Apoptosis 2001; 6: 103116 C 2001 Kluwer Academic Publishers

Apoptosis in AIDS
M. Roshal, Y. Zhu and V. Planelles
Departments of Medicine Microbiology & Immunology, University of Rochester Cancer Center, 601 Elmwood Avenue, Rochester, NY 14642, USA

Infection with the human immunodeciency virus type 1 (HIV-1) leads to progressive immunodeciency and onset of opportunistic infections and neoplasms. The loss of immune competence is associated with declines in both the functionality and the number of CD4+ lymphocytes. Multiple mechanisms have been proposed to explain death and dysfunction of CD4+ T-cells. The mechanisms of HIV-1-mediated cell death which are relevant in vivo are unclear at present. However, in vitro explorations on the cytopathic effects of HIV-1 have yielded a wealth of potential triggering events, and signaling and effector pathways leading to apoptosis. The types of proand anti-apoptotic stimuli that have been associated with HIV-1 are multiple and often appear overlapping or even contradictory. This review focuses on the various molecular determinants from HIV-1 that play a role in induction of apoptosis in T-lymphocytes. Special attention is devoted to the viral genes, env, nef, tat and vpr, for which a signicant body of literature on apotosis-related effects is available.

Keywords: Apoptosis; survival; HIV; vpr; net; tat; env; AIDS

Introduction
Infection with the human immunodeciency virus type 1 (HIV-1) usually leads to progressive immunodeciency and onset of opportunistic infections and neoplasms. The loss of immune competence is associated with declines in both the functionality and the number of CD4+ lymphocytes. Work published in recent years has established that an important component of the decline in numbers of CD4+ T-cells in vivo is death due to direct viral infection.13 The half-life of most HIV-1-infected T-cells in vivo is estimated to be in the range of 12 to 36 hours.13 Key factors in the loss of immune competence are deterioration of CD4+ T-cell function and decrease in numbers of CD4+ T-cells. Multiple mechanisms, including viral and immunological processes, have been proposed to explain death and
Corresponding to: Vicente Planelles, Departments of Medicine and Microbiology & Immunology, University of Rochester Cancer Center, 601 Elmwood Avenue, Box #704, Rochester, NY 14642; Tel: (716) 273-4474; Fax (716) 273-1221; e-mail: vicente planelles@urmc.rochester.edu

dysfunction of CD4+ T-cells. Among the virus-mediated mechanisms proposed are toxicity caused by accumulation of unintegrated viral DNA,4 membrane permeability changes resulting from viral particles budding at the surface of the infected cell,5 and terminal differentiation causing a shortened life span of the CD4+ T-lymphocytes.6 Among the immunological mechanisms that may contribute to death of CD4+ lymphocytes during HIV-1 infection are killing by specic cytotoxic T-lymphocytes (CTL) and signaling through the CD4 molecule, leading to apoptosis.7,8 Various groups have suggested that deleterious immunological interactions lead to the loss of immune cells by apoptosis.7,9,10 Fresh peripheral blood lymphocytes (PBL) from HIV-1 infected patients have an increased propensity to undergo apoptosis following stimulation in vitro compared to those from healthy individuals.11,12 In addition, infection with HIV-1 in vitro leads to enhanced expression of Fas ligand (Fas-L).13,14 Thus, apoptosis may be caused by inappropriate ligation of overexpressed Fas-L with its receptor, Fas, on the surface of CD4+ lymphocytes.

The genetic structure of HIV-1

HIV-1 encodes three structural genes, gag, pol and env, which are common to all replication-competent retroviruses (Figure 1). The product of gag is translated from unspliced mRNA as a precursor poly-protein. This precursor is cleaved by the viral protease (PRO) into the following subunits: matrix (MAp21), capsid (CAp24), nucleocapsid (NCp7), and several additional polypeptides of small size and unknown function, such as p1, p2 and p6. The pol gene also encodes a polyprotein, and it is expressed as a fusion protein with Gag upon ribosomal frameshifting.15 The Gag-Pol polyprotein is also processed by the viral protease. The cleavage of Pol gives rise to three enzymes: PRO, reverse transcriptase (RT) and integrase (IN). RT contains three enzymatic activities: RNA-dependent DNA polymerase, RNAase H and DNA-dependent DNA polymerase. IN is responsible for integration of the viral DNA in the cellular chromosome.
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Figure 1. Genetic structure of HIV-1. HIV-1 encodes nine known genes. Structural genes are shown in black boxes, accessory genes are shown in hatched boxes, and regulatory genes are shown in dotted boxes. The long terminal repeats (LTR; grey boxes) are cis-acting, regulatory sequences anking the coding region. Genes marked with an asterisk are documented to play a central role in the modulation of apoptosis by HIV-1 infection (see text for details).

The env gene is essential for viral binding and entry into the host cells. It encodes the precursor glycoprotein, gp160, which is cleaved into a surface moiety, gp120 (SU), and a transmembrane moiety, gp41 (TM). The surface glycoprotein is required for binding to cellular receptors, whereas the transmembrane glycoprotein is responsible for the fusion with the cellular membrane. In addition to the structural genes, HIV-1 also encodes six small open reading frames (Figure 1). Two of those are regulatory genes, tat and rev, which encode transactivator proteins essential for viral replication. Tat is a transcriptional transactivator. Rev is a post-transcriptional transactivator and allows nuclear export of unspliced and singly spliced mRNAs encoding viral structural proteins. In the absence of Rev the only mRNAs detected in the infected cells are doubly spliced ones, encoding regulatory and accessory genes, but not structural genes. The remaining four small open reading frames (Figure 1) are also known as accessory genes and they include vif, vpr, vpu and nef. These genes were named accessory because they are non-essential for virus replication in cell culture.15 Vif (virion infectivity factor) is not essential for HIV-1 replication in permissive cells such as HeLa-CD4 or SupT1.16,17 However, it is necessary for production of infectious virions by cells that are natural targets for infection, including CD4-positive T-lymphocytes, macrophages, and H9 cells.1820 A recent study has suggested that non-permissive cells contain an endogenous inhibitor of HIV-1 production that is overcome by the virusencoded Vif protein.21 The vpu gene encodes a cytoplasmic viral protein that promotes degradation of CD4 in the endoplasmic reticulum of target cells.2225 Degradation of CD4 allows more efcient transport of the envelope glycoprotein to the cell surface and its incorporation into virions. Vpu is able to stimulate the release of viral particles from certain types of cells including T-lymphocytes, HeLa cells and colonic carcinoma SW480 cells.26,27 Another function of Vpu is to downregulate the expression of MHC I molecules on the surface of infected cells.28 This downregulation may prevent recognition by cytotoxic T-cells.
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General aspects of env, tat, vpr, and nef, as well as their roles in apoptosis will be discussed for each individual gene, below.

HIV-1 induces apoptosis at different stages of its replication cycle

HIV-1 can induce apoptosis from without when the surface glycoprotein, gp120, cross-links CD4 (Figure 2). This interaction primes cells for apoptosis29,30 in the absence of de novo viral gene expression. HIV-1 can also induce apoptosis from within, via expression of certain viral genes, such as tat, nef and vpr (Figure 2). Tat was shown to induce apoptosis when expressed in infected cells.31,32 The Tat protein, however, can be secreted to the medium by infected cells33 and later be taken up at a different site by uninfected lymphocytes via endocytosis.34 Through this route of distribution, Tat can induce apoptosis in trans in bystander cells.35 This review focuses on the various molecular determinants from HIV-1 that play a role in induction of apoptosis in T-lymphocytes. A wealth of scientic work in the last ten years suggests that the connections between HIV-1 and apoptosis induction are highly complex, involving multiple viral genes and diverse signaling pathways. As we will discuss, the types of pro- and anti-apoptotic stimuli that have been associated with HIV-1 are multiple and often may appear overlapping or even contradictory. (See Table 1) This is partly a consequence of the fact that survival and apoptosis pathways are far from being completely understood at present.

