The presence of syncytia-inducing (SI) virus, and theformation of multinucleated cells (syncytia) is supposed toplay a major role in the progression of infection with the humanimmunodeficiency virus (HIV). SI viruses, which use thechemokine receptor CXCR4 to infect cells expressing theCD4 molecule (the main receptor for HIV, present on T-helperlymphocytes, monocytes and macrophages), are found morefrequently in patients with an advanced stage of the diseasethan the non-syncytia-inducing (NSI) type, which are usuallyisolated in the asymptomatic period of the infection. SI HIVisolates have a greater capacity to kill infected cells in culturethan do NSI strains, and the switch from NSI to SI isconsidered a marker of disease progression and an unfavourableprognostic sign. Formation of syncytia causes cell deathwith features of either apoptosis or necrosis. As far aslymphocytes or monocytes are concerned, syncytia areformed by the fusion of infected with uninfected CD4þ cells:in culture, their formation is one of the first signs of HIVinfection of peripheral blood mononuclear cells (PBMC),appearing 2–3 days after adding viral particles or infectedbiological material (such as cells or plasma). In T-cell linessuch as MT-2, the formation of syncytia can occur as early as2 h after the infection.1 Cell fusion is temperature-dependentand does not require DNA, RNA or protein synthesis. Itinvolves carbohydrates and lipids present on the cellmembrane and depends on CD4 and the HIV gp120 andgp41 envelope proteins.2As far as the progressive loss of CD4þ cells occurring inHIVþ patients is concerned, the real role of syncytiaformation is still a matter of debate. Indeed, evidence for thepresence of multinucleated cells in vivo is lacking except in thebrain.3 HIV infects and induces syncytium formation inmicroglia cells from the central nervous system (CNS).Macrophages/microglia cells are the main reservoir for HIVin the CNS, and multinucleated giant cells, the result of fusionof HIV-infected microglia and brain macrophages, are theneuropathologic hallmark of HIV dementia.The formation of a syncytium leads to a cytopathic effectwith a typical balloon degeneration of the cells, which iscaused by a variety of mechanisms. Among these, thoserelated to triggering of apoptotic pathways play a major role.The growing understanding of p53 function helped to establishits fundamental role in the regulation of apoptosis in differentcontexts. Many viruses encode proteins that can specificallyescape p53-mediated apoptotic programmes (i.e. adenovirus,human papilloma viruses, SV40, etc.).4 In contrast, HIV is ableto hijack the apoptotic machinery of the infected cell to induceits demise, possibly to facilitate virus spreading and counteracteither the innate or acquired immune response of thehost.5 Recent reports demonstrated that p53 and its targetgene Bax are involved in the induction of apoptosis triggeredby HIV in infected primary lymphocytes.6,7 The role of p53 inHIV-induced cell death has now been more firmly validated bya recent report by Perfettini et al.8 in the Journal ofExperimental Medicine. The same group had previouslydemonstrated the existence of a sequence of events duringsyncytia formation:9 (i) activation of mammalian target ofrapamycin (mTOR); (ii) mTOR-dependent phosphorylation ofp53 at serine 15 and the induction of its target gene Bax; (iii)activation of a mitochondrial death pathway. This process alsoresults in deregulated mitosis through the induction of cyclinB1. However, the exact mechanisms regulating these eventsare still unclear. To dissect the pathways involved in syncytiaformation, the authors analyse the transcriptional activities ofseveral transcription factors upon cell fusion. The activities ofp53, NF-kB and AP-1 are strongly enhanced by cell fusion,thus suggesting that these factors are functionally involved inthis process. Strikingly, AP-1 activity was abrogated upontransfection of either p53 dominant-negative or NF-kBdominant-negative mutants. Thus, p53 and NF-kB areupstream regulators of AP-1. NF-kB is activated through thephosphorylation and ubiquitin-mediated degradation of