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Signaling Pathways Triggered by HIV-1 Tat in Human Monocytes to Induce TNF-alpha
Abstract
In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.
... There is evidence that extracellular Tat can be taken up by uninfected cells and reach the nucleus rapidly where it can activate a number of transcription factors, including AP-1, Sp1, CCAAT/enhancerbinding protein (C/EBP)-, cAMP-responsive element binding protein (CREB), and NFB (34 -39). The signaling proteins responsible for the induction of these transcription factors have not been elucidated; however, it has been shown that extracellular Tat-induced signaling involves the activation of mitogen-activated protein kinase (MAPK), including c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase, as well as calcium signaling pathways (22,23,37,40,41). These signaling cascades are believed to be activated following interaction of extracellular Tat with a number of cell surface receptors, including integrin receptors, members of the vascular endothelial growth factor receptor family, and the CXCR4 chemokine receptors (12,(42)(43)(44). ...
... In addition, STAT-3 and C/EBP were implicated in the regulation of IL-10 production in different cell systems (47,48). There is evidence to suggest that extracellular recombinant HIV-Tat induces IL-10 in human monocytic cells through the activation of PKC-IIand ␦-dependent pathways (22,23,40). However, the molecular mechanism by which endogenously expressed Tat regulates IL-10 production is not known. ...
... HIV-Tat has been shown to induce the expression of IL-10 in primary human monocytes (22)(23)(24)40). We confirmed these observations by treating human monocytes with highly purified endotoxin-free recombinant Tat for 24 h followed by detection of IL-10 production by ELISA and intracellular staining by flow cytometry. ...
Human immunodeficiency virus (HIV)-Tat plays an important role in virus replication and in various aspects of host immune responses, including dysregulation of cytokine production. IL-10, an anti-inflammatory cytokine, is up-regulated during the course of HIV infection representing an important pathway by which HIV may induce immunodeficiency. Here we show that extracellular as well as intracellular Tat induced IL-10 expression in normal human monocytes and promonocytic THP-1 cells. The signaling pathways involved in the regulation of IL-10 production by endogenous Tat remain unknown. To understand the molecular mechanism underlying intracellular Tat-induced IL-10 transcription, we employed a retroviral expression system to investigate the role of MAPKs and the transcription factor(s) involved. Our results suggest that an inhibitor specific for the ERK1/2, PD98059, selectively blocked intracellular Tat-induced IL-10 expression in THP-1 cells. Furthermore, intracellular Tat activated the CREB-1 transcription factor through Ser¹³³ phosphorylation that was regulated by ERK MAPK as determined by IL-10 promoter analysis and gel shift assays. Overall, our results suggest that intracellular HIV-Tat induces IL-10 transcription by ERK MAPK-dependent CREB-1 transcription factor activation through Ser¹³³ phosphorylation.
... Among many potential HIV-1 components, we chose HIV-1 Tat protein as a potential candidate because of its pivotal role in the physiopathology of HIV-1 infection. In addition to its crucial role in the viral cycle, several additional functions have been attributed to HIV-1 Tat protein including: (i) stimulation of pro-inflammatory cytokine production [23,24,[39][40][41], (ii) establishment of an immunosuppressive state through the induction of PD-L1 [42], IDO [43,44] and IL-10 [22,33,[45][46][47] production associated with a dysfunction in T cell responses, as we recently reported [42,43]; (iii) contribution to the spread of HIV-1 and to pathogenesis [10,39,[48][49][50][51]. ...
... We have previously reported that HIV-1 Tat protein induced TNF-α and IL-10 production by monocytes [22,41,[60][61][62][63]. This production is dependent on the activation of PKC-βII and PKC-δ isoforms and involves classical and alternative NF-κB pathways [64]. ...
... Isolation of cytoplasmic, nuclear and membranes proteins extract were performed as previously described [41,61,63,64]. Briefly, for cytoplasmic and nuclear extraction, cells previously stimulated, were lysed at 4°C with 200 μL of hypotonic buffer A (Hepes 10 mM pH 7.9, KCl 10 mM, EDTA 0.1 mM, EGTA 0.1 mM, DTT 1 mM, PMSF 0.5 mM, Na3VO4 0.2 mM, NaF 0.05 mM) for 15 min. ...
