17–33, 38–50 and 56–77) surrounded by flexible N- and C-terminal domains (Fig. 2). In contrast to a previous structure obtained in the presence of the solvent trifluoroethanol (Wecker et al., 2002), the three alpha-helices are folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues. Interestingly, this structure is highly compatible with the interaction of Vpr with different effector targets. For example, amino acids contained in the N-terminus (e.g. Trp18, Leu 22 and Leu 26, Ile 63, Gln 65, Leu 67) and C-terminus basic amino acids, are accessible for interaction with the structural Gag polyprotein, with the uracil DNA glycosylase, with the nucleocapsid protein NCp7, and for activity to induce G2 arrest, respectively (Morellet et al., 2003; Schuler et al., 1999). Moreover, arginine residues R73, R77, and R80 would be accessible for interaction with the first cytoplasmic loop of the mitochondrial protein ANT (Brenner and Kroemer, 2003) (Fig. 2). Several groups have proposed putative structure– function map of Vpr (Fig. 2). Mutations in the alphahelical region reportedly eliminate the incorporation of Vpr into virions (Di Marzio et al., 1995; Mahalingam et al., 1997; Singh et al., 2000; Yao et al., 1995), as do point deletions at the C terminus
226
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
Fig. 2. Structure/function map of Vpr. The structure of Vpr depicts a folding of the protein characterized by three well-defined alpha-helices I, II and III (amino acids 17–33, 38–50 and 56–77, respectively) surrounded by flexible N- and C-terminal domains. The (72–83) domain, has been identified as the mitochondriotoxic domain, capable of inducing mitochondrial membrane permeabilization. Mutations of aminoacids R73, 77, and R80 abolish the mitochondriotoxic activity of the domain. Key residues for nucleus import of Vpr are localized from aminoacids 17 to 60. Helix I and aminoacids 35–46 are implicated in virion incorporation and aminoacids 84–96, plus R73, R77, R80 and R85 are critical for the Vpr-induced cell cycle arrest in G2.
(Zhou et al., 1998). Amino acids in the core of the protein (amino acids 30–60) have been found to be important for nuclear localization of Vpr (Mahalingam et al., 1997), and the C-terminal basic region is essential for protein stability and induction of cell cycle arrest (Di Marzio et al., 1995; Mahalingam et al., 1997; Yao et al., 1995) (Fig. 2). In addition, a mitochondriotoxic domain has been located in the C-terminal moiety of the protein and key aminoacids identified within the 52–96 domain of Vpr (Jacotot et al., 2000, 2001; Muthumani et al., 2002; Roumier et al., 2002) (Fig. 2). A point mutation in this domain (aminoacid R77) found in long-term non-progressors (that is HIV-1 carriers who do not develop AIDS), generates a mutant protein (aminoacid Q77), which exhibits an impaired pro-apoptotic activity. This observation
established for the first time a link between the mitochondriotoxic properties of Vpr and the clinical outcome of HIV-infected patients (Brenner and Kroemer, 2003; Lum et al., 2003). 3. Vpr targets the mitochondrion to induce MMP After its entry into a cell, a fraction of Vpr localizes to the mitochondrial compartment as well as to the nucleus (Arunagiri et al., 1997; Jacotot et al., 2000). By association with the mitochondrial membrane, Vpr triggers a typical mitochondrial pathway of apoptosis characterized by an early loss of the inner transmembrane potential (DJm), the release of cytochrome c, the loss of respiratory activity and activation of various caspases including caspase 9 and 3 (Jacotot et al., 2000, 2001; Muthumani et al., 2002).