itsinhibitor IkB.10 IkB phosphorylation is present in prekaryogamicor karyogamic syncytia, thus indicating that NF-kB isactive at both stages of syncytia formation. In contrast,phosphorylation of p53 at Ser 15 and 46, which are markers ofits activation, is induced only in early and late karyogamicsyncytia, respectively. Inhibition of NF-kB blocks the transcriptionalactivation of p53 and the emergence of karyogamicsyncytia (Figure 1). The induction of cyclin B1 is also inhibitedin this context. Based on this evidence, NF-kB regulates thecyclin B1-mediated entry into karyogamy that occurs upstreamp53 activation.These experiments are validated by the observation thatIkB phosphorylation is detected in lymph nodes from HIVcarriers, mainly among mitotic cells. The involvement of NFkBin syncytia formation is intriguing, but several questionsremain unanswered. Firstly, it is unclear if NF-kB has anyproapoptotic activity per se and how it regulates p53phosphorylation and activation. One previous report by thegroup of Karen Vousden has proposed that NF-kB is inducedupon p53 activation and is required for apoptosis induced byp53 in tumour cell lines.11 Secondly, which are the NF-kBtarget genes involved in syncytia formation? It is unclear if thecanonical NF-kB-dependent antiapoptotic programme isinduced upon syncytia induction. Finally, it would also beCell Death and Differentiation (2004) 11, 691–692& 2004 Nature Publishing Group All rights reserved 1350-9047/04 $30.00www.nature.com/cddimportant to study the DNA-binding activity of NF-kB byelectrophoretic mobility shift assay and chromatin immunoprecipitationto determine which NF-kB dimers bind to DNAupon syncytia formation.The authors then move on to consider the role of p53 inkaryogamic syncytia. First, they find that syncytia formationinduced by Env-CD4 interaction in Hela cells has a dramaticeffect on gene expression. In all, 82 genes were found to besignificantly up- or downregulated. Pifithrin-alpha, a knownchemical inhibitor of p53,12 prevents 85% of these changes ingene expression. Thus, p53 is an essential modulator of thetranscriptional programme induced by cell fusion. However, itremains to be established if transcription is essential forsyncytia formation. Moreover, it is unknown at present if p53affects transcription of the deregulated genes directly orindirectly. It is unlikely that p53 would act directly in all cases.The striking effect of the p53 inhibitor pifithrin-alpha on thistype of the cell death could have future therapeutic implications.However, inactivation of p53 results in a dramaticincrease in the oncogenic potential of Kaposi’s sarcomaassociatedherpesvirus cyclin, suggesting that inactivation ofp53 might result in increase Kaposi incidence in vivo.13In order to assess which p53 target genes are affected uponcell fusion, Perfettini et al. employ a p53 microarray thatincludes most known p53 target genes. They find that twomain proapoptotic target genes are induced: PUMA and BAX(Figure 1). PUMA is a Bcl-2 family member recently added tothe growing list of BH3-only proteins,14 and is a potentproapoptotic factor that is rapidly induced by ionizing radiationin a p53-dependent fashion.14 Two recent reports demonstratedthat the inactivation of PUMA in the mouse results inno overt effect on development.15,16 By contrast, PUMA/ thymocytes and mouse embryo fibroblasts are resistant toionizing radiation-induced cell death, thus implicating PUMAas the main p53 target for apoptosis.15,16 Perfettini et al.demonstrate that PUMA is induced upon syncytia formation incell lines and that PUMA is expressed at high levels inlymphocytes infected with HIV in vitro. PUMA is alsooverexpressed in lymph nodes from HIV patients and itsexpression levels positively correlate with HIV titres. Moreover,cells from patients treated with HAART show reducedlevels of PUMA. From a clinical point of view, this observationis of great importance as it indicates that variations in PUMAexpression can become a useful marker of the progression ofinfection, and thus could be used to monitor the efficacy ofantiretroviral treatments. PUMA induction correlates