©2010 Taipei Medical University
R E V I E W A R T I C L E
1. Introduction
The crucial role of mitochondria in higher eukaryotes was recognized more than 100 years ago, when these organelles, originally defined “bioplasts”, were renamed because of the typical, threadlike shape assumed during spermatogenesis. Starting with the 1940s, the impor-tance of mitochondria in respiration was clarified, and Mitchell proposed the chemiosmotic theory, according to which the energy obtained by respiration is converted into an electrochemical gradient across the inner mem-brane and used for ATP synthesis.1
Mitochondria are divided into compartments with specialized functions, including the outer mitochondrial membrane, the intermembrane space, the inner mito-chondrial membrane, the cristae and the matrix. Human mitochondria contain more than 600 proteins;1 most of which are codified in the nucleus and transported into mitochondria, whereas only 13 are codified by
mitochondrial DNA (mtDNA), which is a circular DNA molecule of 16,659 bp present in several copies per mito-chondria.2 mtDNA is replicated autonomously and in a unique fashion, and the sole enzyme responsible for its replication is DNA polymerase-γ.2
Some mitochondrial proteins are encoded by mtDNA while others are encoded by nuclear DNA; thus, mito-chondrial dysfunctions can be due to mutations in either nuclear or mitochondrial genes. mtDNA is characterized by a high mutation rate (up to 20 times higher than nu-clear DNA) because of the mutagenic action of reactive oxygen species (ROS) that are produced during elec-tron chain transport, and because of the low capability of mitochondrial enzymes to repair DNA mutations.3 The major tissues affected by mitochondrial dysfunc-tion are tissues with a high energy demand such as the brain, muscles, heart and endocrine glands.
Mitochondria are usually seen as the “power plant” of the cell; indeed, more than 90% of cellular ATP is
Mitochondria play a dual role in the life of the cell, being capable of producing either energy (in the form of ATP) or potentially dangerous reactive oxygen species (ROS), and they also contain molecules that, when released into the cytoplasm, cause apoptosis. There is a growing interest in the importance of these organelles during the infection caused by the human immunodeficiency virus (HIV), as well as during its treatment. Indeed, several drugs that are capable of blocking HIV can also interact with the enzyme responsible for the replication of mitochondrial DNA and inhibit its activity. Cytokines pro-duced by the immune system can alter ROS production. Furthermore, the virus as such can trigger different mechanisms that interfere with mitochondrial functionality and induce alterations, ultimately causing cell death. As a result, mitochondria can be severely altered by HIV infection and by its treatment.
Received: Apr 7, 2010 Revised: Jun 5, 2010 Accepted: Jun 9, 2010 KEY WORDS: AIDS; antiretroviral therapy; HAART; HIV; mitochondria; mitochondrial DNA
The Role of Mitochondria in HIV Infection and
Its Treatment
Marcello Pinti, Milena Nasi, Lara Gibellini, Erika Roat, Sara De Biasi,
Linda Bertoncelli, Andrea Cossarizza*
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
*Corresponding author. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena, Italy. E-mail: andrea.cossarizza@unimore.it
produced by mitochondrial respiration.4 The mitochon-drial microenvironment provides the right context not only for the respiration and synthesis of ATP, but also for many other biochemical reactions that are crucial for cell metabolism, including tricarboxylic acid cycle, beta-oxidation, some steps for steroid synthesis, synthesis of heme group, and some steps of the urea cycle.5 A cru-cial role for the aerobic respiration in the mitochondria is played by the inner mitochondrial membrane, where the protein complexes involved in electron transfer are embedded. The inner mitochondrial membrane is char-acterized by a biochemical composition that renders it impermeable to H+, thus providing the barrier for the generation of proton gradient used for ATP synthesis.
Alongside these crucial functions in cell metabolism, mitochondria play other roles in processes that are not directly related to cell metabolism. In particular, during the 1990s, it was shown that mitochondria are crucial for triggering apoptosis, and in particular that loss of mem-brane potential (⌬ψm) and changes in mitochondrial membrane permeabilization (MMP) often represent cru-cial steps for the execution of apoptosis.6–9 In fact, MMP causes the release of several molecules that are: (1) di-rect activators of caspases (such as cytochrome c);10 (2) indirect activators of caspases (e.g., SMAC/DIABLO; OMI/HTRA2);11 or (3) activator of apoptosis independ-ently from caspases (e.g., apoptosis inducing factor).11
It is well known that the human immunodeficiency virus type-1 (HIV) is able to profoundly act on the regu-lation of apoptotic pathways, either directly (that is, through the action of different viral gene products) or indirectly (through changes in cell metabolism that can alter regulation of extrinsic or intrinsic apoptosis path-ways). Until a few years ago, infection with HIV caused an inexorable decline in immune function, leading to fatal consequences. Until 1995, the only drugs that had some (modest) effects on HIV infection were a few com-pounds in the category of nucleosidic reverse transcrip-tase inhibitors (NRTIs), molecules that act as competitive inhibitors of the viral reverse transcriptase, the enzyme that copies HIV-RNA to cDNA.12 In the mid-nineties, other drugs were developed that were able to inhibit another viral target, the viral protease; this class of drugs is known as protease inhibitors (PIs).13 Nowadays, in most patients the combination of PIs and nucleosidic, non-nucleosidic or nucleotidic reverse transcriptase inhibi-tors can block viral replication, and is defined as highly active antiretroviral therapy (HAART), which is able to restore the immune system and block the onset of op-portunistic infections.14–19 Other categories of drugs, such as inhibitors of viral entry into the cell20 and inhibi-tors of the chemokine receptor CCR5,21 are now available and being used in therapy; other drugs, including in-hibitors of viral integrase, are under testing in Phase III clinical trials.22
HIV + patients on HAART survive by suppression of virus replication, not by total elimination of the virus.
As a consequence, the therapy must be taken through-out life with a very strict adherence in order to avoid the appearance of viral resistance to the drugs. How-ever, in a large number of cases, the chronic consump-tion of antiretroviral drugs causes the onset of several side effects, including metabolic and aesthetic altera-tions, which can have serious consequences for the health of the patient. As mitochondria are involved in so many different metabolic and regulative pathways, it is not surprising that drugs that affect one or more reactions in mitochondria can deeply affect cellular func-tionality, leading to important alterations in different tissues and organs.
In this review, we will focus on the role of mitochon-dria in HIV infection and its treatment, analyzing the effects of drugs used for the treatment of HIV infection on mitochondria metabolism and functionality, the con-sequences of their consumption in treated patients, and the direct effects of HIV on mitochondria and mtDNA.
2. Antiretroviral Therapy and Mitochondria:
The Role of NRTIs
2.1. NRTIs are able to influence mitochondrial metabolism
NRTIs are analogs of endogenous 2⬘-deoxynucleosides and 2’-deoxynucleotides, the molecules necessary for the synthesis of DNA. Since the discovery of the antiret-roviral capabilities of zidovudine (AZT) in 1985,12,23 seven nucleosides and one nucleotide have been approved for the treatment of HIV infection, i.e., AZT in 1987, fol-lowed by didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir diso-proxil fumarate (which is converted in vivo to tenofovir), and emtricitabine (Table 1).23–41
NRTIs are inactive in their parent form, and are phos-phorylated by host cell kinases and phosphotrans-ferases to form deoxynucleoside triphosphate (dNTP) analogs.23,26,29,31,32,37,40–42 In the triphosphate form, NRTIs compete with their corresponding endogenous dNTPs for incorporation by viral reverse transcriptase and, once incorporated, cause the termination of re-verse transcription.