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
This has been shown for primary human lymphoid cells, as well as for T cell lines, independently of the mode of delivery of Vpr, i.e. extracellular addition or adenovirus delivery of Vpr (Jacotot et al., 2000, 2001; Muthumani et al., 2002). However, the implication of caspases is questionable since the knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated cell killing (Roumier et al., 2002). The mitochondrial effect of Vpr is likely to be direct since all criteria of mitochondrial damage depicted above have been reproduced by the addition of synthetic (52–96) Vpr-derived peptide in cell-free systems, such as mice liver-isolated mitochondria or HeLa cell-isolated nuclei (Jacotot et al., 2000, 2001). As a negative control, the synthetic (1–51) Vpr peptide was devoid of any effect on human primary lymphoid cells, on culture cell lines (Jurkat, COS, etc.) or on isolated liver mitochondria (Jacotot et al., 2001). Altogether, this suggested that Vpr requires no extramitochondrial cofactor and/or cytoplasmic second messenger to exert its mitochondriotoxic activity via its C-terminal moiety. Whether the mitochondrial oncoprotein Bcl-2 is able to protect cells from Vpr-driven apoptosis is a matter of debate. Thus, by stable transfection of T cell lines, Bcl-2 prevented the loss of DJm, the exposure of phosphatidylserine residues, the nuclear chromatin condensation and the release of apoptogenic factors from the intermembrane mitochondrial compartment (Jacotot et al., 2000). These effects were comparable with the protective effects of two PTPC inhibitors, cyclosporin A (CsA), a cyclophilin D ligand that inhibits the binding of CypD to ANT, and bongkrekic acid (BA), an ANT ligand. Furthermore, Bcl-2 protection was confirmed in in vitro assays with recombinant Bcl-2. Thus, Bcl-2 blocked Vpr interaction with mitochondrial PTPC proteins, and the channel activity of Vpr in proteoliposomes 3 (Jacotot et al., 2001) (Fig. 3). In contrast, Muthumani, et al. (2002) found that Vpr reduces either the expression level of Bcl-2 or the level of Vpr-induced apoptosis, but does not suppress it, suggesting that experimental conditions (Bcl-2 and Vpr delivery mode, Bcl-2/Vpr ratios, etc.) can critically influence the capacity of Bcl-2 to inhibit Vpr-driven cell death.
227
3.1. Vpr and PTPC components, the lethal interaction The previous functional observations prompted the question of how Vpr can mediate its mitochondrial effects, and notably, the MMP. The first pharmacological clue came from experiments showing that PTPC inhibitors, CsA and BA, can prevent all Vpr-driven mitochondrial damages, in cellular experiments as well as in cell-free systems (Jacotot et al., 2000). This led to the search of an intracellular target of Vpr and resulted in the identification of ANT as a Vpr-cooperating protein critical for the induction of MMP (Jacotot et al., 2000, 2001). Indeed, Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as ANT (Fig. 3) (Jacotot et al., 2001). This effect is prevented by addition of recombinant Bcl-2, CsA and BA, but not a Bcl-2 mutant lacking their pore forming helix nor a non-dimerizing mutant of Bcl-2 (Jacotot et al., 2001). The Vpr COOH terminus binds to purified ANT, as well as a molecular complex containing ANT and VDAC. In addition, Bcl-2 reduces the ANT/Vpr interaction, as determined by affinity purification and plasmon resonance studies (Jacotot et al., 2001). Concomitantly, Bcl-2 suppresses channel formation by the ANT/Vpr complex in synthetic membranes. In contrast to Ko¨nig’s polyanion (PA10) (Jacotot et al., 2001) and to NADH (Brenner, unpublished result), specific pore blockers of VDAC, Bcl-2 failed to prevent (52–96) Vpr from crossing the mitochondrial OM. This finding strongly suggests that VDAC would allow the access of Vpr to the inner mitochondrial membrane (Jacotot et al., 2001). Critical aminoacids for interaction with ANT were identified by point mutation of synthetic peptides (R73, R77 and R80) and the minimal mitochondriotoxic domain was defined to be a dodecapeptide (aminoacids 72–83; FRIGCRHSRIGI) that is located within the C-terminal a-helix. This minimal toxic domain could be engaged in cation–p interactions with aminoacids F109, W111, Y113 and/or F114 within ANT (Brenner and Kroemer, 2003). Indeed, these aminoacids are exposed to the intermembrane space as indicated by the X-ray crystallographic structure of ANT, at least in its ‘c-state’ conformation (Pebay-Peyroula et al., 2003). The Vpr/ANT interaction is direct, since it is abolished by the addition of