with Baxand Bak conformational change and activation. Importantly,syncytial apoptosis is strongly reduced when PUMA, Bax orBak are downregulated, as demonstrated by using antisenseoligonucleotides or RNA interference. Interestingly, anotherBH3-only family member, Bim, has also been implicated inHIV-induced apoptosis.17It remains unclear whether mTOR upregulation has effectsother than on p53 activation, as is also the role of mTOR inapoptosis.18 mTOR is part of the fundamental PI-3K/AKTpathway, which is normally activated by growth factors andmitogenic stimuli.19 Upon activation by AKT, mTOR regulatesprotein synthesis through the modulation of S6 kinase and thesubsequent phosphorylation of the ribosomal subunit S6, andthrough the activation of the initiation factor of translationeIF4E.19 Further investigation is needed to assess whetherthe syncytia-induced activation of mTOR is PI3-K/AKTdependentand which are the targets of mTOR in this process.The identification of major players in syncytia-induced celldeath is a significant step in understanding the molecularmechanisms of HIV cytotoxicity. However, some outstandingquestions require an urgent answer. First, to what extent doessyncytia formation occur in vivo, and are they a cause of deathof infected lymphocytes, as occurs in the CNS? Second, doviral pro-apoptotic proteins such as Tat, vpr and pr play anyrole in syncytia-induced apoptosis in vivo; for example, doesthe secreted form of Tat play any role in bystander cell death?Third, how can PUMA be used as a marker to monitor theprogression of the infection and the efficacy of its treatment?This paper uncovers an emerging role for p53 and p53target genes in HIV-induced apoptosis. Future studies will benecessary to gain a more complete understanding of the roleof p53 in HIV-induced apoptosis in vivo.

HIV: no PUMA no death? / P., Salomoni; Cossarizza, Andrea. - In: CELL DEATH AND DIFFERENTIATION. - ISSN 1350-9047. - STAMPA. - 11:(2004), pp. 691-692.

HIV: no PUMA no death?

COSSARIZZA, Andrea
2004

Abstract

The presence of syncytia-inducing (SI) virus, and theformation of multinucleated cells (syncytia) is supposed toplay a major role in the progression of infection with the humanimmunodeficiency virus (HIV). SI viruses, which use thechemokine receptor CXCR4 to infect cells expressing theCD4 molecule (the main receptor for HIV, present on T-helperlymphocytes, monocytes and macrophages), are found morefrequently in patients with an advanced stage of the diseasethan the non-syncytia-inducing (NSI) type, which are usuallyisolated in the asymptomatic period of the infection. SI HIVisolates have a greater capacity to kill infected cells in culturethan do NSI strains, and the switch from NSI to SI isconsidered a marker of disease progression and an unfavourableprognostic sign. Formation of syncytia causes cell deathwith features of either apoptosis or necrosis. As far aslymphocytes or monocytes are concerned, syncytia areformed by the fusion of infected with uninfected CD4þ cells:in culture, their formation is one of the first signs of HIVinfection of peripheral blood mononuclear cells (PBMC),appearing 2–3 days after adding viral particles or infectedbiological material (such as cells or plasma). In T-cell linessuch as MT-2, the formation of syncytia can occur as early as2 h after the infection.1 Cell fusion is temperature-dependentand does not require DNA, RNA or protein synthesis. Itinvolves carbohydrates and lipids present on the cellmembrane and depends on CD4 and the HIV gp120 andgp41 envelope proteins.2As far as the progressive loss of CD4þ cells occurring inHIVþ patients is concerned, the real role of syncytiaformation is still a matter of debate. Indeed, evidence for thepresence of multinucleated cells in vivo is lacking except in thebrain.3 HIV infects and induces syncytium formation inmicroglia cells from the central nervous system (CNS).Macrophages/microglia cells are the main reservoir for HIVin the CNS, and multinucleated giant cells, the result of fusionof HIV-infected microglia and brain macrophages, are theneuropathologic hallmark of HIV dementia.The formation of a syncytium