We recently reported that the human immunodeficiency virus type-1 (HIV-1) Tat protein induced the expression of programmed death ligand-1 (PD-L1) on dendritic cells (DCs) through a TLR4 pathway. However, the underlying mechanisms by which HIV-1 Tat protein induces the abnormal hyper-activation of the immune system seen in HIV-1 infected patients remain to be fully elucidated. In the present study, we report that HIV-1 Tat protein induced the production of significant amounts of the pro-inflammatory IL-6 and IL-8 cytokines by DCs and monocytes from both healthy and HIV-1 infected patients. Such production was abrogated in the presence of anti-TLR4 blocking antibodies or soluble recombinant TLR4-MD2 as a decoy receptor, suggesting TLR4 was recruited by Tat protein. Tat-induced murine IL-6 and CXCL1/KC a functional homologue of human IL-8 was abolished in peritoneal macrophages derived from TLR4 KO but not from Wt mice, confirming the involvement of the TLR4 pathway. Furthermore, the recruitment of TLR4-MD2-CD14 complex by Tat protein was demonstrated by the activation of TLR4 downstream pathways including NF-κB and SOCS-1 and by down-modulation of cell surface TLR4 by endocytosis in dynamin and lipid-raft-dependent manners. Collectively, these findings demonstrate, for the first time, that HIV-1 Tat interacts with TLR4-MD2-CD14 complex and activates the NF-κB pathway, leading to overproduction of IL-6 and IL-8 pro-inflammatory cytokines by myeloid cells from both healthy and HIV-1 infected patients. This study reveals a novel mechanism by which HIV-1, via its early expressed Tat protein, hijacks the TLR4 pathway, hence establishing abnormal hyper-activation of the immune system.
... Our group has shown that HIV-1 Tat protein, by acting at cell membrane level, induces the production of IL-10, by non infected human monocytes (Badou and Bennasser 2000; Bennasser and Bahraoui 2002). To this end monocytes from healthy donors were purified and stimulated during 24 hours with increasing concentrations of recombinant Tat protein. ...
... To investigate the involvement of PKC activation in Tat-induced IL-10 production, monocytes were incubated with different PKC inhibitors. Ro318220 as well as Gö6983 which inhibits several PKC isoforms including PKC (IC50= 7 nM), PKC- (IC50 = 6 nM), PKC- (IC50 = 10 nM), PKC-(IC50= 60 nM) and PKC- (IC50 = 20 M) totally inhibit IL-10 production (Bennasser and Bahraoui 2002; Leghmari and Bennasser 2008; Contreras 2008). Gö6976 which inhibits classical PKC isoforms - and - has no effect on the capacity of Tat to induce IL-10 production by monocytes. ...
... In these conditions an inhibition reaching 90% was observed. Interestingly, PKC- and PKC inhibitors used separately at 2 and 5 M inhibited IL-10 production induced by Tat by 15 and 45% respectively, but when used in combination, they inhibited IL-10 production by 85% thus suggesting a possible synergistic effect between these two PKC isoforms (Bennasser and Bahraoui 2002). ...
... Both HIV-1 infection and exposure to Tat showed deleterious effects on mesenchymal stromal cell (MSC) growth and differentiation, and suppressed stromal hematopoietic support function. A number of reports have also shown that Tat can alter gene expression in different hematopoietic cell types (34)(35)(36)(37)(38)(39)(40). ...
... The first exon of Tat (amino acids 1-72), especially the lysine-rich core domain, has been implicated in manifesting the intracellular actions of Tat, which may regulate gene expression by altering the function of different transcription factors (46,47). Multiple signal transduction intermediates have been shown to be affected by exposure of different cell types to the Tat protein (36,44,45). Although the effect of Tat on differentiationspecific signaling in HPCs has not been characterized, Tat may play a direct role in altering the function of lineagespecific transcription factors in HPCs. ...
... The present studies contribute to the growing body of evidence demonstrating the effects of exogenously added Tat protein in regulating gene expression in different cell types (22,(33)(34)(35)(36)(37)(38)(39)(40). The paracrine actions of Tat have implicated a crucial role for this viral protein in precipitating HIV-1-induced hematologic dysfunctions (12,15,16,(19)(20)(21)(22)24). ...