228
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
Fig. 3. Experimental methods to measure the capacity of Vpr and derived peptides to functionally interact with PTPC members. ANT, purified from rat heart mitochondria and PTPC, purified from rat brain mitochondria, are reconstituted into phosphatidyl/cardiolipin and phosphatidyl/cholesterol liposomes, repectively, in which 4-methylumbelliferyl phosphate (4-MUP) has been encapsulated. The conversion into a non-specific pore is measured by the release of 4-MUP which reacts with alkaline phosphatase to generate a fluorescent signal. Moreover, ANT is incorporated into planar lipid bilayers to measure its electrophysiological properties, such as single channel activity or macroscopic conductance, opening frequency and ionic specificity. In the two set-ups, ANT and PTPC are in a closed conformation and upon stimulation by sub-micromolar doses of Vpr or Vpr-derived peptides, they can be converted in large non-specific pores. Human recombinant Bcl-2, the peptide (104–116) derived from ANT, cyclosporin A (CsA) and bongrekic acid (BA) are inhibitors of ANT and/or PTPC channel activity and are used as control of the specificity of interaction.
a peptide corresponding to the Vpr binding site of ANT, (104–116) ANT2; DKRTQFWRYFAGN, but not by control peptides such as a scrambled (104–116) ANT2, a mutated (104–116) ANT2 for positively charged aminoacids (K and R), and a topologically equivalent peptide of the human phosphate carrier, a member of mitochondrial carrier family as ANT (Jacotot et al., 2001). Because ANT is expressed as three distinct isoforms, which would exert various roles in apoptosis (Bauer et al., 1999; Schubert and Grimm, 2004), Vpr affinity for each ANT isoform could be slightly different according to differences in their primary sequence. Thus, these isoforms differ in aminoacid composition and in the number of positive charges within the (104–116) domain, i.e. RHK for (106–108) ANT1, KRT for (106–108) ANT2,
and KHT for (106–108) ANT3. In conclusion, this region overlapping with the binding domain of Bax and Bcl-2 to ANT, i.e. aminoacids 105–155 (Marzo et al., 1998b), Vpr modulates MMP through a direct interaction with the apoptogenic region of ANT.
4. Vpr and the yeast model Vpr also harbors multiple activities in yeast cells (for review Zhao and Elder, 2000). This includes nuclear import, induction of cell cycle G2 arrest, mitochondrial damage, morphological changes and cell death. Therefore, Vpr can be expected to target phylogenetically conserved cellular processes and molecules. Thus, studies involving fission yeast
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
(Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) established that Vpr is able to induce a G2 arrest through inhibitory phosphorylation of the cyclin-dependent kinase by a pathway in which protein phosphatase 2A would play a critical role (Zhao et al., 1996). The protein is imported in the nuclear as a viral pre-integration complex by binding to importin-alpha subunit and nucleoporin (Vodicka et al., 1998), and triggers apoptosis by directly permeabilizing the mitochondrial membrane (Macreadie et al., 1997). It should be noted that the Vpr-induced G2 arrest in yeast is independent of cell death induction and of the DNA damage checkpoint and of nuclear localization of Vpr (Chen et al., 1999; Elder et al., 2000). Moreover, the aminoacid residues of Vpr at position 29, 33 and 71 were shown to be important sites for maintaining the overall structure of Vpr (Chen et al., 1999) and the mitochondriotoxic domain defined as the conserved amino acid sequence motif HFRIGCRHSRIG (Macreadie et al., 1997). Taking advantage of knock-out yeasts, invalidated for one or several isoforms of VDAC and ANT, Jacotot et al. (2000) showed that either VDAC or ANT are required for cell killing. This helps to explain how Vpr induces MMP,
Fig. 4. Mechanisms of MMP-induced by viral proteins targeting the PTPC compounds VDAC, ANT and PBR. HBx translocates to mitochondria to interact with VDAC3 and to induce MMP independently of Bcl-2. Vpr enters into mitochondria via VDAC, binds to ANT in a Bcl-2-dependent manner and induces MMP. vMIA interacts with ANT (but not with VDAC) and prevents MMP. M11L inhibits MMP through a physical association with PBR in the OM.