leads to a cytopathic effectwith a typical balloon degeneration of the cells, which iscaused by a variety of mechanisms. Among these, thoserelated to triggering of apoptotic pathways play a major role.The growing understanding of p53 function helped to establishits fundamental role in the regulation of apoptosis in differentcontexts. Many viruses encode proteins that can specificallyescape p53-mediated apoptotic programmes (i.e. adenovirus,human papilloma viruses, SV40, etc.).4 In contrast, HIV is ableto hijack the apoptotic machinery of the infected cell to induceits demise, possibly to facilitate virus spreading and counteracteither the innate or acquired immune response of thehost.5 Recent reports demonstrated that p53 and its targetgene Bax are involved in the induction of apoptosis triggeredby HIV in infected primary lymphocytes.6,7 The role of p53 inHIV-induced cell death has now been more firmly validated bya recent report by Perfettini et al.8 in the Journal ofExperimental Medicine. The same group had previouslydemonstrated the existence of a sequence of events duringsyncytia formation:9 (i) activation of mammalian target ofrapamycin (mTOR); (ii) mTOR-dependent phosphorylation ofp53 at serine 15 and the induction of its target gene Bax; (iii)activation of a mitochondrial death pathway. This process alsoresults in deregulated mitosis through the induction of cyclinB1. However, the exact mechanisms regulating these eventsare still unclear. To dissect the pathways involved in syncytiaformation, the authors analyse the transcriptional activities ofseveral transcription factors upon cell fusion. The activities ofp53, NF-kB and AP-1 are strongly enhanced by cell fusion,thus suggesting that these factors are functionally involved inthis process. Strikingly, AP-1 activity was abrogated upontransfection of either p53 dominant-negative or NF-kBdominant-negative mutants. Thus, p53 and NF-kB areupstream regulators of AP-1. NF-kB is activated through thephosphorylation and ubiquitin-mediated degradation of itsinhibitor IkB.10 IkB phosphorylation is present in prekaryogamicor karyogamic syncytia, thus indicating that NF-kB isactive at both stages of syncytia formation. In contrast,phosphorylation of p53 at Ser 15 and 46, which are markers ofits activation, is induced only in early and late karyogamicsyncytia, respectively. Inhibition of NF-kB blocks the transcriptionalactivation of p53 and the emergence of karyogamicsyncytia (Figure 1). The induction of cyclin B1 is also inhibitedin this context. Based on this evidence, NF-kB regulates thecyclin B1-mediated entry into karyogamy that occurs upstreamp53 activation.These experiments are validated by the observation thatIkB phosphorylation is detected in lymph nodes from HIVcarriers, mainly among mitotic cells. The involvement of NFkBin syncytia formation is intriguing, but several questionsremain unanswered. Firstly, it is unclear if NF-kB has anyproapoptotic activity per se and how it regulates p53phosphorylation and activation. One previous report by thegroup of Karen Vousden has proposed that NF-kB is inducedupon p53 activation and is required for apoptosis induced byp53 in tumour cell lines.11 Secondly, which are the NF-kBtarget genes involved in syncytia formation? It is unclear if thecanonical NF-kB-dependent antiapoptotic programme isinduced upon syncytia induction. Finally, it would also beCell Death and Differentiation (2004) 11, 691–692& 2004 Nature Publishing Group All rights reserved 1350-9047/04 $30.00www.nature.com/cddimportant to study the DNA-binding activity of NF-kB byelectrophoretic mobility shift assay and chromatin immunoprecipitationto determine which NF-kB dimers bind to DNAupon syncytia formation.The authors then move on to consider the role of p53 inkaryogamic syncytia. First, they find that syncytia formationinduced by Env-CD4 interaction in Hela cells has a dramaticeffect on gene expression. In all, 82 genes were found to besignificantly up- or downregulated. Pifithrin-alpha, a knownchemical inhibitor of p53,12 prevents 85% of these changes ingene expression. Thus, p53 is an essential modulator of thetranscriptional