General aspects of apoptosis

Apoptosis is a form of cell death that was initially characterized at a morphological level.36 Distinctive features of apoptosis include cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. Recently, apoptosis has been characterized at the genetic and

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Figure 2. HIV-induced apoptosis is triggered at multiple steps of the viral life cyle. A. Induction of apoptosis from without. Virioncontained protein, such as Vpr and the envelope glycoprotien, may trigger apoptotic signals prior to full completion of the viral life cycle. Envelope binding to CD4 and/or a co-receptor may trigger early signals. Immediately after virus entry, release of Vpr in the cytosol may lead to activation of the apoptotic machinery. B. Induction of apoptosis from within. After completion of the entry, reverse trancription and integration steps, expression of viral mRNAs is followed by de novo production of viral proteins in the target cell. These proteins may display pro- and anti- apoptotic abilities. C. Induction of apoptosis in trans. Tat can be secreted into the medium and taken up by endocytosis by bystander, uninfected cells. The biological effects of Tat have been demonstrated to occur in bystander cells.

biochemical levels, and a plethora of genes and proteins involved in the positive and negative regulation of apoptosis have become known. Three major types of stimuli induce apoptosis. Each of these stimuli triggers a specic signaling cascade, although a certain degree of overlap exists among different cascades. The rst group of stimuli includes binding of certain ligands (TNF-, Fas-L, TRAIL) to death-inducing receptors (DR) on the surface of cells. The second type

of stimulus is DNA damage. DNA damage is a natural signal that may induce cell cycle arrest and apoptosis. Failure to repair mutations will lead to activation of the apoptosis signaling cascade and commitment of the cell to death.37,38 The third type of stimulus that may lead to apoptosis is delivered by cytotoxic T-cells when they recognize a target cell. Dissection of apoptosis signaling pathways can be accomplished based on analysis of the presence, localization and activity of various proteins (such as caspases, and cytochrome C), physical properties
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Table 1. Apoptosis-related molecular determinants of HIV-1 Molecular determinant Pro-apoptotic (A) or pro-survival (S)/Mode of action (A) Activation of caspase 8 (A/S) Transcriptional activation of NFB (A) Upregulation of FasL expression (A)Enhancement of TNF-secretion and toxicity (A/S) Change concentrations of Bcl-2 family members (S) Activation of Akt (A) Abnormal centrosome duplication (A) Induction of genomic instabilty (A) Induction of mitochondrial abnormalities (A/S) Suppression of NF b activity (S) Increase concentration of Bcl-2 and decrease concentration of Bax (A) Upregulation of Fas (A) Fas-L (A) Downmodulation of caspase activation inhibitor (A) Decrease in Bcl-2 and increase in Bax protein concentrations (A) Induction of TNF- and TNF Receptor-II expression (A) Upregulation of Fas-L (A) Activation of PAKs (A) Upregulation of Fas (S) Downregulation of MHC-I presentation References 31 42, 43 52, 14 56, 53, 57, 54, 55, 58 35, 63, 62, 60, 61 69 105, 96 108, 109 118, 97 119 121 140, 141 136, 138 137 134, 137 147, 141 160163 167173 163 151153

tat

vpr

env

nef

of the cells (such as depolarization of the mitochondria), and susceptibility to inhibition by cellular anti-apoptotic proteins (such as Bcl-2).

The Tat transactivator has proand anti-apoptotic properties

The viral protein Tat upregulates viral transcription at the level of elongation via interaction with the Tat activation region (TAR) located at the 5 end of all viral mRNAs. In addition to the interaction with TAR, Tat also interacts with a cellular kinase, termed Tat-associated kinase (TAK), which is a complex between cyclin-T and the cyclin-dependent kinase-9, cdk-9.3941 Both of the above interactions are required for the transactivation function of Tat. In addition to transactivating the HIV-1 LTR, Tat has been shown to also transactivate a number of cellular promoters (see below). HIV-1 Tat can be secreted by virus-infected cells33 and can be taken up by bystander cells via endocytosis.34 Because of this property, Tats biological activities can be exerted in uninfected cells. Tat transactivates cellular genes involved in apoptosis signaling Tat increases the activity of a number of cellular transcription regulators including nuclear factor kappa-B
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(NFB),42,43 nuclear factor of activated T-cells (NFAT)4446 and AP-1.47 Tat can also inuence a number of cellular signaling networks affecting, among others, cytokine secretion proles,4850 MAP/SAPK kinase cascades47,45 and tyrosine kinases of the Src superfamily.51 These interactions are thought to increase viral replication in the infected cells. Because of the variety of the interactions with cellular genes, the effects of Tat on the survival/apoptosis pathways are likely to be multiple. The activation of NFB, for example, was shown to increase the cellular survival in certain paradigms, but to lead to apoptosis in others. Tat leads to transcriptional activation of both pro- and anti-apoptotic genes, as well as changes in inammatory cytokine secretion proles. Similarly, the effect Tat on the MAP kinase family member, Jun N-terminal kinase (JNK) and consequent activation of the AP-1 transcription factor may have both pro- and anti-apoptotic effects. Little is known about he cross talk between the various pathways affected by Tat. For the purposes of this review, we will focus on those effects of Tat that have been linked to a well-described apoptosis pathway. Effects of Tat in receptor-mediated apoptosis It has been suggested that expression of Tat renders cells more susceptible to apoptosis via death receptors.14,52 The effect of Tat on Fas and tumor necrosis factor-

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(TNF-)-induced cell death has been observed at several signaling steps. Exogenous Tat can upregulate the expression of the Fas ligand (Fas-L) mRNA and increase the susceptibility of the Tat-expressing cells to CD4 crosslinking-induced cell death.14 Tat-responsive sites in the Fas ligand (Fas-L) promoter are identical to NFB binding sites, which suggests that upregulation of Fas-L by Tat is NFB-dependent.52 Tat may affect various pro-apoptotic factors that act downstream of Fas-L/Fas interaction. Five membrane receptors (Fas, TNF R1, DR3, DR4 and DR5) can act as sensors for their respective ligands and trigger proapoptotic signaling via a shared pathway. This pathway involves the formation of an active complex known as death-induced signaling complex (DISC; see Figure 3). Death receptors share intracytoplasmic domains that bind to the death domain of Fas-associated death domain protein (FADD/MORT-I). FADD acts as an adaptor by recruiting caspases 8 and 10 through its death domains. The recruitment of caspases 8 and 10 by FADD leads to the generation of the DISC in which pro-caspases 8 and 10 are cleaved to give rise to their activated forms. Barz and Emerman31 showed that upon expression of Tat, the levels of caspase 8 mRNA, protein and enzymatic

activity increased. The upregulation of caspase 8, in turn, renders cells susceptible to apoptosis via FAS signaling. The previous observations raise the possibility that the infected cells would also be more susceptible to apoptosis signaling through other death receptors that utilize the DISC. Additionally tat can accentuate TNF- signaling by both increasing TNF- secretion5355 and shifting the cellular redox potential towards oxidation.56,57 This may be accomplished by decreasing transcription of the mitochondrial enzyme, manganese-dependent superoxide dismutase (Mn-SOD).57 The pro-oxidative state of the cells expressing Tat may render them susceptible to TNF toxicity.58

Effects of Tat on Bcl-2 and related proteins In an early report, Zauli et al. suggested that lymphoblastoid (Jurkat), epithelial (293) and neuronal (PC12) cell lines stably expressing Tat were protected from apoptotic death induced by serum withdrawal.59 This protection was associated with increased levels of the anti-apoptotic protein, Bcl-2. The increase in Bcl-2 was found to be due to an increase in the bcl-2 promoter activity.60,61 Both endogenously expressed and recombinant Tat were shown to increase Bcl-2 promoter activity. However the mechanism for this transactivation has not yet been elucidated. On the other hand, other groups have observed a Tatdependent decrease in Bcl-2 protein levels in hematopoietic cell lines62 or have not observed any change.35,63 Thus, the effects of Tat in Bcl-2 levels remain unresolved. Finally, a Bcl-2-related protein with pro-apoptotic effects, Bax, was shown to increase in levels following Tat expression.62

Figure 3. The death receptor signaling pathway. Schematic representation of the main signaling steps involving apoptisis signaling via death receptors.