229
possibly via a VDAC-mediated import of Vpr for a direct interaction with ANT (Fig. 4). Because of the great versatility of yeast and the existence of wide mutant libraries, future studies of Vpr in yeast should lead to important advances in understanding the mechanisms of Vpr activities. Moreover, yeast may be instrumental for the screening of inhibitory molecules of Vpr inhibitors (Yao et al., 2002). 4.1. Open questions and perspectives Vpr represents probably one of the viral protein for which the lethal effects are the best characterized (Boya et al., 2001, 2003). However, it is interesting to note that Vpr is not the sole viral protein affecting mitochondrial function (Fig. 4). Recently at least, three proteins, vMIA from cytomagalovirus (Goldmacher, 2002; Goldmacher et al., 1999), M11L from myxoma virus (Everett et al., 2000, 2002; Everett and McFadden, 2001) and HBx from Hepatitis B (Rahmani et al., 2000; Shirakata and Koike, 2003), have been shown to modulate MMP by targeting PTPC members. vMIA reportedly interacts with ANT and M11L with the peripheral benzodiazepine receptor to inhibit cell death, whereas HBx would interact with VDAC to promote apoptosis. As to Vpr, numerous issues should be addressed to elucidate the precise molecular mechanisms of its pro-apoptotic activity. Uncertainties remain on the mechanisms allowing the targeting of Vpr to the mitochondrial compartment, the precise hierarchy of mitochondrial events, and the existence of one single or several submitochondrial targets. Moreover, it will be important to determine the exact place of Vpr in AIDS pathogenesis. In HIV-1 carriers, the envelope glycoprotein complex has recently been shown to stimulate a p53-dependent transcriptional program leading to the overexpression of the BH3-only protein Puma, a pro-apoptotic protein that can activate pro-opapototic Bcl-2 multidomain family members such as Bax and Bak (Castedo et al., 2001, 2002; Perfettini et al., 2004). This suggests that several pro-apoptotic proteins, be they virus encoded (such as Vpr) or from endogenous origin (such as Puma, Bax and Bak) all of which can act on the PTPC cooperate in the patient to induce apoptosis of a specific subset of vulnerable cells. Whether these
230
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
effects are simply additive or whether they interact synergistically to trigger AIDS remains on open conundrum.
Acknowledgements Our work is supported by grants from CNRS (to GK and CB), ANRS (to GK), EC (QLG1-CT-199900739, to GK), LNC (to GK), ARC (to GK and CB), FRM (to CB), and Ministry of Science (to GK and CB). Aure´lien Deniaud recieves a fellowship from the French ministry of Science.