The human immunodeficiency virus type 1 (HIV-1) Tat protein regulates transcription factor functions and alters cellular gene expression. Because hematopoietic progenitor cell (HPC) differentiation requires activation of lineage-specific transcription factors, Tat may affect hematopoiesis in HIV-1-infected micro-environments. We have monitored the molecular effects of Tat on megakaryocytic differentiation in the HPC line, K562. Flow cytometry analysis of CD61 indicated that phorbol myristate acetate (PMA) (16 nM) stimulated megakaryocytic commitment of K562 cells was increased (3- to 4-fold) following exposure to Tat (1-100 ng/ml). Activation of the megakaryocytic transcription factor cAMP regulatory element binding protein (CREB) and its coactivation by the CREB binding protein (CBP) was subsequently monitored. CREB phosphorylation and DNA binding were measured by Western immunodetection and electrophoretic mobility shift assays (EMSA), respectively. Within 2 hrs after stimulation, Tat increased both CREB phosphorylation and DNA binding by 7- to 10-fold. Transient cotransfection with CREB reporter and CBP expression plasmids demonstrated that Tat treatment increases (3- to 4-fold) both PMA-stimulated and CBP-mediated transcription via the cAMP regulatory element. Histone acetyl transferase (HAT) activity was increased (8- to 10-fold) in Tat-stimulated cells, which suggested increased chromosomal accessibility of transcription factors. Two-hybrid cotransfection assays using reporter plasmid containing the GAL4 DNA-binding domain and expression plasmid coding for the GAL4-CBP fusion protein, showed that Tat increases (2-fold) CBP-mediated coactivation of CREB. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis showed that Tat treatment increases CBP gene expression (7- to 9-fold) and protein levels (5- to 7-fold) within 6-12 hrs after stimulation. Our findings indicated that Tat treatment increases both CREB function and CREB coactivation by CBP, which may facilitate megakaryocytic commitment of K562 cells. Induction of this molecular signaling by HIV-1 Tat protein may have relevance in understanding the HIV-induced hematologic manifestations and possibly in regulation of viral infectivity parameters in progenitor cell reservoirs.
... There is evidence that extracellular Tat can be taken up by uninfected cells and reach the nucleus rapidly where it can activate a number of transcription factors, including AP-1, Sp1, CCAAT/enhancerbinding protein (C/EBP)-, cAMP-responsive element binding protein (CREB), and NFB (34 -39). The signaling proteins responsible for the induction of these transcription factors have not been elucidated; however, it has been shown that extracellular Tat-induced signaling involves the activation of mitogen-activated protein kinase (MAPK), including c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase, as well as calcium signaling pathways (22,23,37,40,41). These signaling cascades are believed to be activated following interaction of extracellular Tat with a number of cell surface receptors, including integrin receptors, members of the vascular endothelial growth factor receptor family, and the CXCR4 chemokine receptors (12,(42)(43)(44). ...
... In addition, STAT-3 and C/EBP were implicated in the regulation of IL-10 production in different cell systems (47,48). There is evidence to suggest that extracellular recombinant HIV-Tat induces IL-10 in human monocytic cells through the activation of PKC-IIand ␦-dependent pathways (22,23,40). However, the molecular mechanism by which endogenously expressed Tat regulates IL-10 production is not known. ...
... HIV-Tat has been shown to induce the expression of IL-10 in primary human monocytes (22)(23)(24)40). We confirmed these observations by treating human monocytes with highly purified endotoxin-free recombinant Tat for 24 h followed by detection of IL-10 production by ELISA and intracellular staining by flow cytometry. ...
Human immunodeficiency virus (HIV)-Tat plays an important role in virus replication and in various aspects of host immune
responses, including dysregulation of cytokine production. IL-10, an anti-inflammatory cytokine, is up-regulated during the
course of HIV infection representing an important pathway by which HIV may induce immunodeficiency. Here we show that extracellular
as well as intracellular Tat induced IL-10 expression in normal human monocytes and promonocytic THP-1 cells. The signaling
pathways involved in the regulation of IL-10 production by endogenous Tat remain unknown. To understand the molecular mechanism
underlying intracellular Tat-induced IL-10 transcription, we employed a retroviral expression system to investigate the role
of MAPKs and the transcription factor(s) involved. Our results suggest that an inhibitor specific for the ERK1/2, PD98059,
selectively blocked intracellular Tat-induced IL-10 expression in THP-1 cells. Furthermore, intracellular Tat activated the
CREB-1 transcription factor through Ser133 phosphorylation that was regulated by ERK MAPK as determined by IL-10 promoter analysis and gel shift assays. Overall, our
results suggest that intracellular HIV-Tat induces IL-10 transcription by ERK MAPK-dependent CREB-1 transcription factor activation
through Ser133 phosphorylation.