Since all these drugs act as competitive inhibitors of nucleotides during the synthesis of cDNA from HIV-1 RNA by reverse transcriptase, they can also potentially act as competitive inhibitors of the DNA polymerases responsible for the replication of the host cell DNA. While such an inhibitory effect is negligible for the DNA polymerases that replicate or repair nuclear DNA, this is biologically significant for DNA polymerase-γ (pol-γ), the sole enzyme that replicates mtDNA. As a conse-quence, NRTIs can inhibit replication of mtDNA.
Within mitochondria, a wide set of enzymes (such as deoxyguanosine kinase and thymidine kinase)
phosphorylate nucleosides to nucleotides, which are necessary for mtDNA replication, in a way similar to that present in the cytoplasm. Differently from their cyto-plasmic counterparts, mitochondrial enzyme expres-sion is not linked to the cell cycle, and their levels are not reduced in post mitotic cells.43 The traffic of nucleo-sides and nucleotides from and toward mitochondria is allowed by the transmembrane transporters “equilibra-tive nucleoside transporter” (ENT) and “deoxynucleotide carrier”.44–46 Such enzymes work in both directions, al-lowing the direct import of dNTPs when the concentra-tion of cytoplasmic dNTP exceeds that of mitochondria, and de novo synthesis of dNTPs through ENT nucleoside import, followed by phosphorylation inside the mito-chondria, when cytoplasmic concentration of dNTPs is low. Thus, the intramitochondrial concentration of nucle-otides derived from nucleoside analogs can be greatly affected by the dNTP pool present in the cytoplasm (whose concentration is strictly linked to the cell cycle) and by the activity of ENT and deoxynucleotide carrier, as well as that of mitochondrial kinases.
The capability of NRTIs to inhibit DNA pol-γ is di-rectly correlated with their rate of incorporation into DNA during mtDNA replication. The hierarchy of inhibi-tion is ddC > ddA (that is, the active metabolite of ddI) > d4T > 3TC > AZT > ABC;30 thus, drugs of the so-called “d” category, i.e., dideoxy-NRTIs such as ddC, ddI and d4T are the most potent inhibitors of pol-γ, as reported by several basic and clinical studies.47–55 The order re-ported above is quite similar to the level of toxicity of the drugs, with the notable exception of AZT, whose tox-icity is quite high despite the low rate of incorporation into DNA by DNA pol-γ. This discrepancy is likely due to the reduced capability of the enzyme to remove AZT from DNA using its proofreading activity.56 Recognition of the role of d4T and AZT in the onset of lipoatrophy
has led to a shift in the prescription of NRTIs with a lower impact on mtDNA, such as tenofovir disoproxil fuma-rate, 3TC or ABC, with consequent benefits for patients who can switch therapy.
2.2. AZT, the first drug used in the treatment of HIV infection, alters mitochondria
As far as mitochondrial metabolism is concerned, AZT is likely the most studied NRTI. AZT is a derivative of the natural nucleoside thymidine, and was the first drug used for the treatment of HIV infection. Since its intro-duction, it has been observed that AZT displays several side effects, including general myopathy and cardio-myopathy.57,58 which were attributed to a toxic activity on mitochondria. The first description of the toxic effects of AZT was reported in 1999.59 A 57-year-old patient who had been on AZT for 3 years showed muscular and hepatic disturbances and lactic acidosis, fatigue and weight loss. He became confused and febrile and died 8 days after detection of high blood lactatemia. Liver biopsy showed diffuse macrovacuolar and microvacu-olar steatosis; muscles had mitochondrial abnormalities with ragged-red fibers and lipid droplet accumulation. Southern blot analysis revealed depletion of mtDNA, af-fecting skeletal muscle and liver tissue, suggesting that AZT can induce mitochondrial multisystem disease.59
In the following years, intensive studies were per-formed to elucidate the interactions of this drug with DNA pol-γ, the enzyme responsible for the replication of mtDNA.60 Indeed, AZT is able to inhibit the activity of DNA pol-γ in mitochondria in at last three ways: (1) once AZT is incorporated into a growing strand, DNA replication is halted; (2) AZT competes with natural nucleotides to be incorporated into growing DNA chains; and (3) AZT inhibits mtDNA reparatory exonuclease activity as it
Table 1 List of nucleosidic reverse transcription inhibitors (NRTIs) approved for the treatment of HIV infection and their main effects on mitochondria
NRTI Analog of Effects on mitochondria References
AZT (zidovudine) Thymidine Inhibits mtDNA pol-γ; depletes mtDNA; reduces dTTP pool; 23–25 increases incorporation error rate in mtDNA.
ddI (didanosine) Adenosine Inhibits mtDNA pol-γ; depletes mtDNA; impairs complex II 26–28 and IV activity; increases lactate production.
ddC (zalcitabine) Cytidine Inhibits mtDNA pol-γ. 29, 30
d4T (stavudine) Thymidine Inhibits mtDNA pol-γ; depletes mtDNA; increases ROS; 31–36 impairs complex I, II, IV activity.
3TC (lamivudine) Cytidine No significant alterations of mitochondria in vitro; no morphological 37–39 changes of mitochondria.
ABC (abacavir) Guanosine Increases lactate production in vitro. 40
TDF (tenofovir) Adenosine No significant alterations of mitochondria in vitro; increases 39, 41
lactate production.
FTC (emtricitabine) Cytidine No significant alterations in mtDNA content; no morphological 37 changes of mitochondria.
can resist exonucleolytic removal by the exonuclease activity of pol-γ because of the lack of a 3-OH group.61 Once incorporated in the nascent chain of mtDNA, compared to other NRTIs, AZT is removed at a slower rate by the proofreading activity of DNA pol-γ.27
Apart from its direct effect on DNA pol-γ, AZT is able to affect mitochondrial metabolism in other ways. First, AZT can compete with thymidine for its phosphoryla-tion, so reducing the capability of thymidine kinase-2 to maintain the deoxythymidine triphosphate pool in the mitochondria.24,25 Second, it can alter the relative ratio in the dNTP pool, so increasing the incorporation error rate during mtDNA replication.62 Third, the increase in the error rate during mtDNA synthesis by DNA pol-γ causes an increased number of mutations in mtDNA that in turn leads to a reduced capability of mitochon-dria to produce the mtDNA-encoded subunits of respi-ratory chain complexes. Finally, the observation that polymorphisms in three amino acid residues of pol-γ can lead to a reduction in mtDNA copies in peripheral blood mononuclear cells (PBMCs) and favor the onset of lipodystrophy63 is in agreement with the observation that the capability of NRTIs to interact with DNA pol-γ is crucial for the side effects of antiretroviral therapy mediated by mitochondrial toxicity.