References Arunagiri, C., Macreadie, I., Hewish, D., Azad, A., 1997. A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4C lymphocytes. Apoptosis 2, 69–76. Azad, A., 2000. Could Nef and Vpr proteins contribute to disease progression by promoting depletion of bystander cells and prolonged survival of HIV-infected cells? Biochem. Biophys. Res. Commun. 267, 677–685. Bauer, M.K., Schubert, A., Rocks, O., Grimm, S., 1999. Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis. J. Cell Biol. 147 (7), 1493–1502. Belzacq, A.S., El Hamel, C., Vieira, H.L., Cohen, I., Haouzi, D., Metivier, D., et al., 2001a. Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20 (52), 7579–7587. Belzacq, A.S., Jacotot, E., Vieira, H.L., Mistro, D., Granville, D.J., Xie, Z., et al., 2001b. Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target. Cancer Res. 61 (4), 1260–1264. Belzacq, A.S., Vieira, H.L., Kroemer, G., Brenner, C., 2002. The adenine nucleotide translocator in apoptosis. Biochimie 84 (2/3), 167–176. Beutner, G., Ru¨ck, A., Riede, B., Welte, W., Brdiczka, D., 1996. Complexes between kinases, mitochondrial porin, and adenylate translocator in rat brain resemble the permeability transition pore. Fed. Eur. Biochem. Soc. Lett. 396, 189–195. Boya, P., Roques, B., Kroemer, G., 2001. Viral and bacterial proteins regulating apoptosis at the mitochondrial level. Eur. Mol. Biol. Org. J. 20 (16), 4325–4331. Boya, P., Roumier, T., Andreau, K., Gonzalez-Polo, R., Zamzami, N., Castedo, M., Kroemer, G., 2003. Mitochondrion-targeted apoptosis regulators of viral origin. Biochem. Biophys. Res. Commun. 2003;, 575–581.
Brenner, C., Kroemer, G., 2003. The mitochondriotoxic domain of Vpr determines HIV-1 virulence. J. Clin. Invest. 111, 1455– 1457. Brustovetsky, N., Klingenberg, M., 1996. Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2C. Biochemistry 35 (26), 8483–8488. Bukrinsky, M., Adzhubei, A., 1999. Viral protein R of HIV-1. Rev. Med. Virol. 9, 39–49. Castedo, M., Ferri, K.F., Blanco, J., Roumier, T., Larochette, N., Barretina, J., et al., 2001. Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J. Exp. Med. 194 (8), 1097–1110. Castedo, M., Roumier, T., Blanco, J., Ferri, K., Barretina, J., Tintignac, L., et al., 2002. Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope. Eur. Mol. Biol. Org. J. 21, 4070–4080. Chen, M., Elder, R., Yu, M., O’Gorman, M., Selig, L., Benarous, R., et al., 1999. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast. J. Virol. 73, 3236–3245. Cohen, E., Terwilliger, E., Jalinoos, Y., Proulx, J., Sodroski, J., Haseltine, W., 1990. Identification of HIV-1 vpr product and function. J. Acquir. Immune Defic. Syndr., 11–18. Costantini, P., Belzacq, A.S., Vieira, H.L., Larochette, N., de Pablo, M.A., Zamzami, N., et al., 2000. Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis. Oncogene 19 (2), 307–314. De Oliveira Pinto, L., Lecoeur, H., Ledru, E., Rapp, C., Patey, O., Gougeon, M., 2002. Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro. Acquir. Immune Defic. Syndr. 16, 329–339. Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R., Landau, N., 1995. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69, 7909–7916. Doerner, A., Pauschinger, M., Badorff, A., Noutsias, M., Giessen, S., Schulze, K., et al., 1997. Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans. Fed. Eur. Biochem. Soc. Lett. 414 (2), 258–262. Doerner, A., Olesch, M., Giessen, S., Pauschinger, M., Schultheiss, H.P., 1999. Transcription of the adenine nucleotide translocase isoforms in various types of tissues in the rat. Biochim. Biophys. Acta 1417 (1), 16–24. Elder, R., Yu, M., Chen, M., Edelson, S., Zhao, Y., 2000. Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint. Virus Res. 68, 161–173. Everett, H., McFadden, G., 2001. Viral proteins and the mitochondrial apoptotic checkpoint. Cytokine Growth Factor Rev. 12 (2/3), 181–188. Everett, H., Barry, M., Lee, S.F., Sun, X., Graham, K., Stone, J., et al., 2000. M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J. Exp. Med. 191 (9), 1487–1498.