... We and others have demonstrated previously that Tat can increase TNF-α release from macrophages and glial cells (Chen et al., 1997;Nath et al., 1999;Mayne et al., 2000;Bruce-Keller et al., 2001;Bennasser et al., 2002;Fotheringham et al., 2004). Tat is encoded by two exons, is up to 104 amino acids in length, and has sequences classified into 5 distinct domains; N-terminal (Tat 1-21 ), cysteine-rich (Tat 22-37 ), core (Tat 38-48 ), basic (Tat 49-57 ), and C-terminal (Tat 58-104 ). ...
... Tat derived from the first exon (Tat 1-72 ) is sufficient to induce TNF-α production (Chen et al., 1997). The N-terminal region of Tat has been identified as the one that contains the active domain for TNF-α production, since Tat 1-72 , Tat 1-55 and Tat 1-101 induce similar amounts of TNF-α, but the regions between amino acids 20-72 and 30-72 cannot stimulate TNF-α production by monocytes (Bennasser et al., 2002). Other studies suggest that the basic region and the C-terminal region of Tat seem not to be involved in the production of TNF-α (New et al., 1998). ...
... The transactivating HIV-1 protein Tat is thought to play an important role in the pathogenesis of HIV-1 associated dementia in part because of findings that Tat has been detected in brains of people with HIV-dementia (Hudson et al., 2000), is actively released from HIV-infected lymphoid and glial cells (Ensoli et al., 1990), can cause neuroinflammation (Philippon et al., 1994;Jones et al., 1998;Aksenov et al., 2003;Pu et al., 2003), and can cause neuronal cell death through direct and indirect mechanisms. In addition to the direct toxic effects of Tat on neurons, Tat promotes the release of neurotoxic agents, including TNF-α, from macrophages and glial cells (Philippon et al., 1994;Chen et al., 1997;Nath et al., 1999;Mayne et al., 2000;Bruce-Keller et al., 2001;Johnston et al., 2001;Bennasser et al., 2002). Here, we found that Tat 1-72 caused neuronal cell death through increased production of substances by immune cells independently of its direct neurotoxic actions on neurons, and that Tat Δ31-61 , a mutant Tat peptide lacking the neurotoxic domain that does not kill neurons when applied directly to them, elicited the release of similarly-regulated toxic substances from immune cells which can then kill neurons. ...
HIV-1 infection causes, with increasing prevalence, neurological disorders characterized in part by neuronal cell death. The HIV-1 protein Tat has been shown to be directly and indirectly neurotoxic. Here, we tested the hypothesis that a non-neurotoxic epitope of Tat can, through actions on immune cells, increase neuronal cell death. Tat(1-72) and a mutant Tat(1-72) lacking the neurotoxic epitope (Tat(Delta31-61)) concentration-dependently and markedly increased TNF-alpha production in macrophage-like differentiated human U937 and THP-1 cells, in mouse peritoneal macrophages and in mouse brain microglia. Tat(1-72) was but Tat(Delta31-61) was not neurotoxic when applied directly to neurons. Supernatants from U937 cells treated with either Tat(1-72) or Tat(Delta31-61) were neurotoxic and their immunoneutralization with an anti-TNF-alpha antibody decreased Tat(1-72)- and Tat(Delta31-61)-induced neurotoxicity. Together, these results demonstrate that the neurotoxic epitope of Tat(1-72) is different from the epitope that is indirectly neurotoxic following production of TNF-alpha from immune cells, and suggest that therapeutic interventions against TNF-alpha might be beneficial against HIV-1 associated neurological disorders.
... This cell-free protein exerts bystander effects on other cells whether or not they are infected, leading to the modulation of cellular genes expression. In this field, HIV-1 Tat, by acting at the cell membrane surface, stimulates IL-10 and TNF-α secretion by human monocytes and macrophages [10,11]. Different domains in the HIV-1 Tat protein have been implicated in interactions with various cell receptors: (1) the N-terminal region in Tat binds the CD26 receptor expressed at the lymphocyte cell membrane; (2) the tripeptide RGD (Arginine-Glycine-Aspartate) motif interacts with α v β 3 and α 5 β 1 integrins at the surface of dendritic cells; (3) the basic region recruits membrane lipids and the VEGF receptor expressed by endothelial cells [12]; (4), Albini et al. reported the interaction of the cysteine-rich region in Tat (24-51) with CCR2, CCR3 and CXCR4 chemokines receptors [13]. ...