Interactions of Tat with members of the survival pathway Cell survival cannot be explained solely on the basis of failure of apoptosis to be activated. An increasing body of evidence supports the notion that survival, like apoptosis, is an active process. The principal intracellular mediators of survival (Figure 4) are Akt/protein kinase B64 and NFB/Rel.65 The apoptosis-survival pathway is particularly relevant to the biology of T-lymphocytes because T-cell activation is accompanied by certain pro-apoptotic signals.66 To prevent apoptosis, T-lymphocytes upregulate the activity of NFB (an anti-apoptotic factor) in response to activation signals. Thus, activation-induced apoptosis of T-cells appears to be dictated by the balance between pro- and anti-apoptotic forces. Constitutive activation of NFB in certain T-cell lymphomas causes resistance to activationinduced apoptosis.67 Remarkably, the HIV-1 promoter,
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Figure 4. The survival signaling pathway. Schematic representation of signaling steps which determine suppression of apoptosis.

Tat and the apoptotic machinery are likely to be multiple. The type of cell, the of exposure to Tat (exogenous versus endogenous),71 its expression levels as well as the levels of the polypeptide in the medium,32,35,59,70,72 the state of activation of cells73 and the cytokine milieu may all play a role in the response.4850 Further exploration of the effects of Tat on the HIV-1 infected as well as bystander cells is bound to provide important clues to the mechanism of cell depletion in HIV-1 pathogenesis.

The vpr gene induces transactivation, cell cycle arrest and apoptosis
HIV-1 vpr encodes a 96-amino acid protein with multiple functions in the viral life cycle. The rst reported role for vpr was a moderate transactivation effect on the viral promoter, the long terminal repeat (LTR).7476 HIV-1 mutants with deletions in vpr replicate with slower kinetics than wild-type viruses.77,78 Vpr is encapsidated into virions in signicant amounts.74,79,80 The presence of vpr in the viral particle facilitates efcient infection of macrophages and other non-dividing cells78,81,82 by mediating active nuclear import of pre-integration complexes.83,84 In addition, the presence of vpr enhances the transcriptional activity of the viral LTR in macrophages and T-cells, allowing for the production of a larger viral progeny.8587 HIV-1 vpr contributes to the multiple cytopathic effects iduced by HIV-1 by inducing cell cycle arrest in G2 8892 and apoptosis.9396 Interestingly, the vpr-induced growth arrest and cell enlargement phenotypes are observed in yeast, which suggest the involvement a rather conserved pathway.97,98 The apoptogenic and cytostatic properties of Vpr have been mapped to its basic C-terminal domain.99104 The mechanism of apoptosis induction by Vpr is not fully understood. A recent report suggests that Vpr-expressing cells die during the M phase of the cell cycle, which they enter after a prolonged G2 delay. The researchers hypothesized that the death was due to an abnormal multipolar mitosis due an aberrant centrosome duplication.96,105 Caspases have been shown to be involved becase both caspase inhibition with either pharmacological or viral inhibitors results in abrogation of Vpr-induced apoptosis.94,106

the long terminal repeat (LTR), contains NFB binding sites that confer high levels of transcriptional activity in activated T-cells. A role for Tat in activation and translocation of NFB to the nucleus is well established.43,68 Tat and Akt Low levels of extracellular Tat were shown to activate phosphatidylinositol-3-kinase (PI-3-K) and Akt/PKB kinases in CD4+ T-lumphoblastoid Jurkat cells, leading to increased cell survival following serum starvation.69 Interestingly, when cells were pre-treated with PI-3-K inhibitors, the percentage of apoptotic cells was dramatically increased in the presence of Tat.69 These observations suggest that the activation of PI-3-K is an important antiapoptotic modulator of the cellular response to exogenous Tat. It was also found that protection from apoptosis in Jurkat cells expressing Tat was, at least in part, due to an autocrine loop, which could be blocked by adding anti-Tat antibodies to the growth medium.50,70 Tat may play a pleiotropic role in cell survival. Both incresed and decreased apoptosis were observed in response to Tat often under similar conditions. Reasons for these discrepancies are not clear. Because Tat affects numerous pathways involved in cell growth, activation and survival, the points of interaction between
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Vpr and DNA repair pathways It has been hypothesized that vpr utilizes the DNA damage pathway, which can also arrest cells in G2 and cause apoptosis.107 Several lines of evidence support the previous hypothesis. Vpr expression in certain cell lines leads to genomic instability cromosome breaks, micronuclei

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formation, aneuploidy and gene amplication.108,109 Hyperphosphorylation of Cdc-2 observed in Vpr-arrested cells is reminiscent of cells treated with a DNA-alkylating agent, nitrogen mustard.107 Furthermore, the arrest by Vpr can be reversed by methylxanthines that can also reverse cell cycle arrest induced by DNA damage.107 Although a direct binding of Vpr to DNA has been reported,110 the possibility that Vpr activates DNA damagedependent cellular pathways by directly causing alterations in the structure or the integrity of DNA has not been demonstrated. Vpr has been shown to physically interact two proteins possibly involved in DNA repair: Uracil DNA glycosylase (UDG)111,112 and HHR23-A,113 a human homologue of the yeast rad 23 gene, involved in repair of radiationinduced damage. Mutagenesis experiments demostrated that the interaction of Vpr with UNG is dispensable for Vpr-induced cell cycle arrest and apoptosis.112 Rather, Vpr-UNG interaction appears to be involved in determination the HIV-1 genomic integrity.114 Overexpression of HHR23-A was shown to partially abrogate Vpr-induced G2 arrest.113,115 However it is not clear at present how the interaction with HHR23-A may be involved in Vpr-induced G2 arrest. Other lines of evidence argue against the DNA damage pathway as a primary pathway for Vprs cytostatic and cytotoxic action. Cell cycle arrest and apoptosis induced by genotoxic stress are often mediated through p53 and ATM (mutated in ataxia-telangiectasia) gene products.116 Bartz and colleagues have shown that Vprinduced G2 arrest is independent of p53 and ATM function.117 p53 is also not involved in vpr-mediated apoptosis.94 Furthermore genetic studies in the yeast S. pombe have suggested that in this organism the machinery that arrests cells in G2 /M in response to DNA damage is, at least in part, dispensable for Vpr-induced G2 arrest.98 Furthermore, the aberrant mitotic spindle formation that has been observed in Vpr-expressing cells has not been observed in cells treated with DNA-damaging agents.96

Figure 5. Relationships among various functions of HIV-1 Vpr. HIV-1 vpr triggers G2 /M arrest as a priymary effect. Transactivation of the viral LTR and induction of apoptosis appear to be secondary effects which are dependent on the induction of cell cycle arrest.