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233 Everett, H., Barry, M., Sun, X., Lee, S.F., Frantz, C., Berthiaume, L.G., et al., 2002. The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J. Exp. Med. 196 (9), 1127–1139. Fouchier, R., Meyer, B., Simon, J., Fischer, U., Albright, A., Gonzalez-Scarano, F., Malim, M., 1998. Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J. Virol. 1998;, 6004–6013. Goldmacher, V.S., 2002. vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie 84 (2/3), 177–185. Goldmacher, V.S., Bartle, L.M., Skaletskaya, A., Dionne, C.A., Kedersha, N.L., Vater, C.A., et al., 1999. A cytomegalovirusencoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl Acad. Sci. USA 96 (22), 12536–12541. Gougeon, M., 2003. Apoptosis as an HIV strategy to escape immune attack. Nat. Rev. Immunol. 3, 392–404. Gragerov, A., Kino, T., Ilyina-Gragerova, G., Chrousos, G., Pavlakis, G., 1998. HHR23A, the human homologue of the yeast repair protein RAD23, interacts specifically with Vpr protein and prevents cell cycle arrest but not the transcriptional effects of Vpr. Virology 245, 323–330. Halestrap, A., Brenner, C., 2003. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10, 1507–1525. Huang, M., Weeks, O., Zhao, L., Saltarelli, M., Bond, V., 2000. Effects of extracellular human immunodeficiency virus type 1 vpr protein in primary rat cortical cell cultures. J. Neurovirol. 6, 202–220. Jacotot, E., Ravagnan, L., Loeffler, M., Ferri, K.F., Vieira, H.L., Zamzami, N., et al., 2000. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J. Exp. Med. 191 (1), 33–46. Jacotot, E., Ferri, K.F., El Hamel, C., Brenner, C., Druillennec, S.Q, Hoebeke, J., et al., 2001. Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein R and Bcl-2. J. Exp. Med. 193 (4), 509–520. Jan, G., Belzacq, A.S., Haouzi, D., Rouault, A., Metivier, D., Kroemer, G., Brenner, C., 2002. Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria. Cell Death Differ. 9 (2), 179–188. Kroemer, G., Reed, J., 2000. Mitochondrial control of cell death. Nat. Med. 6, 513–519. Le Rouzic, E., Mousnier, A., Rustum, C., Stutz, F., Hallberg, E., Dargemont, C., Benichou, S., 2002. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J. Biol. Chem. 277, 45091–45098. Levy, D., Refaeli, Y., MacGregor, R., Weiner, D., 1994. Serum Vpr regulates productive infection and latency of human immunodeficiency virus type 1. Proc. Natl Acad. Sci. USA 91, 10873– 10877. Levy, D., Refaeli, Y., Weiner, D., 1995. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency
231
virus replication and reactivates virus from latency. J. Virol. 69, 1243–1252. Lum, J., Cohen, J., Nie, Z., Weaver, J., Gomez, T., Yao, X., et al., 2003. Vpr R77Q is associated with long-term non progressive HIV infection and impaired apoptosis induction. J. Clin. Invest. 111, 1547–1554. Luo, Z., Butcher, D., Murali, R., Srinivasan, A., Huang, Z., 1998. Structural studies of synthetic peptide fragments derived from the HIV-1 Vpr protein. Biochem. Biophys. Res. Commun. 244, 732–736. Macreadie, I., Castelli, L., Hewish, D., Kirkpatrick, A., Ward, A., Azad, A., 1995. A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc. Natl Acad. Sci. USA 92, 2770–2774. Macreadie, I., Thorburn, D., Kirby, D., Castelli, L., de Rozario, N., Azad, A., 1997. HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae. Fed. Eur. Biochem. Soc. Lett. 410, 145–149. Mahalingam, S., Ayyavoo, V., Patel, M., Kieber-Emmons, T., Weiner, D., 1997. Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J. Virol. 71, 6339–6347. Mansky, L., Preveral, S., Selig, L., Benarous, R., Benichou, S., 2000. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate. J. Virol. 