programme induced by cell fusion. However, itremains to be established if transcription is essential forsyncytia formation. Moreover, it is unknown at present if p53affects transcription of the deregulated genes directly orindirectly. It is unlikely that p53 would act directly in all cases.The striking effect of the p53 inhibitor pifithrin-alpha on thistype of the cell death could have future therapeutic implications.However, inactivation of p53 results in a dramaticincrease in the oncogenic potential of Kaposi’s sarcomaassociatedherpesvirus cyclin, suggesting that inactivation ofp53 might result in increase Kaposi incidence in vivo.13In order to assess which p53 target genes are affected uponcell fusion, Perfettini et al. employ a p53 microarray thatincludes most known p53 target genes. They find that twomain proapoptotic target genes are induced: PUMA and BAX(Figure 1). PUMA is a Bcl-2 family member recently added tothe growing list of BH3-only proteins,14 and is a potentproapoptotic factor that is rapidly induced by ionizing radiationin a p53-dependent fashion.14 Two recent reports demonstratedthat the inactivation of PUMA in the mouse results inno overt effect on development.15,16 By contrast, PUMA/ thymocytes and mouse embryo fibroblasts are resistant toionizing radiation-induced cell death, thus implicating PUMAas the main p53 target for apoptosis.15,16 Perfettini et al.demonstrate that PUMA is induced upon syncytia formation incell lines and that PUMA is expressed at high levels inlymphocytes infected with HIV in vitro. PUMA is alsooverexpressed in lymph nodes from HIV patients and itsexpression levels positively correlate with HIV titres. Moreover,cells from patients treated with HAART show reducedlevels of PUMA. From a clinical point of view, this observationis of great importance as it indicates that variations in PUMAexpression can become a useful marker of the progression ofinfection, and thus could be used to monitor the efficacy ofantiretroviral treatments. PUMA induction correlates with Baxand Bak conformational change and activation. Importantly,syncytial apoptosis is strongly reduced when PUMA, Bax orBak are downregulated, as demonstrated by using antisenseoligonucleotides or RNA interference. Interestingly, anotherBH3-only family member, Bim, has also been implicated inHIV-induced apoptosis.17It remains unclear whether mTOR upregulation has effectsother than on p53 activation, as is also the role of mTOR inapoptosis.18 mTOR is part of the fundamental PI-3K/AKTpathway, which is normally activated by growth factors andmitogenic stimuli.19 Upon activation by AKT, mTOR regulatesprotein synthesis through the modulation of S6 kinase and thesubsequent phosphorylation of the ribosomal subunit S6, andthrough the activation of the initiation factor of translationeIF4E.19 Further investigation is needed to assess whetherthe syncytia-induced activation of mTOR is PI3-K/AKTdependentand which are the targets of mTOR in this process.The identification of major players in syncytia-induced celldeath is a significant step in understanding the molecularmechanisms of HIV cytotoxicity. However, some outstandingquestions require an urgent answer. First, to what extent doessyncytia formation occur in vivo, and are they a cause of deathof infected lymphocytes, as occurs in the CNS? Second, doviral pro-apoptotic proteins such as Tat, vpr and pr play anyrole in syncytia-induced apoptosis in vivo; for example, doesthe secreted form of Tat play any role in bystander cell death?Third, how can PUMA be used as a marker to monitor theprogression of the infection and the efficacy of its treatment?This paper uncovers an emerging role for p53 and p53target genes in HIV-induced apoptosis. Future studies will benecessary to gain a more complete understanding of the roleof p53 in HIV-induced apoptosis in vivo.
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HIV: no PUMA no death? / P., Salomoni; Cossarizza, Andrea. - In: CELL DEATH AND DIFFERENTIATION. - ISSN 1350-9047. - STAMPA. - 11:(2004), pp. 691-692.
P., Salomoni; Cossarizza, Andrea
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