appears to be caused by the same carboxy-terminal domain of Vpr that is involved in mammalian cell apoptosis. A recent report has shown that exogenous Vpr can directly cause the loss of mitochondrial potential and trigger apoptotic changes in cell-free system as well as in intact cells.118 In a cell-free system, Vpr interacted with the permeability trasition pore complex (PTPC) to cause increased ion permeability and swelling of mitochondria, leading to the rapid release of cytochrome C and induction of apoptosis within several hours of Vpr exposure.118 A model that integrates the transactivation, G2 arrest and apoptosis induction properties of Vpr is presented in Figure 5. Inhibition of G2 arrest by caffeine also inhibits the downstream effects, transactivation and induction of apoptosis. The higher transcriptional activity of the viral LTR during the abnormally long G2 phase leads to a high progeny output leading to the next cycle of infection. The taget cell, then, undergoes apoptosis, a relatively non-immunogenic form of cell death. It has been proposed that induction of apoptosis by Vpr may decrease the immunogenicity of virus-infected cells.106 Vpr as a pro-survival protein Several investigators have suggested that expression of vpr may protect cells from apoptosis. Ayyavoo et al. have suggested that Vpr may protect T-cells from apoptosis in the presence of T-cell receptor (TCR) activation.119 It was hypothesized that Vpr acted as a general suppressor of NFB activity. However this observation was recently questioned.120 Other groups have found that cells that stably express low levels of Vpr are protected from apoptosis.121,122 HIV-1 vpr protected stably transfected cells from sorbitol-induced122 as well as Fas-L-, TNF-and serum starvation-induced apoptosis.121 A putative mechanism for the previous protection effects is the upregulation of bcl-2 and downregulation of bax.121 A recent report by Conti et al. has suggested that in the course of acute HIV infection Vpr plays a cytoprotective role early on, and increases apoptosis at later stages of infection.123
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Vpr and the mitochondria Given the importance of mitochondria in most mechanisms of apoptosis, it would be logical to propose that mitochondria are involved in Vpr-induced apoptosis. Indeed, several groups have suggested that mitochondria are the primary site for Vprs apoptogenic and cytostatic actions.97,118 Results from Macreadie et al. indicate that yeast cells constitutively expressing vpr show gross mitochondrial defects including loss of several respiratory chain complexes and inability to utilize unconventional carbon sources such as ethanol or glycerol.97 This defect

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Binding of the HIV-1 envelope glycoprotein induces signaling events in the target cells
In order to enter a target cell HIV-1 must rst bind two molecules on the cell surface. Depending on the cell type and the tropism of the virus, gp120 interacts with its primary receptor CD4 and an accessory receptor (typically, but not exclusively, CXCR4 or CCR5).124,125 The binding of gp120 to the cell receptors can induce signaling events.29,126128 These signals have been implicated in HIV-1-induced apoptosis of both lymphoid and nonlymphoid cells.

TNF on the macrophages. The TNFR-II interaction with TNF then leads to increased apoptosis of the CD8+ positive cells.

Nef
The nef gene is essential for viral infectivity in vivo, but not in vitro.148 Nef reduces interactions between Env and intracellular CD4 by inducing internalization and degradation of CD4.149,150 Nef also downregulates cell surface expression of MHC I molecules and protects infected cells from killing by cytotoxic T-lymphocytes.151153 Nef was also shown to enhance the infectivity of viral particles, independently of the effects on CD4.154,155 Nef-dependent enhancement of infectivity occurs at the level of proviral DNA synthesis, early in the viral life cycle.156158 Nef has been shown to interact with a number of cellular signal transduction molecules including members of the Src family of tyrosine kinases; p21-activated kinases (PAKs); mitogen-activated kinases (MAPs); the G protein, Raf1; and a proto-oncogene and guanine-nucleotide exchange factor, Vav, involved in activation of T-cells.159 Nef may play an important role in the immune dysregulation and lymphoid tissue apoptosis in HIV-1 infected patients. Several mechanisms for Nef-dependent apoptosis have been postulated. Nef was shown to induce activation of lymphocytes leading to upregulation of Fas-L which, in turn, can induce apoptosis of both CD4+ and CD8+ positive cells.160163 In order to activate lymphocytes, Nef interacts with and signals through the tyrosine-kinase activation motifs of the T-cell receptor zeta chain.161,164166 A downstream signal involves activation of p21-activated kinases 1 and 2167173 and formation of the Nef-activated kinase complex, NAK, between PAK and Nef. Interestingly PAK2 is cleaved and activated by caspases [Rudel, 1997 #331; Walter, 1998 #328; Chan, 1999 #327] and plays an important role in the regulation of morphology and biochemistry of apoptotic cells. Thus, it would be logical to suspect that Nef may induce certain pro-apoptotic events through the interaction with PAK2.174,175 Another mechanism that may be involved in Nef-dependent apoptosis include upregulation of Fas.163 Our understanding of the mechanisms involved in lymphocyte depletion in HIV has improved dramatically over the past decade. In 1991 several groups proposed apoptosis as a mechanism for cell depletion in HIV infection.7,10 An extensive body of literature since then has supported this hypothesis. Despite this large and ever-growing body of literature, many questions still need to be answered. Relatively little information is available about the specic interactions of the HIV-encoded proteins and cellular ones that modulate apoptosis pathways. In particular, the specic mechanisms of HIV-induced cell death in vivo are poorly understood.

Binding of gp120 and CD4 Both soluble gp120 as well gp120 on the surface of the infected cells can induce apoptosis independently of syncytium formation.29,129132 Cross-linking of bound gp120 on human CD4+ T-cells followed by signaling in through the T-cell receptor was found to result in activation-dependent apoptosis.29 Alternatively T-cells may be induced to undergo apoptosis in the absence of TCR engagement when monocyte/macrophages are present.129 The mechanism for the induction of apoptosis by gp120 has been extensively studied in recent years. Both Fasdependent and Fas-independent pathways have been reported.133139 Premature CD4 engagement results in the increased susceptibility of the CD4+ cells to Fasinduced apoptosis via two mechanisms: (1) upregulation of Fas140,141 and Fas-ligand;136,138 and (2) downmodulation of a caspase inhibitor, the FLICE-like inhibitory protein (FLIP).137 Fas-independent mechanisms of gp120-induced apoptosis have also been demonstrated. It appears that gp120 binding to CD4 may induce downregulation of bcl-2134 and upregulation of the pro-apoptotic gene, bax,134,137 leading to mitochondria-dependent apoptosis. Both Fas and Bcl-2-dependent pathways are caspase-dependent and therefore it is not surprising that caspase inhibition results in downmodulation of gp120-induced apoptosis.142,143

HIV-1 co-receptors and apoptosis Fas-independent, gp120-induced apoptosis of CD4+ lymphocytes appears to be enhanced by the interaction of gp120 with CXCR4.144146 The gp120 interaction with CXCR4 has also been implicated in the increased apoptosis of CD8+ positive cells in the presence of macrophages.147 The binding of gp120 to CXCR4 can signal through the receptor in both CD8+ cells and macrophages, leading to the induction of TNF receptor-II (TNFR-II) on the CD8+ cells and membrane-bound
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It is perhaps no accident that much research on HIVinduced apoptosis has focused on the Fas pathway. The Fas pathway is relatively well understood. Because the apoptotic machinery of the cell is intricately linked to other cellular programs, and HIV clearly modulates a number of these programs, the triggers of apoptosis are likely to be multiple. Understanding of the apoptosis determinants in HIV infection will have profound implications in anti-HIV vaccine design. For instance, knowing that expression of certain viral genes in antigen-expressing cells will induce apoptosis will prompt investigators to re-design immunogens so that such genes are removed or modied. A clear example of this is the Nef-dependent upregulation of Fas-L, which results in decreased anti-HIV cytotoxic T-lymphocyte (CTL) activity.162 One may rationalize that only a modied version of nef that cannot trigger Fas-L expression should be used as part of an HIV vaccine. Other determinants of apoptosis, such as vpr, may decrease the life span of an antigen-presenting cell, perhaps hindering the production of an effective immune response. Although a better understanding of apoptosis will help us dissect AIDS progression at the cellular and molecular levels, it should be noted that prevention of bystander cell killing could prove a rational strategy in slowing or preventing immune deterioration.
Acknowledgments

This work was supported by a grant from the National Institutes of Health (R29-AI41407) to VP.
References
1. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373: 123 126. 2. Perelson AS, Neumann AU, Markowitz M, Leonard JM, Ho DD. HIV-1 dynamics in vivo: Virion clearance rate, infected cell life-span, and viral generation time. Science 1996; 271: 15821586. 3. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human immunodeciency virus type 1 infection. Nature 1995; 373: 117122. 4. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deciency syndrome. Science 1984; 226: 11651171. 5. Fauci AS. The human immunodeciency virus: Infectivity and mechanisms of pathogenesis. Science 1988; 239: 617 622. 6. Zagury D, Bernard J, Leonard R, et al. Long-term cultures of HTLV-IIIinfected T cells: A model of cytopathology of T-cell depletion in AIDS. Science 1986; 231: 850853. 7. Ameisen JC, Capron A. Cell dysfunction and depletion in AIDS: The programmed cell death hypothesis. Immunol Today 1991; 12: 102105.