74, 7039–7047. Martinou, J.C., Desagher, S., Antonsson, B., 2000. Cytochrome c release from mitochondria: all or nothing. Nat. Cell Biol. 2 (3), E41–E43. Marzo, I., Brenner, C., Zamzami, N., Susin, S.A., Beutner, G., Brdiczka, D., et al., 1998a. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl2-related proteins. J. Exp. Med. 187 (8), 1261–1271. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., et al., 1998b. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281 (5385), 2027–2031. Matsuda, M., Matsuda, N., Watanabe, A., Fujisawa, R., Yamamoto, K., Masuda, M., 2003. Cell cycle arrest induction by an adenoviral vector expressing HIV-1 Vpr in bovine and feline cells. Biochem. Biophys. Res. Commun. 311, 748–753. Morellet, N., Bouaziz, S., Petitjean, P., Roques, B., 2003. NMR structure of the HIV-1 regulatory protein Vpr. J. Mol. Biol. 327, 215–227. Muthumani, K., Hwang, D., Desai, B., Zhang, D., Dayes, N., Green, D., et al., 2002. HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells. J. Biol. Chem. 277, 37820–37831. Muthumani, K., Choo, A., Hwang, D., Chattergoon, M., Dayes, N., Zhang, D., et al., 2003. Mechanism of HIV-1 viral protein Rinduced apoptosis. Biochem. Biophys. Res. Commun. 304, 583–592. Nishizawa, M., Kamata, M., Mojin, T., Nakai, Y., Aida, Y., 2000. Induction of apoptosis by the Vpr protein of human
232
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233
immunodeficiency virus type 1 occurs independently of G(2) arrest of the cell cycle. Virology 276, 16–26. Pebay-Peyroula, E., Dahout-Gonzalez, C., Kahn, R., Trezeguet, V., Lauquin, G., Brandolin, G., 2003. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39–44. Perfettini, J., Roumier, T., Castedo, M., Larochette, N., Boya, P., Reynal, B., et al., 2004. NF-kB and p53 are the dominant apoptosis-inducing transcription factors elicited by the HIV-1 enveloppe. J. Exp. Med. 199, 629. Pfaff, E., Klingenberg, M., 1968. Adenine nucleotide translocation of mitochondria. 1. Specificity and control. Eur. J. Biochem. 6 (1), 66–79. Pfaff, E., Heldt, H.W., Klingenberg, M., 1969. Adenine nucleotide translocation of mitochondria. Kinetics of the adenine nucleotide exchange. Eur. J. Biochem. 10 (3), 484–493. Poon, B., Grovit-Ferbas, K., Stewart, S., Chen, I., 1998. Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents. Science 281, 266–269. Rahmani, Z., Huh, K.W., Lasher, R., Siddiqui, A., 2000. Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J. Virol. 74 (6), 2840–2846. Roumier, T., Vieira, H., Castedo, M., Ferri, K., Boya, P., Andreau, K., et al., 2002. The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway. Cell Death Differ. 9, 1212–1219. Ru¨ck, A., Dolder, M., Wallimann, T., Brdiczka, D., 1998. Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore. Fed. Eur. Biochem. Soc. Lett. 426 (1), 97–101. Schubert, A., Grimm, S., 2004. Cyclophilin d, a component of the permeability transition-pore, is an apoptosis repressor. Cancer Res. 64, 85–93. Schuler, W., Wecker, K., de Rocquigny, H., Baudat, Y., Sire, J., Roques, B.P., 1999. NMR structure of the (52–96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions. J. Mol. Biol. 285 (5), 2105–2117. Shirakata, Y., Koike, K., 2003. Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J. Biol. Chem. Singh, S., Tomkowicz, B., Lai, D., Cartas, M., Mahalingam, S., Kalyanaraman, V., et al., 2000. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr. J. Virol. 74, 10650–10657. Stark, L., Hay, R., 1998. Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription. J. Virol. 72, 3037–3044. Stewart, S., Poon, B., Jowett, J., Chen, I., 1997. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J. Virol. 71, 5579–5592. Tsujimoto, Y., Shimizu, S., 2000. Bcl-2 family: life-or-death switch. Fed. Eur. Biochem. Soc. Lett. 466 (1), 6–10.