2.3. d4T is extremely potent in altering mitochondrial functionality
d4T (2⬘,3⬘-didehydro-3⬘-deoxythymidine) is a pyrimidine nucleosidic analog, approved for the treatment of HIV-1 infection in 1994. The first studies showed that its prin-cipal adverse effect was peripheral neuropathy.64–66 Further reports published after the advent of HAART showed that d4T is one of the most toxic drugs for mito-chondria, and clarified its mechanism of action. Studies in animal models have shown that d4T causes im-pairment of complexes I, II and IV in mitochondria,33 along with mtDNA depletion in liver; in patients, such mitochondrial alterations are often accompanied by hyperlactatemia.33,34 In vitro studies on human cells demonstrated that d4T not only causes marked mtDNA depletion, but also induces an increase in the level of ROS, causing mitochondrial oxidative stress.35 Our group has further confirmed such results in cells of hepatic and adipocytic origin, and showed that d4T-induced oxidative stress activates compensatory mechanisms through the upregulation of Lon protease, a mitochondrial protein that is able to degrade oxidized substrate proteins.36
In the HAART era, several clinical studies have sug-gested that the mitochondrial toxicity of d4T causes serious side effects, particularly the loss of fat tissue. Indeed, d4T is strongly correlated with lipoatrophy,67–70 and its consumption is associated with a reduction in the mtDNA content of lipoatrophic adipose tis-sue.71,72 Furthermore, it has been shown that its substi-tution with other NRTIs that have a lower impact on
mitochondria significantly reduces or even reverses this side effect.73,74
2.4. ddI has been studied in vitro and in vivo
ddI (2’,3’-dideoxyinosine) was approved for the treat-ment of HIV infection in 1991, becoming the second drug available for therapy. While first studies on animal models did not report any significant evidence of mito-chondrial toxicity,75 further analyses indicated that ddI exerts a cytotoxic effect on human cultured muscular cells, evidenced by increased lactate production; such an effect is accompanied by a decrease in mtDNA content, and a reduction in the activity of complex IV, and part of complex II, of the respiratory chain.27 Interestingly, the same effect was observed in cultured CD4 and CD8 T lymphocytes, in a dose- and time-dependent manner.28
Recently, a single-blind clinical study of 49 HIV + patients naïve for antiretroviral therapy and treated for a mean of 15 months with ddI-containing regimens showed that those taking such drugs had a lower amount of mtDNA in highly purified peripheral blood CD4+ and CD8+ T lymphocytes.28 In the same cells, the expression of different mitochondrial RNAs showed sig-nificant differences that were dependent on the drug used. However, no alterations in mitochondrial mem-brane potential and mitochondrial mass in peripheral lymphocytes were noted, and the viroimmunological efficacy of ddI-containing HAART was equivalent to that of regimens devoid of this drug.
2.5. Other NRTIs have a low impact on mitochondria
NRTIs other than d-drugs have a lower, if at all, toxic ef-fect on mitochondria and mtDNA.38,76 In vitro studies on hepatoma HepG2 cells did not show any significant ef-fect of tenofovir or 3TC on cell growth, lactate produc-tion, mtDNA levels and expression of proteins encoded by mtDNA, such as COXII. On the contrary, ABC im-paired proliferation and increased lactate, but did not induce mtDNA depletion.39
These results are in agreement with several clinical observations reporting that tenofovir and ABC have a lower impact on adipose tissue, and do not signifi-cantly affect lactatemia. Moreover, the switch from d-drugs to non d-drugs typically leads to a general amel-ioration of a number of symptoms present in patients with lipodystrophy.55,73,77–79
3. Antiretroviral Therapy and Mitochondria:
Low Mitochondrial Toxicity of PIs
While a large number of studies exist on the effects of NRTIs on mitochondria, less attention has been de-voted to the effects of PIs on the organelle. At present,
nine PIs are approved for clinical use (saquinavir, ritona-vir, indinaritona-vir, nelfinaritona-vir, amprenaritona-vir, lopinaritona-vir, atazanaritona-vir, tipanavir, darunavir) and others are in development.80 This class of antiretroviral agent targets the aspartyl pro-tease of HIV, inhibiting the cleavage of viral polypro-teins and the subsequent generation of individual viral proteins, which are required for virion assembly.
As reported above, CD4+ T lymphocytes from HIV + patients are characterized by an enhanced tendency to undergo apoptosis. Since PI-based HAART significantly increased CD4 T cell count in treated patients, research-ers asked if PIs were, in a direct or indirect way, able to inhibit apoptosis, so contributing to counteract CD4 T cell loss, and found a positive answer.81 Several different mechanisms have been proposed to explain the effect, including the direct inhibition of caspases by PIs,82 the capability of PIs to reduce expression of proapoptotic molecules or increase that of proliferative ones,83–86 the capability to inhibit calpain,87–89 and finally, the capability to counteract the loss of mitochondrial membrane potential.84
It is of note that contrasting results have been ob-tained for all but the latest mechanism, and different
in vitro studies have demonstrated that a possible major
role in such action is played by mitochondria. Indeed, a study on Jurkat T cell lines have shown that nelfinavir inhibits apoptotic signaling by preventing loss of ⌬ψm and the consequent release of apoptotic mediators such as cytochrome c.84 These data were confirmed by obser-vations on resting lymphocytes treated with different PIs (lopinavir, indinavir, saquinavir), whose mitochondria were protected from ⌬ψm loss compared to control lymphocytes after challenge with different apoptotic stimuli (Fas, tumor necrosis factor-α, TRAIL or UV irra-diation). Of note, treatment with PIs reduced the toxic effect of AZT on mitochondria.90,91 Data from patients on HAART regimens that include nelfinavir corroborated these observations,92 and studies in different mouse models further confirmed these results, showing that nelfinavir and ritonavir are able to reduce cytochrome c and apoptosis-inducing factor release.93
4. HAART-related Lipodystrophy is a Main
Clinical Manifestation of Mitochondrial
Toxicity
In 1998, 2 years after the introduction of HAART, it was shown that such treatment can have a significant side effect, i.e., lipodystrophy,94 which rapidly became an important problem in the treatment of HIV infection. Depending on the criteria used for defining the syn-drome, its overall prevalence was estimated to be from 18% to 83%.95 As in lipodystrophy syndromes of genetic origin, in HAART-related lipodystrophy fat redistribu-tion may precede the development of metabolic alter-ations96 that include (but are not limited to) increased
serum total and low-density lipoprotein, cholesterol and triglyceride levels (observed in about 70% of patients), insulin resistance and type 2 diabetes mellitus (ob-served in 8–10% of patients),97–99 and lactic acidemia.100 HAART-related lipodystrophy is characterized by an increase in visceral adipose tissue and a reduction in subcutaneous adipose tissue; as a consequence, fat is completely lost in the periphery (cheeks, arms, legs) and accumulates in the abdomen (including vessel walls) and behind the neck, in the so-called buffalo hump.101
The pathogenesis of lipodystrophy is extremely com-plex, as pharmacological, immunological, metabolic, genetic and environmental factors can contribute to its onset. The data regarding mtDNA depletion during lipodystrophy are quite controversial and sometimes misleading, because of the different origins of the cells where mtDNA content was analyzed, and because of the different techniques used for the quantification of mtDNA. In the first study, the mtDNA content of subcu-taneous fat tissue from the neck, abdomen and thigh was measured, and a decrease in mtDNA content was found in HAART-treated HIV + patients with peripheral fat wasting compared to HIV + patients without lipo-dystrophy but with a similar treatment history.71 Fur-thermore, HIV + patients naïve to antiretroviral therapy and healthy controls had similar amounts of mtDNA, suggesting that lipodystrophy with peripheral fat wast-ing is associated with a decrease in subcutaneous adi-pose tissue mtDNA content.71 In the following years, several studies have indicated that depletion of mtDNA and consequent mitochondrial impairment is the pri-mary cause of lipodystrophy.53,71,102
However, data from several groups suggest that the pathogenesis of lipodystrophy is not based merely on NRTI-induced depletion of mtDNA. The first study on mtDNA content in peripheral blood lymphocytes ana-lyzed mitochondria function and apoptosis in the cells of HIV-infected children, with or without lipodystrophy, who were receiving HAART.48 No significant changes were detected in the lymphocytes from children with lipodystrophy, suggesting that normal mitochondrial function and tendency to undergo apoptosis were present in these cells.