8. Finkel TH, Banda NK. Indirect mechanisms of HIV pathogenesis: How does HIV kill T cells? Curr Opin Immunol 1994; 6: 605615. 9. Laurent-Crawford AG, Krust B, Muller S, et al. The cytopathic effect of HIV is associated with apoptosis. Virology 1991; 185: 829839. 10. Terai C, Kornbluth RS, Pauza CD, Richman DD, Carson DA. Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1. J Clin Invest 1991; 87: 17101715. 11. Groux H, Torpier G, Monte D, Mouton Y, Capron A, Ameisen JC. Activation-induced death by apoptosis in CD4+ T cells from human immunodeciency virus-infected asymptomatic individuals. J Exp Med 1992; 175: 331340. 12. Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RP, Miedema F. Programmed death of T cells in HIV-1 infection. Science 1992; 257: 217219. 13. Badley AD, McElhinny JA, Leibson PJ, Lynch DH, Alderson MR, Paya CV. Upregulation of Fas ligand expression by human immunodeciency virus in human macrophages mediates apoptosis of uninfected T lymphocytes. J Virol 1996; 70: 199206. 14. Westendorp MO, Frank R, Ochsenbauer C, et al. Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 1995; 375: 497500. 15. Luciw PA. Human immunodeciency viruses and their replication. In: Fields BN, Knippe DM, Howley PM, ed. Fields Virology. Philadelphia: Lippincott-Raven 1996; 1881 1975. 16. Gabuzda DH, Lawrence K, Langhoff E, et al. Role of vif in replication of human immunodeciency virus type 1 in CD4+ T lymphocytes. J Virol 1992; 66: 64896495. 17. Sakai H, Shibata R, Sakuragi J, Sakuragi S, Kawamura M, Adachi A. Cell-dependent requirement of human immunodeciency virus type 1 Vif protein for maturation of virus particles. J Virol 1993; 67: 16631666. 18. Blanc D, Patience C, Schulz TF, Weiss R, Spire B. Transcomplementation of VIF-HIV-1 mutants in CEM cells suggests that VIF affects late steps of the viral life cycle. Virology 1993; 193: 186192. 19. Fouchier RA, Simon JH, Jaffe AB, Malim MH. Human immunodeciency virus type 1 Vif does not inuence expression or virion incorporation of gag-, pol-, and env-encoded proteins. J Virol 1996; 70: 82638269. 20. von Schwedler U, Song J, Aiken C, Trono D. Vif is crucial for human immunodeciency virus type 1 proviral DNA synthesis in infected cells. J Virol 1993; 67: 49454955. 21. Madani N, Kabat D. An endogenous inhibitor of human immunodeciency virus in human lymphocytes is overcome by the viral Vif protein. J Virol 1998; 72: 1025110255. 22. Geleziunas R, Bour S, Wainberg MA. Human immunodeciency virus type 1-associated CD4 downmodulation. Adv Virus Res 1994; 44: 203266. 23. Vincent MJ, Raja NU, Jabbar MA. Human immunodeciency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: Role of the cytoplasmic domain in Vpuinduced degradation in the endoplasmic reticulum. J Virol 1993; 67: 55385549. 24. Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeciency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol 1992; 66: 71937200. 25. Yao XJ, Friborg J, Checroune F, et al. Degradation of CD4 induced by human immunodeciency virus type 1 Vpu protein: A predicted alpha-helix structure in the proximal cytoplasmic

Apoptosis Vol 6 No 1/2 2001

111

M. Roshal et al.
region of CD4 contributes to Vpu sensitivity. Virology 1995; 209: 615623. Gottlinger HG, Dorfman T, Cohen EA, Haseltine WA. Vpu protein of human immunodeciency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses. Proc Natl Acad Sci USA 1993; 90: 73817385. Klimkait T, Strebel K, Hoggan MD, Martin MA, Orenstein JM. The human immunodeciency virus type 1-specic protein vpu is required for efcient virus maturation and release. J Virol 1990; 64: 621629. Kerkau T, Bacik I, Bennink JR, et al. The human immunodeciency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules. J Exp Med 1997; 185: 12951305. Banda NK, Bernier J, Kurahara DK, et al. Crosslinking CD4 by human immunodeciency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med 1992; 176: 1099 1106. Lu YY, Koga Y, Tanaka K, Sasaki M, Kimura G, Nomoto K. Apoptosis induced in CD4+ cells expressing gp160 of human immunodeciency virus type 1. J Virol 1994; 68: 390399. Bartz SR, Emerman M. Human immunodeciency virus type 1 Tat induces apoptosis and increases sensitivity to apoptotic signals by up-regulating FLICE/caspase-8. J Virol 1999; 73: 19561963. Purvis SF, Jacobberger JW, Sramkoski RM, Patki AH, Lederman MM. HIV type 1 Tat protein induces apoptosis and death in Jurkat cells. AIDS Res Hum Retroviruses 1995; 11: 443450. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposis sarcoma lesions of AIDS patients. Nature 1990; 345: 8486. Mann DA, Frankel AD. Endocytosis and targeting of exogenous HIV-1 Tat protein. Embo J 1991; 10: 17331739. Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 1995; 268: 429431. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239257. Hartwell LH, Weinert TA. Checkpoints: Controls that ensure the order of cell cycle events. Science 1989; 246: 629634. Paulovich AG, Toczyski DP, Hartwell LH. When checkpoints fail. Cell 1997; 88: 315321. Ivanov D, Kwak YT, Nee E, Guo J, Garcia-Martinez LF, Gaynor RB. Cyclin T1 domains involved in complex formation with Tat and TAR RNA are critical for tat- activation. J Mol Biol 1999; 288: 4156. Romano G, Kasten M, De Falco G, Micheli P, Khalili K, Giordano A. Regulatory functions of Cdk9 and of cyclin T1 in HIV tat transactivation pathway gene expression. J Cell Biochem 1999; 75: 357368. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV1 Tat and mediates its high-afnity, loop-specic binding to TAR RNA. Cell 1998; 92: 451462. Conant K, Ma M, Nath A, Major EO. Extracellular human immunodeciency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes. J Virol 1996; 70: 13841389. Demarchi F, dAdda di Fagagna F, Falaschi A, Giacca M. Activation of transcription factor NF-kappaB by the Tat protein of human immunodeciency virus type 1. J Virol 1996; 70: 44274437. Kinoshita S, Su L, Amano M, Timmerman LA, Kaneshima H, Nolan GP. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity 1997; 6: 235244. Li X, Multon MC, Henin Y, et al. Grb3-3 is up-regulated in HIV-1 infected T-cells and can potentiate cell activation through NFATc. J Biol Chem 2000; 275: 3092530933. Macian F, Rao A. Reciprocal modulatory interaction between human immunodeciency virus type 1 Tat and transcription factor NFAT1. Mol Cell Biol 1999; 19: 36453653. Kumar A, Manna SK, Dhawan S, Aggarwal BB. HIV-Tat protein activates c-Jun N-terminal kinase and activator protein1. J Immunol 1998; 161: 776781. Ambrosino C, Ruocco MR, Chen X, et al. HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancerbinding protein beta (NF-IL6) transcription factors. J Biol Chem 1997; 272: 1488314892. Scala G, Ruocco MR, Ambrosino C, et al. The expression of the interleukin 6 gene is induced by the human immunodeciency virus 1 TAT protein. J Exp Med 1994; 179: 961971. Zauli G, Gibellini D, Celeghini C, et al. Pleiotropic effects of immobilized versus soluble recombinant HIV-1 Tat protein on CD3-mediated activation, induction of apoptosis, and HIV-1 long terminal repeat transactivation in puried CD4+ T lymphocytes. J Immunol 1996; 157: 22162224. Manna SK, Aggarwal BB. Differential requirement for p56lck in HIV-tat versus TNF- induced cellular responses: Effects on NF-kappa B, activator protein-1, c-Jun N-terminal kinase, and apoptosis. J Immunol 2000; 164: 51565166. Li-Weber M, Laur O, Dern K, Krammer PH. T cell activationinduced and HIV tat-enhanced CD95(APO-1/Fas) ligand transcription involves NF-kappaB. Eur J Immunol 2000; 30: 661670. Buonaguro L, Barillari G, Chang HK, et al. Effects of the human immunodeciency virus type 1 Tat protein on the expression of inammatory cytokines. J Virol 1992; 66: 7159 7167. Sastry KJ, Reddy HR, Pandita R, Totpal K, Aggarwal BB. HIV-1 tat gene induces tumor necrosis factor-beta (lymphotoxin) in a human B-lymphoblastoid cell line. J Biol Chem 1990; 265: 2009120093. Shatrov VA, Ratter F, Gruber A, Droge W, Lehmann V. HIV type 1 glycoprotein 120 amplies tumor necrosis factorinduced NF-kappa B activation in Jurkat cells. AIDS Res Hum Retroviruses 1996; 12: 12091216. Banki K, Hutter E, Gonchoroff NJ, Perl A. Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase. J Biol Chem 1998; 273: 1194411953. Flores SC, Marecki JC, Harper KP, Bose SK, Nelson SK, McCord JM. Tat protein of human immunodeciency virus type 1 represses expression of manganese superoxide dismutase in HeLa cells. Proc Natl Acad Sci USA 1993; 90: 7632 7636. Westendrop MO, Shatrov VA, Schulze-Osthoff K, et al. HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. Embo J 1995; 14: 546554. Zauli G, Gibellini D, Milani D, et al. Human immunodeciency virus type 1 Tat protein protects lymphoid, epithelial, and neuronal cell lines from death by apoptosis. Cancer Res