Tungaturthi, P., Sawaya, B., Singh, S., Tomkowicz, B., Ayyavoo, V., Khalili, K., et al., 2003. Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr. Biomed. Pharmacother. 20–24. Vander Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T., Thompson, C.B., 1997. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91 (5), 627–637. Vieira, H.L., Haouzi, D., Hamel, C.E., Jacotot, E., Belzacq, A.S., Brenner, C., Kroemer, G., 2000. Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator. Cell Death Differ. 7 (12), 1146–1154. Vodicka, M., Koepp, D., Silver, P., Emerman, M., 1998. HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 12, 175–185. Waldhuber, M., Bateson, M., Tan, J., Greenway, A., McPhee, D., 2003. Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization. Virology 313, 91–104. Wang, L., Mukherjee, S., Jia, F., Narayan, O., Zhao, L., 1995. Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and transactivation of viral long terminal repeat. J. Biol. Chem. 270, 25564–25569. Wecker, K., Morellet, N., Bouaziz, S., Roques, B., 2002. NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol. Comparison with the Vpr N-terminal (1–51) and Cterminal (52–96) domains. Eur. J. Biochem. 269, 3779–3788. Yamaguchi, T., Watanabe, N., Nakauchi, H., Koito, A., 1999. Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways. Microbiol. Immunol. 437–447. Yao, X., Subbramanian, R., Rougeau, N., Boisvert, F., Bergeron, D., Cohen, E., 1995. Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alphahelical structure in Vpr nuclear localization and virion incorporation. J. Virol. 69, 7032–7044. Yao, X., Lemay, J., Rougeau, N., Clement, M., Kurtz, S., Belhumeur, P., Cohen, E., 2002. Genetic selection of peptide inhibitors of human immunodeficiency virus type 1 Vpr. J. Biol. Chem. 277, 48816–48826. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssie`re, J.L., Petit, P.X., Kroemer, G., 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661–1672. Zamzami, N., Hamel, C.E., Maisse, C., Brenner, C., MunozPinedo, C., Belzacq, A.S., et al., 2000. Bid acts on the permeability transition pore complex to induce apoptosis. Oncogene 19 (54), 6342–6350. Zander, K., Sherman, M., Tessmer, U., Bruns, K., Wray, V., Prechtel, A., et al., 2003. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J. Biol. Chem. 278, 43202–43213. Zhang, S., Feng, Y., Narayan, O., Zhao, L., 2001. Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein–protein
A. Deniaud et al. / Mitochondrion 4 (2004) 223–233 interaction with a novel human cytoplasmic protein VprBP. Gene 263, 131–140. Zhao, Y., Elder, R., 2000. Yeast perspectives on HIV-1 Vpr. Front. Biosci. 5, 905–916. Zhao, Y., Cao, J., O’Gorman, M.R., Yu, M., Yogev, R., 1996. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J. Virol. 70, 5821–5826.
233
Zhou, Y., Lu, Y., Ratner, L., 1998. Arginine residues in the Cterminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest. Virology 242, 414–424. Zoratti, M., Szabo, I., 1994. Electrophysiology of the inner mitochondrial membrane. J. Bioenerg. Biomembr. 26 (5), 543–553. Zoratti, M., Szabo, I., 1995. The mitochondrial permeability transition. Biochim. Biophys. Acta 1241 (2), 139–176.