Then, analysis of mtDNA content in isolated T cells from adult lipodystrophic patients showed a para-doxical increase in the mtDNA content of CD4+ T cells, while no change was observed in CD8+ T cells.103 It is of note, however, that in the aforementioned cases, the quantification of mtDNA had been performed in circu-lating CD4+ T lymphocytes that were likely to have been newly formed cells, and thus were exposed to damaging drugs for only a relatively short period of time. Studies on fat cells are always needed to better clarify mitochondrial damage. In any case, it is crucial to avoid the use of the most toxic antiretrovirals, particularly d4T.104
5. HIV Exerts a Direct Effect on
Mitochondria and mtDNA
In past years, the majority of this type of study has been devoted to the analysis of the effects of antiretro-viral drugs on mitochondria. However, it must be noted that HIV per se can directly affect mitochondrial func-tionality. While the capability of HIV to alter mitochon-drial functionality by impairing the functionality of complex I of the electron transport chain has been shown,105 the capability of HIV to alter mtDNA content remains a matter of debate.
Some authors reported a depletion of mtDNA in HIV + patients naïve for therapy compared to normal controls, at least in PBMCs,106,107 suggesting that the reduction in mtDNA can occur with HIV infection alone and precedes the use of NRTIs, and that HIV products or cytokines released in response to HIV infection may make mitochondria more vulnerable to NRTIs.108 How-ever, other authors did not observe any alteration in mtDNA that could be attributed to HIV in adipose tis-sue from lipoatrophic patients.72 The discrepancy in such observations can probably be attributed to the different tissues used for this kind of analysis, as HIV-1 could exert a greater impact on blood cells than on adipocytes.
6. HIV Exerts Direct Effects on
Mitochondria-mediated Apoptosis
A large number of studies over the last two decades have demonstrated that during HIV infection, apopto-sis is one of the major mechanisms of CD4 T cell deple-tion.109–112 Indeed, in response to a variety of different stimuli, both CD4+ and CD8+ T cells from HIV patients display a high susceptibility to ⌬ψm loss and subsequent apoptosis. Such a tendency can also be observed in the earliest phases of the infection, i.e., in patients with acute infection, and the capability of HIV to alter ⌬ψm and induce apoptosis in peripheral blood cells plays an important role in this phenomenon.113,114 Intensive analyses of the proapoptotic activities of HIV products have shown an important role for viral proteins that are the products of the env, nef, tat and vpr genes.
6.1. Env-induced apoptosis
Env proteins (such as gp120, a glycoprotein exposed on the surface of the HIV envelope, and its precursor gp160) induce apoptosis in a Fas-independent manner, through the so-called intrinsic pathway of apoptosis; in different cellular models, this activity of Env proteins has been correlated with caspase 3 activation,115,116 in-creased expression of Bax,117 and downregulation of Bcl-2.118 Indeed, the interaction of Env proteins on the surface of HIV-infected cells with the CD4/CXCR4 or
CD4/CCR5 complex of uninfected cells could mediate a rapid ⌬ψm loss that in turn initiates apoptosis in lym-phocytes,119,120 probably by altering the ion fluxes through the plasma membrane, and the global ion equilibrium of the cell.121
Furthermore, Env proteins can induce apoptosis not only in infected cells, but also in bystander T lymphocytes through the formation of syncytia, when the gp120/gp41 complex on infected cells interacts with the CD4/CXCR4 complex on bystander cells. Syncytia then undergo ap-optosis through a caspase-3 dependent mechanism, mediated by mitochondrial depolarization.122
6.2. Nef-induced apoptosis
Nef (negative regulatory factor) is a protein required for the efficient replication of virus. It has been shown that Nef can be both proapoptotic and antiapoptotic, depending on the situation,123 since it can promote the killing of bystander (non-infected) cells through the ac-tivation of the Fas/FasL (CD95/CD178) pathway,124 while simultaneously preserving viral replication in the HIV-infected host cell by blocking apoptosis in HIV-infected T cells.125 When exerting its proapoptotic activity, Nef acts mainly by triggering apoptosis via Fas/FasL interactions; however, Nef can also trigger apoptosis through the intrinsic pathway, being able to decrease the expres-sion of Bcl-2 and Bcl-XL in infected cells.126
6.3. Tat-induced apoptosis
The protein Tat (transactivator of transcription) is nec-essary for many functions of the virus, including gene transcription and virus replication. As far as apoptosis is concerned, Tat triggers Fas-independent pathways by reducing Bcl-2 expression, and increasing both Bax expression127 and the levels of caspase 8 and 10.128,129 Tat can translocate into mitochondria where it pro-motes MMP and downregulates mitochondrial super-oxide dismutase (SOD2), either at the transcriptional or post-transcriptional level.130–132 In turn, SOD2 down-regulation reduces the capability of the cell to cope with ROS produced within mitochondria, sensitizing the cell to ROS-induced apoptosis.132
6.4. Vpr-induced apoptosis
Viral protein R (Vpr) is a 14-kilodalton molecule that plays an important role in regulating nuclear import of the HIV-1 pre-integration complex. Vpr is essential for productive infection of macrophages133 and resting PBMCs.134,135
Like other viral proteins, Vpr can induce apoptosis by downregulating Bcl-2 and Bcl-XL.136 However, it is also able to directly interact with the mitochondria: Vpr binds adenine nucleotide translocator, which in turn causes the permeabilization of the inner mitochondrial
membrane, swelling of mitochondria and breaking of the outer mitochondrial membrane.137,138
7. Conclusions
During HIV infection and its treatment, a variety of molecular and cellular mechanisms are triggered to cope with the virus and to eliminate dangerous virus-producing cells. The immune system actively fights the infection, and, in the case of viral infections, has to remove infected cells. A large number of immune mech-anisms are triggered, including the production of proin-flammatory cytokines, among which is tumor necrosis factor-α, which has mitochondria as a main target and is capable of provoking an increase in ROS production.
As reported above and illustrated in Figure 1, in healthy, uninfected cells (such as, for example, adipocytes or hepatocytes), the functionality of mitochondria can be altered either by viral products or by different antiretro-virals of the NRTI category, especially d-drugs. Further-more, molecules produced by the immune system, such as proinflammatory cytokines, are often capable of in-fluencing mitochondria in different ways. As a result, a striking number of non-infected cells belonging to dif-ferent tissues and organs, which are clearly innocent
bystanders, are involved in HIV infection and its treat-ment and can be irreversibly damaged or even killed.
Acknowledgments
This work was partially supported by VI Programma Nazionale di Ricerca sull’AIDS 2006 (Istituto Superiore di Sanita’, Rome, Italy), grants No. 30G.62 and 40G.62 to Andrea Cossarizza, and by a grant from MIUR–PRIN 2008 (project: “Regulation of Lon protease expression in response to oxidative damage to mitochondria”) to Andrea Cossarizza.
References
1. Taylor SW, Fahy E, Zhang B, Glenn GM, Warnock DE, Wiley S, Murphy AN, et al. Characterization of the human heart mito-chondrial proteome. Nat Biotechnol 2003;21:281–6.
2. Wallace DC. The mitochondrial genome in human adaptive radi-ation and disease: on the road to therapeutics and performance enhancement. Gene 2005;354:169–80.
3. Kujoth GC, Bradshaw PC, Haroon S, Prolla TA. The role of mito-chondrial DNA mutations in mammalian aging. PLoS Genet 2007;3:e24.