26.

44.

27.

45. 46. 47. 48.

28.

29.

30. 31.

49. 50.

32.

51.

33.

52.

34. 35. 36. 37. 38. 39.

53.

54.

55.

56.

40.

57.

41.

58.

42.

59.

43.

112 Apoptosis Vol 6 No 1/2 2001

Apoptosis in AIDS
1993; 53: 44814485. 60. Wang Z, Morris GF, Reed JC, Kelly GD, Morris CB. Activation of Bcl-2 promoter-directed gene expression by the human immunodeciency virus type-1 Tat protein. Virology 1999; 257: 502510. 61. Zauli G, Gibellini D, Caputo A, et al. The human immunodeciency virus type-1 Tat protein upregulates Bcl-2 gene expression in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Blood 1995; 86: 38233834. 62. Sastry KJ, Marin MC, Nehete PN, McConnell K, el-Naggar AK, McDonnell TJ. Expression of human immunodeciency virus type I tat results in down- regulation of bcl-2 and induction of apoptosis in hematopoietic cells. Oncogene 1996; 13: 487493. 63. Macho A, Calzado MA, Jimenez-Reina L, Ceballos E, Leon J, Munoz E. Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms. Oncogene 1999; 18: 75437551. 64. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 1995; 376: 599602. 65. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS, Jr. NF-kappaB antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c- IAP2 to suppress caspase-8 activation. Science 1998; 281: 16801683. 66. Kolenko V, Bloom T, Rayman P, Bukowski R, Hsi E, Finke J. Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3. J Immunol 1999; 163: 590598. 67. Giri DK, Aggarwal BB. Constitutive activation of NFkappaB causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. Autocrine role of tumor necrosis factor and reactive oxygen intermediates. J Biol Chem 1998; 273: 1400814014. 68. Demarchi F, Gutierrez MI, Giacca M. Human immunodeciency virus type 1 tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR. J Virol 1999; 73: 70807086. 69. Borgatti P, Zauli G, Colamussi ML, et al. Extracellular HIV-1 Tat protein activates phosphatidylinositol 3- and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells. Eur J Immunol 1997; 27: 28052811. 70. Zauli G, La Placa M, Vignoli M, et al. An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity. J Acquir Immune Dec Syndr Hum Retrovirol 1995; 10: 306316. 71. McCloskey TW, Ott M, Tribble E, et al. Dual role of HIV Tat in regulation of apoptosis in T cells. J Immunol 1997; 158: 10141019. 72. Ranki A, Lagerstedt A, Ovod V, Aavik E, Krohn KJ. Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines. Arch Virol 1994; 139: 365378. 73. Katsikis PD, Garcia-Ojeda ME, Torres-Roca JF, Greenwald DR, Herzenberg LA. HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals. Int Immunol 1997; 9: 835841. 74. Cohen EA, Dehni G, Sodroski JG, Haseltine WA. Human immunodeciency virus vpr product is a virion-associated regulatory protein. J Virol 1990; 64: 30973099. 75. Ogawa K, Shibata R, Kiyomasu T, et al. Mutational analysis of the human immunodeciency virus vpr open reading frame. J Virol 1989; 63: 41104114. Sawaya BE, Khalili K, Gordon J, Taube R, Amini S. Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome. J Biol Chem 2000; 275: 3520935214. Balliet JW, Kolson DL, Eiger G, et al. Distinct effects in primary macrophages and lymphocytes of the human immunodeciency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate. Virology 1994; 200: 623631. Connor RI, Chen BK, Choe S, Landau NR. Vpr is required for efcient replication of human immunodeciency virus type-1 in mononuclear phagocytes. Virology 1995; 206: 935944. Accola MA, Bukovsky AA, Jones MS, Gottlinger HG. A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeciency viruses SIV(mac) and SIV(agm). J Virol 1999; 73: 9992 9999. Yuan X, Matsuda Z, Matsuda M, Essex M, Lee TH. Human immunodeciency virus vpr gene encodes a virionassociated protein. AIDS Res Hum Retroviruses 1990; 6: 1265 1271. Gallay P, Stitt V, Mundy C, Oettinger M, Trono D. Role of the karyopherin pathway in human immunodeciency virus type 1 nuclear import. J Virol 1996; 70: 10271032. Heinzinger NK, Bukinsky MI, Haggerty SA, et al. The Vpr protein of human immunodeciency virus type 1 inuences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994; 91: 73117315. Fouchier RA, Meyer BE, Simon JH, et al. Interaction of the human immunodeciency virus type 1 Vpr protein with the nuclear pore complex. J Virol 1998; 72: 60046013. Popov S, Rexach M, Zybarth G, et al. Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. Embo J 1998; 17: 909917. Goh WC, Rogel ME, Kinsey CM, et al. HIV-1 Vpr increases viral expression by manipulation of the cell cycle: A mechanism for selection of Vpr in vivo. Nat Med 1998; 4: 6571. Gummuluru S, Emerman M. Cell cycle- and Vpr-mediated regulation of human immunodeciency virus type 1 expression in primary and transformed T-cell lines. J Virol 1999; 73: 54225430. Subbramanian RA, Yao XJ, Dilhuydy H, et al. Human immunodeciency virus type 1 Vpr localization: Nuclear transport of a viral protein modulated by a putative amphipathic helical structure and its relevance to biological activity. J Mol Biol 1998; 278: 1330. He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR. Human immunodeciency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 1995; 69: 67056711. Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, Chen IS. The human immunodeciency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J Virol 1995; 69: 63046313. Planelles V, Jowett JB, Li QX, Xie Y, Hahn B, Chen IS. Vpr-induced cell cycle arrest is conserved among primate lentiviruses. J Virol 1996; 70: 25162524. Re F, Braaten D, Franke EK, Luban J. Human immunodeciency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B. J Virol 1995; 69: 68596864. Rogel ME, Wu LI, Emerman M. The human immunodeciency virus type 1 vpr gene prevents cell proliferation during

76.

77.

78. 79.

80.

81. 82.

83. 84. 85. 86.

87.

88.

89.

90. 91.

92.

Apoptosis Vol 6 No 1/2 2001

113

M. Roshal et al.
chronic infection. J Virol 1995; 69: 882888. 93. Chang L, Chen C, Urlacher V, Lee T. Differential apoptosis effects of primate lentiviral vpr and vpx in mammalian cells. J Biomed Sci 2000; 7: 322333. 94. Shostak LD, Ludlow J, Fisk J, et al. Roles of p53 and caspases in the induction of cell cycle arrest and apoptosis by HIV-1 vpr. Exp Cell Res 1999; 251: 156165. 95. Stewart SA, Poon B, Jowett JB, Chen IS. Human immunodeciency virus type 1 Vpr induces apoptosis following cell cycle arrest. J Virol 1997; 71: 55795592. 96. Watanabe N, Yamaguchi T, Akimoto Y, Rattner JB, Hirano H, Nakauchi H. Induction of M-phase arrest and apoptosis after HIV-1 vpr expression through uncoupling of nuclear and centrosomal cycle in HeLa cells. Exp Cell Res 2000; 258: 261269. 97. Macreadie IG, Thorburn DR, Kirby DM, Castelli LA, de Rozario NL, Azad AA. HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae. FEBS Lett 1997; 410: 145149. 98. Masuda M, Nagai Y, Oshima N, et al. Genetic studies with the ssion yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeciency virus type 1 Vpr. J Virol 2000; 74: 26362646. 99. Berglez JM, Castelli LA, Sankovich SA, Smith SC, Curtain CC, Macreadie IG. Residues within the HFRIGC sequence of HIV-1 vpr involved in growth arrest activities. Biochem Biophys Res Commun 1999; 264: 287290. 100. Chen M, Elder RT, Yu M, et al. Mutational analysis of Vprinduced G2 arrest, nuclear localization, and cell death in ssion yeast. J Virol 1999; 73: 32363245. 101. Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau NR. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeciency virus type 1 Vpr. J Virol 1995; 69: 79097916. 102. Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, Ward AC, Azad AA. A domain of human immunodeciency 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 1995; 92: 27702774. 103. Mahalingam S, Collman RG, Patel M, Monken CE, Srinivasan A. Role of the conserved dipeptide Gly75 and Cys76 on HIV-1 Vpr function. Virology 1995; 210: 495500. 104. Zhou Y, Lu Y, Ratner L. Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest. Virology 1998; 242: 414424. 105. Minemoto Y, Shimura M, Ishizaka Y, Masamune Y, Yamashita K. Multiple centrosome formation induced by the expression of Vpr gene of human immunodeciency virus. Biochem Biophys Res Commun 1999; 258: 379384. 106. Stewart SA, Poon B, Song JY, Chen IS. Human immunodeciency virus type 1 vpr induces apoptosis through caspase activation. J Virol 2000; 74: 31053111. 107. Poon B, Jowett JB, Stewart SA, Armstrong RW, Rishton GM, Chen IS. Human immunodeciency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA alkylating agent, nitrogen mustard. J Virol 1997; 71: 39613971. 108. Shimura M, Onozuka Y, Yamaguchi T, Hatake K, Takaku F, Ishizaka Y. Micronuclei formation with chromosome breaks and gene amplication caused by Vpr, an accessory gene of human immunodeciency virus. Cancer Res 1999; 59: 2259 2264. 109. Shimura M, Tanaka Y, Nakamura S, et al. Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeciency virus type 1. Faseb J 1999; 13: 621637. 110. Zhang S, Pointer D, Singer G, Feng Y, Park K, Zhao LJ. Direct binding to nucleic acids by Vpr of human immunodeciency virus type 1. Gene 1998; 212: 157166. 111. Bouhamdan M, Benichou S, Rey F, et al. Human immunodeciency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme. J Virol 1996; 70: 697704. 112. Selig L, Benichou S, Rogel ME, et al. Uracil DNA glycosylase specically interacts with Vpr of both human immunodeciency virus type 1 and simian immunodeciency virus of scooty mangabeys, but binding does not correlate with cell cycle arrest. J Virol 1997; 71: 48424846. 113. Withers-Ward ES, Jowett JB, Stewart SA, et al. Human immunodeciency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair. J Virol 1997; 71: 97329742. 114. Mansky LM, Preveral S, Selig L, Benarous R, Benichou S. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeciency virus type 1 In vivo mutation rate. J Virol 2000; 74: 70397047. 115. Gragerov A, Kino T, Ilyina-Gragerova G, Chrousos GP, Pavlakis GN. HHR23A, the human homologue of the yeast repair protein RAD23, interacts specically with Vpr protein and prevents cell cycle arrest but not the transcriptional effects of Vpr. Virology 1998; 245: 323330. 116. Morgan SE, Kastan MB. p53 and ATM: Cell cycle, cell death, and cancer. Adv Cancer Res 1997; 71: 125. 117. Bartz SR, Rogel ME, Emerman M. Human immunodeciency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J Virol 1996; 70: 2324 2331. 118. Jacotot E, Ravagnan L, Loefer M, 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: 3346. 119. Ayyavoo V, Mahboubi A, Mahalingam S, et al. HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B. Nat Med 1997; 3: 11171123. 120. Roux P, Aleri C, Hrimech M, Cohen EA, Tanner JE. Activation of transcription factors NF-kappaB and NF-IL-6 by human immunodeciency virus type 1 protein R (Vpr) induces interleukin-8 expression. J Virol 2000; 74: 46584665. 121. Conti L, Rainaldi G, Matarrese P, et al. The HIV-1 vpr protein acts as a negative regulator of apoptosis in a human lymphoblastoid T cell line: Possible implications for the pathogenesis of AIDS. J Exp Med 1998; 187: 403413. 122. Fukumori T, Akari H, Iida S, et al. The HIV-1 Vpr displays strong anti-apoptotic activity. FEBS Lett 1998; 432: 1720. 123. Conti L, Matarrese P, Varano B, et al. Dual role of the HIV-1 vpr protein in the modulation of the apoptotic response of T cells. J Immunol 2000; 165: 32933300. 124. Dittmar MT, McKnight A, Simmons G, Clapham PR, Weiss RA, Simmonds P. HIV-1 tropism and co-receptor use. Nature 1997; 385: 495496. 125. Moore JP. Coreceptors: Implications for HIV pathogenesis and therapy. Science 1997; 276: 5152. 126. Davis CB, Dikic I, Unutmaz D, et al. Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. J Exp Med 1997; 186: 17931798. 127. Neudorf SM, Jones MM, McCarthy BM, Harmony JA, Choi EM. The CD4 molecule transmits biochemical information important in the regulation of T lymphocyte activity. Cell Immunol 1990; 125: 301314. 128. Weissman D, Rabin RL, Arthos J, et al. Macrophage-tropic