4. Pedersen PL. The cancer cell’s “power plants” as promising thera-peutic targets: an overview. J Bioenerg Biomembr 2007;39:1–12. Figure 1 Mechanisms of mitochondrial toxicity in uninfected cells during HIV infection and its treatment. Several antiretroviral compounds of the category of nucleosidic reverse transcription inhibitors (NRTIs), especially those defined as “d-drugs”, can influence the activity of DNA polymerase-γ and cause a decrease in mtDNA content. This results in a decrease of proteins devoted to oxidative phosphorylation (OXPHOS), coded by mtDNA, and a consequent decreased capacity to produce ATP. The infection can trigger several immune responses, including the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), which increases the production of intramitochondrial reactive oxygen species (ROS). Even proteins derived from the virus can directly affect mitochondria. The result of all the aforementioned mechanisms can be an irreversible altera-tion of the organelle, with a decrease in mitochondrial membrane potential (⌬ψm) and the release into the cytoplasm of
mole-cules such as cytochrome c (cyt-c) and apoptosis inducing factor (AIF), which activate caspases and lead to cell death.
Apoptogenic molecules (AIF, cyt-c) Irreversible alteration ATP ROS Bax Bcl-2, BclXL HIV proteins mtDNA TNF-α OXPHOS proteins Therapy with NRTIs (d-drugs) Proinflammatory cytokines Immune response HIV infection Δψm
5. Reichert AS, Neupert W. Mitochondriomics or what makes us breathe. Trends Genet 2004;20:555–62.
6. Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane per-meabilization in cell death. Physiol Rev 2007;87:99–163. 7. Barbieri D, Abbracchio MP, Salvioli S, Monti D, Cossarizza A,
Ceruti S, Brambilla R, et al. Apoptosis by 2-chloro-2 ⬘-deoxy-adenosine and 2-chloro-⬘-deoxy-adenosine in human peripheral blood mononuclear cells. Neurochem Int 1998;32:493–504.
8. Ceruti S, Barbieri D, Veronese E, Cattabeni F, Cossarizza A, Giammarioli AM, Malorni W, et al. Different pathways of apopto-sis revealed by 2-chloro-adenosine and deoxy-D-ribose in mam-malian astroglial cells. J Neurosci Res 1997;47:372–83.
9. Salvioli S, Dobrucki J, Moretti L, Troiano L, Fernandez MG, Pinti M, Pedrazzi J, et al. Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level. Cytometry 2000;40:189–97. 10. Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G.
Mechanisms of cytochrome c release from mitochondria. Cell Death Differ 2006;13:1423–33.
11. Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 2006; 25:4812–30.
12. Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, Bolognesi D, et al. 3’-Azido-3’-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/ lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci USA 1985;82:7096–100.
13. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lym-phocytes in HIV-1 infection. Nature 1995;373:123–6.
14. Perelson AS, Essunger P, Ho DD. Dynamics of HIV-1 and CD4 + lymphocytes in vivo. AIDS 1997;11 Suppl A:S17–24.
15. Ho DD. Time to hit HIV, early and hard. N Engl J Med 1995;333: 450–1.
16. Palella FJ Jr, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, Aschman DJ, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998; 338:853–60.
17. Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R, Katlama C, et al. Positive effects of combined antiretroviral ther-apy on CD4 + T cell homeostasis and function in advanced HIV disease. Science 1997;277:112–6.
18. Mussini C, Pezzotti P, Govoni A, Borghi V, Antinori A, d’Arminio Monforte A, De Luca A, et al. Discontinuation of primary prophy-laxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis in human immunodeficiency virus type I-infected patients: the changes in opportunistic prophylaxis study. J Infect Dis 2000;181:1635–42.
19. Mussini C, Pezzotti P, Miro JM, Martinez E, de Quiros JC, Cinque P, Borghi V, et al. Discontinuation of maintenance therapy for cryp-tococcal meningitis in patients with AIDS treated with highly active antiretroviral therapy: an international observational study. Clin Infect Dis 2004;38:565–71.
20. Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, Alldredge L, et al. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 1998;4:1302–7.
21. Fatkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, et al. Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1. Nat Med 2005;11:1170–2.
22. Hicks C, Gulick RM. Raltegravir: the first HIV type 1 integrase inhibitor. Clin Infect Dis 2009;48:931–9.
23. Furman PA, Fyfe JA, St Clair MH, Weinhold K, Rideout JL, Freeman GA, Lehrman SN, et al. Phosphorylation of 3’-azido-3’-deoxythy-midine and selective interaction of the 5’-triphosphate with
human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci USA 1986;83:8333–7.
24. Lynx MD, McKee EE. 3’-Azido-3’-deoxythymidine (AZT) is a com-petitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria. Biochem Pharmacol 2006;72:239–43. 25. Lynx MD, Bentley AT, McKee EE. 3’-Azido-3’-deoxythymidine
(AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: a possible mechanism of AZT hepatotoxicity. Biochem Pharmacol 2006;71:1342–8.
26. Johnson MA, Fridland A. Phosphorylation of 2’,3’-dideoxyinosine by cytosolic 5’-nucleotidase of human lymphoid cells. Mol Pharmacol 1989;36:291–5.
27. Benbrik E, Chariot P, Bonavaud S, Ammi-Said M, Frisdal E, Rey C, Gherardi R, et al. Cellular and mitochondrial toxicity of zidovu-dine (AZT), didanosine (ddI) and zalcitabine (ddC) on cultured human muscle cells. J Neurol Sci 1997;149:19–25.
28. Setzer B, Schlesier M, Walker UA. Effects of of didanosine-related depletion of mtDNA in human T lymphocytes. J Infect Dis 2005; 191:848–55.
29. Balzarini J, Cooney DA, Dalal M, Kang GJ, Cupp JE, DeClercq E, Broder S, et al. 2’,3’-Dideoxycytidine: regulation of its metabo-lism and anti-retroviral potency by natural pyrimidine nucleo-sides and by inhibitors of pyrimidine nucleotide synthesis. Mol Pharmacol 1987;32:798–806.
30. Lee H, Hanes J, Johnson KA. Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 2003;42:14711–9.
31. Becher F, Landman R, Mboup S, Kane CN, Canestri A, Liegeois F, Vray M, et al. Monitoring of didanosine and stavudine intracellu-lar trisphosphorylated anabolite concentrations in HIV-infected patients. AIDS 2004;18:181–7.
32. Ho HT, Hitchcock MJ. Cellular pharmacology of 2’,3’-dideoxy-2’,3’-didehydrothymidine, a nucleoside analog active against human immunodeficiency virus. Antimicrob Agents Chemother 1989;33:844–9.
33. Gerschenson M, Nguyen VT, St Claire MC, Harbaugh SW, Harbaugh JW, Proia LA, Poirier MC. Chronic stavudine exposure induces hepatic mitochondrial toxicity in adult Erythrocebus patas monkeys. J Hum Virol 2001;4:335–42.
34. Gaou I, Malliti M, Guimont MC, Letteron P, Demeilliers C, Peytavin G, Degott C, et al. Effect of stavudine on mitochondrial genome and fatty acid oxidation in lean and obese mice. J Pharmacol Exp Ther 2001;297:516–23.
35. Velsor LW, Kovacevic M, Goldstein M, Leitner HM, Lewis W, Day BJ. Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine. Toxicol Appl Pharmacol 2004;199:10–9. 36. Pinti M, Gibellini L, Guaraldi G, Orlando G, Gant TW, Morselli E,
Nasi M, et al. Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipo-dystrophy: implications for the pathogenesis of the disease. AIDS 2010;24:841–50.
37. Shewach DS, Liotta DC, Schinazi RF. Affinity of the antiviral enantiomers of oxathiolane cytosine nucleosides for human 2’-deoxycytidine kinase. Biochem Pharmacol 1993;45:1540–3. 38. Cui L, Schinazi RF, Gosselin G, Imbach JL, Chu CK, Rando RF,
Revankar GR, et al. Effect of beta-enantiomeric and racemic nucl-eoside analogues on mitochondrial functions in HepG2 cells. Implications for predicting drug hepatotoxicity. Biochem Phar-macol 1996;52:1577–84.