114 Apoptosis Vol 6 No 1/2 2001

Apoptosis in AIDS
HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor. Nature 1997; 389: 981 985. Cottrez F, Manca F, Dalgleish AG, Arenzana-Seisdedos F, Capron A, Groux H. Priming of human CD4+ antigenspecic T cells to undergo apoptosis by HIV-infected monocytes. A two-step mechanism involving the gp120 molecule. J Clin Invest 1997; 99: 257266. Laurent-Crawford AG, Krust B, Riviere Y, et al. Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells. AIDS Res Hum Retrovirusus 1993; 9: 761773. Nardelli B, Gonzalez CJ, Schechter M, Valentine FT. CD4+ blood lymphocytes are rapidly killed in vitro by contact wit autologous human immunodeciency virus-infected cells. Proc Natl Acad Sci USA 1995; 92: 73127316. Oyaizu N, McCloskey TW, Coronesi M, Chirmule N, Kalyanaraman VS, Pahwa S. Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeciency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals. Blood 1993; 82: 33923400. Bottarel F, Feito MJ, Bragardo M, et al. The cell deathinducing ability of glycoprotein 120 from different HIV strains correlates with their ability to induce CD4 lateral association with CD95 on CD4+ T cells. AIDS Res Hum Retroviruses 1999; 15: 12551263. 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 immunodeciency virus infection and for rescue of apoptosis by interleukin-2. Blood 1997; 90: 745753. 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: 52465252. Oyaizu N, Adachi Y, Hashimoto F, et al. Monocytes express Fas ligand upon CD4 cross-linking and induce CD4+ T cells apoptosis: A possible mechanism of bystander cell death in HIV infection. J Immunol 1997; 158: 24562463. Somma F, Tuosto L, Montani MS, Di Somma MM, Cundari E, Piccolella E. Engagement of CD4 before TCR triggering regulates both Bax- and Fas (CD95)-mediated apoptosis. J Immunol 2000; 164: 50785087. Tateyama M, Oyaizu N, McCloskey TW, Than S, Pahwa S. CD4 T lymphocytes are primed to express Fas ligand by CD4 cross-linking and to contribute to CD8 T-cell apoptosis via Fas/FasL death signaling pathway. Blood 2000; 96: 195202. Yagi T, Sugimoto A, Tanaka M, et al. Fas/FasL interaction is not involved in apoptosis of activated CD4+ T cells upon HIV-1 infection in vitro. J Acquir Immune Dec Syndr Hum Retrovirol 1998; 18: 307315. Desbarats J, Freed JH, Campbell PA, Newell MK. Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells. Proc Natl Acad Sci USA 1996; 93: 1101411018. Oyaizu N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, Pahwa S. Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferongamma and tumor necrosis factor- alpha secretion. Blood 1994; 84: 26222631. Cicala C, Arthos J, Rubbert A, et al. HIV-1 envelope induces activation of caspase-3 and cleavage of focal adhesion kinase in primary human CD4(+) T cells. Proc Natl Acad Sci USA 2000; 97: 11781183. 143. Ullrich CK, Groopman JE, Ganju RK. HIV-1 gp120- and gp 160-induced apoptosis in cultured endothelial cells is mediated by caspases. Blood 2000; 96: 14381442. 144. 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: 1255612561. 145. Biard-Piechaczyk M, Robert-Hebmann V, Richard V, Roland J, Hipskind RA, Devaux C. Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeciency virus type 1 envelope glycoprotein (gp120). Virology 2000; 268: 329344. 146. Blanco J, Jacotot E, Cabrera C, et al. The implication of the chemokine receptor CXCR4 in HIV-1 envelope proteininduced apoptosis is independent of the G protein-mediated signalling. Aids 1999; 13: 909917. 147. Herbein G, Mahlknecht U, Batliwalla F, et al. Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. Nature 1998; 395: 189194. 148. Kestler HW, Ringler DJ, Panicaly DL, Sehgal PK, Daniel MD, Desrosiers RC. Importance of the nef gene for maintenance of high virus load and for development of AIDS. Cell 1991; 65: 651662. 149. Aiken C, Konner J, Landau NR, Lenburg ME, Trono D. Nef induces CD4 endocytosis: Requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 1994; 76: 853864. 150. Garcia JV, Miller AD. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991; 350: 508511. 151. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 1998; 391: 397 401. 152. Piguet V, Chen YL, Mangasarian A, Foti M, Carpentier JL, Trono D. Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes. Embo J 1998; 17: 24722481. 153. Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 1996; 2: 338342. 154. Goldsmith MA, Warmerdam MT, Atchison RE, Miller MD, Greene WC. Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeciency virus type 1 Nef. J Virol 1995; 69: 41124121. 155. Miller MD, Warmerdam MT, Page KA, Feinberg MB, Greene WC. Expression of the human immunodeciency virus type 1 (HIV-1) nef gene during HIV-1 Production increases progeny particle infectivity independently of gp160 or viral entry. J Virol 1995; 69: 579584. 156. Aiken C, Trono D. Nef stimulates human immunodeciency virus type 1 proviral DNA synthesis. J Virol 1995; 69: 5048 5056. 157. Chowers MY, Pandori MW, Spina CA, Richman DD, Guatelli JC. The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation. Virology 1995; 212: 451457. 158. Schwartz O, Marechal V, Danos O, Heard JM. Human immunodeciency virus type 1 Nef increases the efciency of reverse transcription in the infected cell. J Virol 1995; 69: 40534059. 159. Renkema HG, Saksela K. Interactions of HIV-1 NEF with

129.

130. 131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

Apoptosis Vol 6 No 1/2 2001

115

M. Roshal et al.
cellular signal transducing proteins. Front Biosci 2000; 5: D268283. Hodge S, Novembre FJ, Whetter L, Gelbard HA, Dewhurst S. Induction of fas ligand expression by an acutely lethal simian immunodeciency virus, SIVsmmPBj14. Virology 1998; 252: 354363. Xu XN, Laffert B, Screaton GR, 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: 14891496. Xu XN, Screaton GR, Gotch FM, et al. Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeciency virus-infected cells. J Exp Med 1997; 186: 716. Zauli G, Gibellini D, Secchiero P, et al. Human immunodeciency virus type 1 Nef protien sensitizes CD4(+) T lymphoid cells to apoptosis via functional upregulation of the CD95/CD95 ligand pathway. Blood 1999; 93: 10001010. Bell I, Ashman C, Maughan J, Hooker E, Cook F, Reinhart TA. Association of simian immunodeciency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR downmodulation. J Gen Virol 1998; 79: 27172727. Howe AY, Jung JU, Desrosiers RC. Zeta chain of the T-cell receptor interacts with nef of simian immunodeciency virus and human immunodeciency virus type 2. J Virol 1998; 72: 98279834. Schaefer TM, Bell I, Fallert BA, Reinhart TA. The T-cell receptor zeta chain contains two homologous domains with which simian immunodeciency virus Nef interacts and mediates down-modulation. J Virol 2000; 74: 32733283. Barber SA, Flaherty MT, Plafker SM, Clements JE. A novel kinase activity associated with Nef derived from neurovirulent simian immunodeciency virus. Virology 1998; 251: 165 175. Brown A, Wang X, Sawai E, Cheng-Mayer C. Activation of the PAK-related kinase by human immunodeciency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex. J Virol 1999; 73: 98999907. Facker OT, Lu X, Frost JA, et al. p21-activated kinase 1 plays a critical role in cellular activation by Nef. Mol Cell Biol 2000; 20: 26192627. Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K. SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology 1998; 250: 273282. Nunn MF, Marsh JW. Human immunodeciency virus type 1 Nef associates with a member of the p21-activated kinase family. J Virol 1996; 70: 61576161. Renkema GH, Manninen A, Mann DA, Harris M, Saksela K. Identication of the Nef-associated kinase as p21-activated kinase 2. Curr Biol 1999; 9: 14071410. Sawai ET, Baur A, Struble H, Peterlin BM, Levy JA, ChengMayer C. Human immunodecieny virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci USA 1994; 91: 15391543. Bokoch GM. Caspase-mediated activation of PAK2 during apoptosis: Proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Differ 1998; 5: 637645. Rudel T, Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 1997; 276: 15711574.

160.

168.

161. 162.

169. 170.

163.

171. 172. 173.

164.

165.

174.

166.

175.

167.

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