39. Venhoff N, Setzer B, Melkaoui K, Walker UA. Mitochondrial toxicity of tenofovir, emtricitabine and abacavir alone and in combina-tion with addicombina-tional nucleoside reverse transcriptase inhibitors. Antivir Ther 2007;12:1075–85.
40. Harris M, Back D, Kewn S, Jutha S, Marina R, Montaner JS. Intra-cellular carbovir triphosphate levels in patients taking abacavir once a day. AIDS 2002;16:1196–7.
41. Hawkins T, Veikley W, St Claire RL 3rd, Guyer B, Clark N, Kearney BP. Intracellular pharmacokinetics of tenofovir diphosphate, carbovir
triphosphate, and lamivudine triphosphate in patients receiving triple-nucleoside regimens. J Acquir Immune Defic Syndr 2005;39: 406–11.
42. Faletto MB, Miller WH, Garvey EP, St Clair MH, Daluge SM, Good SS. Unique intracellular activation of the potent anti-human immu-nodeficiency virus agent 1592U89. Antimicrob Agents Chemother 1997;41:1099–107.
43. Rylova SN, Mirzaee S, Albertioni F, Eriksson S. Expression of deoxynucleoside kinases and 5’-nucleotidases in mouse tissues: implications for mitochondrial toxicity. Biochem Pharmacol 2007; 74:169–75.
44. Govindarajan R, Leung GP, Zhou M, Tse CM, Wang J, Unadkat JD. Facilitated mitochondrial import of antiviral and anticancer nucl-eoside drugs by human equilibrative nuclnucl-eoside transporter-3. Am J Physiol Gastrointest Liver Physiol 2009;296:G910–22. 45. Leung GP, Tse CM. The role of mitochondrial and plasma
mem-brane nucleoside transporters in drug toxicity. Expert Opin Drug Metab Toxicol 2007;3:705–18.
46. Lai Y, Tse CM, Unadkat JD. Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. J Biol Chem 2004;279:4490–7.
47. Foli A, Benvenuto F, Piccinini G, Bareggi A, Cossarizza A, Lisziewicz J, Lori F. Direct analysis of mitochondrial toxicity of antiretroviral drugs. AIDS 2001;15:1687–94.
48. Cossarizza A, Pinti M, Moretti L, Bricalli D, Bianchi R, Troiano L, Fernandez MG, et al. Mitochondrial functionality and mitochon-drial DNA content in lymphocytes of vertically infected human immunodeficiency virus-positive children with highly active anti-retroviral therapy-related lipodystrophy. J Infect Dis 2002;185: 299–305.
49. Walker UA, Setzer B, Venhoff N. Increased long-term mitochon-drial toxicity in combinations of nucleoside analogue reverse-transcriptase inhibitors. AIDS 2002;16:2165–73.
50. McComsey G, Tan DJ, Lederman M, Wilson E, Wong LJ. Analysis of the mitochondrial DNA genome in the peripheral blood leu-kocytes of HIV-infected patients with or without lipoatrophy. AIDS 2002;16:513–8.
51. Walker UA, Bickel M, Lutke Volksbeck SI, Ketelsen UP, Schofer H, Setzer B, Venhoff N, et al. Evidence of nucleoside analogue reverse transcriptase inhibitor—associated genetic and struc-tural defects of mitochondria in adipose tissue of HIV-infected patients. J Acquir Immune Defic Syndr 2002;29:117–21.
52. Miro O, Lopez S, Pedrol E, Rodriguez-Santiago B, Martinez E, Soler A, Milinkovic A, et al. Mitochondrial DNA depletion and respiratory chain enzyme deficiencies are present in peripheral blood mononuclear cells of HIV-infected patients with HAART-related lipodystrophy. Antivir Ther 2003;8:333–8.
53. Cote HC, Brumme ZL, Craib KJ, Alexander CS, Wynhoven B, Ting L, Wong H, et al. Changes in mitochondrial DNA as a marker of nucl-eoside toxicity in HIV-infected patients. N Engl J Med 2002;346: 811–20.
54. Lopez S, Miro O, Martinez E, Pedrol E, Rodriguez-Santiago B, Milinkovic A, Soler A, et al. Mitochondrial effects of antiretroviral therapies in asymptomatic patients. Antivir Ther 2004;9:47–55. 55. Maggiolo F, Roat E, Pinti M, Nasi M, Gibellini L, De Biasi S, Airoldi M,
et al. Mitochondrial changes during D-drug-containing once-daily therapy in HIV-positive treatment-naive patients. Antivir Ther 2010;15:51–9.
56. Lim SE, Copeland WC. Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma. J Biol Chem 2001;276:23616–23.
57. Dalakas MC, Illa I, Pezeshkpour GH, Laukaitis JP, Cohen B, Griffin JL. Mitochondrial myopathy caused by long-term zidovudine ther-apy. N Engl J Med 1990;322:1098–105.
58. Lewis W, Papoian T, Gonzalez B, Louie H, Kelly DP, Payne RM, Grody WW. Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. Lab Invest 1991;65:228–36.
59. Chariot P, Drogou I, de Lacroix-Szmania I, Eliezer-Vanerot MC, Chazaud B, Lombes A, Schaeffer A, et al. Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion. J Hepatol 1999;30:156–60.
60. Kaguni LS. DNA polymerase gamma, the mitochondrial repli-case. Annu Rev Biochem 2004;73:293–320.
61. Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nat Rev Drug Discov 2003;2:812–22.
62. Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK. DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity. Proc Natl Acad Sci USA 2005;102: 4990–5.
63. Chiappini F, Teicher E, Saffroy R, Debuire B, Vittecoq D, Lemoine A. Relationship between polymerase gamma (POLG) polymorphisms and antiretroviral therapy-induced lipodystrophy in HIV-1 in-fected patients: a case-control study. Curr HIV Res 2009;7:244–53. 64. Riddler SA, Anderson RE, Mellors JW. Antiretroviral activity of sta-vudine (2’,3’-didehydro-3’-deoxythymidine, D4T). Antiviral Res 1995;27:189–203.
65. Rana KZ, Dudley MN. Clinical pharmacokinetics of stavudine. Clin Pharmacokinet 1997;33:276–84.
66. Villalba N, Soriano V, Gomez-Cano M, Castilla J, Mas A, Gonzalez-Lahoz J. Short-term efficacy and safety of stavudine in pre-treated HIV-infected patients. Antivir Ther 1997;2:185–9. 67. van Griensven J, De Naeyer L, Mushi T, Ubarijoro S, Gashumba D,
Gazille C, Zachariah R. High prevalence of lipoatrophy among patients on stavudine-containing first-line antiretroviral therapy regimens in Rwanda. Trans R Soc Trop Med Hyg 2007;101:793–8. 68. Joly V, Flandre P, Meiffredy V, Leturque N, Harel M, Aboulker JP,
Yeni P. Increased risk of lipoatrophy under stavudine in HIV-1-infected patients: results of a substudy from a comparative trial. AIDS 2002;16:2447–54.
69. Lowe SH, Hassink EA, van Eck-Smit BL, Borleffs JC, Lange JM, Reiss P. Stavudine but not didanosine as part of HAART contrib-utes to peripheral lipoatrophy: a substudy from the Antiretroviral Regimen Evaluation Study (ARES). HIV Clin Trials 2007;8:337–44. 70. ter Hofstede HJ, Koopmans PP, Burger DM. Stavudine plasma
concentrations and lipoatrophy. J Antimicrob Chemother 2008; 61:933–8.
71. Shikuma CM, Hu N, Milne C, Yost F, Waslien C, Shimizu S, Shiramizu B. Mitochondrial DNA decrease in subcutaneous adi-pose tissue of HIV-infected individuals with peripheral lipoatro-phy. AIDS 2001;15:1801–9.
72. McComsey GA, Libutti DE, O’Riordan M, Shelton JM, Storer N, Ganz J, Jasper J, et al. Mitochondrial RNA and DNA alterations in HIV lipoatrophy are linked to antiretroviral therapy and not to HIV infection. Antivir Ther 2008;13:715–22.
73. McComsey GA, Paulsen DM, Lonergan JT, Hessenthaler SM, Hoppel CL, Williams VC, Fisher RL, et al. Improvements in lipoat-rophy, mitochondrial DNA levels and fat apoptosis after replac-ing stavudine with abacavir or zidovudine. AIDS 2005;19:15–23. 74. Ribera E, Paradineiro JC, Curran A, Sauleda S, Garcia-Arumi E,
Castella E, Puiggros C, et al. Improvements in subcutaneous fat, lipid profile, and parameters of mitochondrial toxicity in patients with peripheral lipoatrophy when stavudine is switched to ten-ofovir (LIPOTEST study). HIV Clin Trials 2008;9:407–17.
75. Pedrol E, Masanes F, Fernandez-Sola J, Cofan M, Casademont J, Grau JM, Urbano-Marquez. A lack of muscle toxicity with didano-sine (ddI). Clinical and experimental studies. J Neurol Sci 1996; 138:42–8.
76. Vidal F, Domingo JC, Guallar J, Saumoy M, Cordobilla B, Sanchez de la Rosa R, Giralt M, et al. In vitro cytotoxicity and mitochon-drial toxicity of tenofovir alone and in combination with other antiretrovirals in human renal proximal tubule cells. Antimicrob Agents Chemother 2006;50:3824–32.
77. Lonergan JT, Barber RE, Mathews WC. Safety and efficacy of switch-ing to alternative nucleoside analogues followswitch-ing symptomatic hyperlactatemia and lactic acidosis. AIDS 2003;17:2495–9. 78. Carr A, Workman C, Smith DE, Hoy J, Hudson J, Doong N,
Martin A, et al. Abacavir substitution for nucleoside analogs in patients with HIV lipoatrophy: a randomized trial. JAMA 2002; 288:207–15.
79. Birkus G, Hitchcock MJ, Cihlar T. Assessment of mitochondrial toxicity in human cells treated with tenofovir: comparison with other nucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother 2002;46:716–23.
80. Wensing AM, van Maarseveen NM, Nijhuis M. Fifteen years of HIV protease inhibitors: raising the barrier to resistance. Antiviral Res 2010;85:59–74.
81. Badley AD. In vitro and in vivo effects of HIV protease inhibitors on apoptosis. Cell Death Differ 2005;12 Suppl 1:924–31. 82. Weichold FF, Bryant JL, Pati S, Barabitskaya O, Gallo RC, Reitz MS
Jr. HIV-1 protease inhibitor ritonavir modulates susceptibility to apoptosis of uninfected T cells. J Hum Virol 1999;2:261–9. 83. Sloand EM, Young NS, Sato T, Kim S, Maciejewski JP. Inhibition of
interleukin-1beta-converting enzyme in human hematopoietic progenitor cells results in blockade of cytokine-mediated apop-tosis and expansion of their proliferative potential. Exp Hematol 1998;26:1093–9.
84. Phenix BN, Lum JJ, Nie Z, Sanchez-Dardon J, Badley AD. Antiap-optotic mechanism of HIV protease inhibitors: preventing mito-chondrial transmembrane potential loss. Blood 2001;98:1078–85. 85. Sloand EM, Kumar PN, Kim S, Chaudhuri A, Weichold FF, Young NS.
Human immunodeficiency virus type 1 protease inhibitor mod-ulates activation of peripheral blood CD4(+) T cells and decreases their susceptibility to apoptosis in vitro and in vivo. Blood 1999; 94:1021–7.
86. Chavan S, Kodoth S, Pahwa R, Pahwa S. The HIV protease inhibitor indinavir inhibits cell-cycle progression in vitro in lymphocytes of HIV-infected and uninfected individuals. Blood 2001;98:383–9. 87. Ghibelli L, Mengoni F, Lichtner M, Coppola S, De Nicola M,
Bergamaschi A, Mastroianni C, et al. Anti-apoptotic effect of HIV protease inhibitors via direct inhibition of calpain. Biochem Pharmacol 2003;66:1505–12.
88. Cuerrier D, Nie Z, Badley AD, Davies PL. Ritonavir does not inhibit calpain in vitro. Biochem Biophys Res Commun 2005;327:208–11. 89. Vanderklish PW, Bahr BA. The pathogenic activation of calpain: a
marker and mediator of cellular toxicity and disease states. Int J Exp Pathol 2000;81:323–39.
90. Matarrese P, Gambardella L, Cassone A, Vella S, Cauda R, Malorni W. Mitochondrial membrane hyperpolarization hijacks activated T lymphocytes toward the apoptotic-prone phenotype: homeo-static mechanisms of HIV protease inhibitors. J Immunol 2003; 170:6006–15.
91. Matarrese P, Tinari A, Gambardella L, Mormone E, Narilli P, Pierdominici M, Cauda R, et al. HIV protease inhibitors prevent mitochondrial hyperpolarization and redox imbalance and decrease endogenous uncoupler protein-2 expression in gp 120-activated human T lymphocytes. Antivir Ther 2005;10 Suppl 2: M29–45.
92. Miro O, Villarroya J, Garrabou G, Lopez S, Rodriguez de la Concepcion M, Pedrol E, Martinez E, et al. In vivo effects of highly active antiretroviral therapies containing the protease inhibitor nelfinavir on mitochondrially driven apoptosis. Antivir Ther 2005;10:945–51.
93. Hisatomi T, Nakazawa T, Noda K, Almulki L, Miyahara S, Nakao S, Ito Y, et al. HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice. J Clin Invest 2008;118:2025–38.
94. Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, Cooper DA. A syndrome of peripheral lipodystrophy, hyperlipi-daemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998;12:F51–8.
95. Samaras K, Wand H, Law M, Emery S, Cooper D, Carr A. Preva-lence of metabolic syndrome in HIV-infected patients receiv-ing highly active antiretroviral therapy usreceiv-ing Interna tional Diabetes Foun dation and Adult Treatment Panel III criteria: associations with insulin resistance, disturbed body fat com-partmentalization, elevated C-reactive protein, and [corrected] hypoadiponectinemia. Diabetes Care 2007;30:113–9. Erratum in: Diabetes Care 2007;30:455.
96. Barbaro G, Iacobellis G. Metabolic syndrome associated with HIV and highly active antiretroviral therapy. Curr Diab Rep 2009;9:37–42.
97. Grinspoon S, Carr A. Cardiovascular risk and body-fat abnor-malities in HIV-infected adults. N Engl J Med 2005;352:48–62. 98. Wand H, Calmy A, Carey DL, Samaras K, Carr A, Law MG, Cooper
DA, et al. Metabolic syndrome, cardiovascular disease and type 2 diabetes mellitus after initiation of antiretroviral therapy in HIV infection. AIDS 2007;21:2445–53.
99. Carr A, Samaras K, Thorisdottir A, Kaufmann GR, Chisholm DJ, Cooper DA. Diagnosis, prediction, and natural course of HIV-1 protease-inhibitor-associated lipodystrophy, hyperlipi-daemia, and diabetes mellitus: a cohort study. Lancet 1999;353: 2093–9.
100. Carr A, Miller J, Law M, Cooper DA. A syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with HIV nucl-eoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome. AIDS 2000;14:F25–32. 101. Carr A, Cooper DA. Adverse effects of antiretroviral therapy.
Lancet 2000;356:1423–30.
102. Shiramizu B, Shikuma KM, Kamemoto L, Gerschenson M, Erdem G, Pinti M, Cossarizza A, et al. Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy. J Acquir Immune Defic Syndr 2003;32:370–4.
103. Cossarizza A, Riva A, Pinti M, Ammannato S, Fedeli P, Mussini C, Esposito R, et al. Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy. Antivir Ther 2003;8:315–21.
104. Calmy A, Hirschel B, Cooper DA, Carr A. A new era of antiretro-viral drug toxicity. Antivir Ther 2009;14:165–79.
105. Ladha JS, Tripathy MK, Mitra D. Mitochondrial complex I activity is impaired during HIV-1-induced T-cell apoptosis. Cell Death Differ 2005;12:1417–28.
106. Maagaard A, Holberg-Petersen M, Kvittingen EA, Sandvik L, Bruun JN. Depletion of mitochondrial DNA copies/cell in periph-eral blood mononuclear cells in HIV-1-infected treatment-naive patients. HIV Med 2006;7:53–8.
107. Miura T, Goto M, Hosoya N, Odawara T, Kitamura Y, Nakamura T, Iwamoto A. Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy. J Med Virol 2003;70:497–505.
108. Prakash O, Teng S, Ali M, Zhu X, Coleman R, Dabdoub RA, Chambers R, et al. The human immunodeficiency virus type 1 Tat protein potentiates zidovudine-induced cellular toxicity in transgenic mice. Arch Biochem Biophys 1997;343:173–80. 109. Banda NK, Bernier J, Kurahara DK, Kurrle R, Haigwood N, Sekaly
RP, Finkel TH. Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med 1992;176:1099–106.
110. Groux H, Torpier G, Monte D, Mouton Y, Capron A, Ameisen JC. Activation-induced death by apoptosis in CD4 + T cells from human immunodeficiency virus-infected asymptomatic indi-viduals. J Exp Med 1992;175:331–40.
111. Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RP, Miedema F. Programmed death of T cells in HIV-1 infection. Science 1992; 257:217–9.
112. Lopes MF, da Veiga VF, Santos AR, Fonseca ME, DosReis GA. Activation-induced CD4 + T cell death by apoptosis in experi-mental Chagas’ disease. J Immunol 1995;154:744–52.
113. Franceschi C, Franceschini MG, Boschini A, Trenti T, Nuzzo C, Castellani G, Smacchia C, et al. Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV + long term non progressors. Cell Death Differ 1997;4: 815–23.
114. Cossarizza A, Mussini C, Mongiardo N, Borghi V, Sabbatini A, De Rienzo B, Franceschi C. Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lym-phocytes during acute HIV syndrome. AIDS 1997;11:19–26. 115. Ohnimus H, Heinkelein M, Jassoy C. Apoptotic cell death upon
contact of CD4 + T lymphocytes with HIV glycoprotein-expressing cells is mediated by caspases but bypasses CD95 (Fas/Apo-1) and TNF receptor 1. J Immunol 1997;159:5246–52.
116. Kaul M, Lipton SA. Chemokines and activated macrophages in HIV gp120-induced neuronal apoptosis. Proc Natl Acad Sci USA 1999;96:8212–6.
117. Ferri KF, Jacotot E, Blanco J, Este JA, Zamzami N, Susin SA, Xie Z, et al. Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex: role of mitochondria and caspases. J Exp Med 2000;192:1081–92.
118. Hashimoto F, Oyaizu N, Kalyanaraman VS, Pahwa S. Modulation of Bcl-2 protein by CD4 cross-linking: a possible mechanism for lymphocyte apoptosis in human immunodeficiency virus infec-tion and for rescue of apoptosis by interleukin-2. Blood 1997;90: 745–53.
119. Herbein G, Mahlknecht U, Batliwalla F, Gregersen P, Pappas T, Butler J, O’Brien WA, et al. Apoptosis of CD8 + T cells is mediated by macrophages through interaction of HIV gp120 with chem-okine receptor CXCR4. Nature 1998;395:189–94.
120. Berndt C, Mopps B, Angermuller S, Gierschik P, Krammer PH. CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells. Proc Natl Acad Sci USA 1998;95:12556–61. 121. Fermin CD, Garry RF. Membrane alterations linked to early
inter-actions of HIV with the cell surface. Virology 1992;191:941–6. 122. Garg H, Blumenthal R. HIV gp41-induced apoptosis is mediated
by caspase-3-dependent mitochondrial depolarization, which is inhibited by HIV protease inhibitor nelfinavir. J Leukoc Biol 2006;79:351–62.
123. Cossarizza A. Apoptosis and HIV infection: about molecules and genes. Curr Pharm Des 2008;14:237–44.
124. Xu XN, Laffert B, Screaton GR, Kraft M, Wolf D, Kolanus W, Mongkolsapay J, et al. Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med 1999;189:1489–96.
125. Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC. HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature 2001; 410:834–8.
126. Rasola A, Gramaglia D, Boccaccio C, Comoglio PM. Apoptosis en-hancement by the HIV-1 Nef protein. J Immunol 2001;166:81–8. 127. Sastry KJ, Marin MC, Nehete PN, McConnell K, el-Naggar AK,
McDonnell TJ. Expression of human immunodeficiency virus type I tat results in down-regulation of bcl-2 and induction of apoptosis in hematopoietic cells. Oncogene 1996;13:487–93. 128. Bartz SR, Emerman M. Human immunodeficiency virus type 1
Tat induces apoptosis and increases sensitivity to apoptotic sig-nals by up-regulating FLICE/caspase-8. J Virol 1999;73:1956–63. 129. Gibellini D, Re MC, Ponti C, Vitone F, Bon I, Fabbri G, Grazia Di
Iasio M, et al. HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity. J Cell Physiol 2005;203:547–56.
130. Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 1995;268:429–31.
131. Creaven M, Hans F, Mutskov V, Col E, Caron C, Dimitrov S, Khochbin S. Control of the histone-acetyltransferase activity of Tip60 by the HIV-1 transactivator protein, Tat. Biochemistry 1999;38:8826–30.
132. Westendorp MO, Shatrov VA, Schulze-Osthoff K, Frank R, Kraft M, Los M, Krammer PH, et al. HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. EMBO J 1995;14:546–54.
133. Connor RI, Chen BK, Choe S, Landau NR. Vpr is required for effi-cient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995;206:935–44.
134. Westervelt P, Henkel T, Trowbridge DB, Orenstein J, Heuser J, Gendelman HE, Ratner L. Dual regulation of silent and produc-tive infection in monocytes by distinct human immunodefi-ciency virus type 1 determinants. J Virol 1992;66:3925–31. 135. Levy DN, Refaeli Y, Weiner DB. Extracellular Vpr protein
increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency. J Virol 1995; 69:1243–52.
136. Muthumani K, Hwang DS, Desai BM, Zhang D, Dayes N, Green DR, Weiner DB. HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells. J Biol Chem 2002;277:37820–31.
137. Jacotot E, Ferri KF, El Hamel C, Brenner C, Druillennec S, Hoebeke J, Rustin P, et al. Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med 2001;193:509–19. 138. Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami
N, Costantini P, et al. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 2000;191:33–46.