... Next, we determined in vitro effects of the HIV-1 Tat protein on IL-10 production by human monocytes. Recombinant HIV-1 Tat protein 1-86 (obtained from the Agence Nationale de la Recherche sur le SIDA, Paris, France) or recombinant GST-Tat 1-45 produced from our laboratory as previously described [21] and controlled for endotoxin contamination using the Limulus amebocyte lysate (LAL) assay (Bio-Sepra, France) [10,[21][22][23] were added to primary human monocytes pre-incubated or not of with the HTA125 anti-TLR4 mAb or with a nonspecific isotype-matched IgG (1 μg/ml). The supernatant was collected 24 h post-stimulation and analyzed for human IL-10 content as previously described [21]. ...
... This cell-free protein exerts bystander effects on other cells whether or not they are infected, leading to the modulation of cellular genes expression. In this field, HIV-1 Tat, by acting at the cell membrane surface, stimulates IL-10 and TNF-α secretion by human monocytes and macrophages [10,11]. Different domains in the HIV-1 Tat protein have been implicated in interactions with various cell receptors: (1) the N-terminal region in Tat binds the CD26 receptor expressed at the lymphocyte cell membrane; (2) the tripeptide RGD (Arginine-Glycine-Aspartate) motif interacts with α v β 3 and α 5 β 1 integrins at the surface of dendritic cells; (3) the basic region recruits membrane lipids and the VEGF receptor expressed by endothelial cells [12]; (4), Albini et al. reported the interaction of the cysteine-rich region in Tat (24-51) with CCR2, CCR3 and CXCR4 chemokines receptors [13]. ...
... Next, we determined in vitro effects of the HIV-1 Tat protein on IL-10 production by human monocytes. Recombinant HIV-1 Tat protein 1-86 (obtained from the Agence Nationale de la Recherche sur le SIDA, Paris, France) or recombinant GST-Tat 1-45 produced from our laboratory as previously described [21] and controlled for endotoxin contamination using the Limulus amebocyte lysate (LAL) assay (Bio-Sepra, France) [10,[21][22][23] were added to primary human monocytes pre-incubated or not of with the HTA125 anti-TLR4 mAb or with a nonspecific isotype-matched IgG (1 μg/ml). The supernatant was collected 24 h post-stimulation and analyzed for human IL-10 content as previously described [21]. ...
Background
In HIV-1 infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with progression of infection toward AIDS. HIV-1 Tat protein, by interacting with TLR4-MD2 at the membrane level, induces IL-10 production by primary human monocytes and macrophages. In the present study we evaluated the effect of the TLR4 antagonist Eritoran tetrasodium (E5564) on HIV-1 Tat-induced IL-10 production.FindingsHere, we confirm that the recombinant HIV-1 Tat protein and the GST-Tat 1¿45 fusion protein efficiently stimulate IL-10 production by primary monocytes and macrophages and that this stimulation is inhibited by blocking anti-TLR4 mAbs. We show that a similar inhibition is observed by preincubating the cells with the TLR4 antagonist E5564.Conclusion
This study provides compelling data showing for the first time that the TLR4 antagonist E5564 inhibits the immunosuppressive cytokine IL-10 production by primary human monocytes and macrophages incubated in the presence of HIV-1 Tat protein.
... However, Eritoran treatment of EBOV-infected mice did significantly decrease the levels of certain cytokines and chemokines (79). Human Immunodeficiency Virus (HIV), another globally important virus, is known to dysregulate the immune system with the loss of T cell proliferation, shifting Th1 cells to Th2 cells and associated cytokines, and high levels of secreted IL-10 (122)(123)(124)(125)(126). The HIV Tat protein has been shown to play a role in the host immune system modulation by acting at the cell surface to stimulate cytokine secretion, particularly IL-10, on monocytes and macrophages (127,128). The N-terminal sequence of Tat was reported to interact with TLR4 (129). ...
Respiratory viral infections have been a long-standing global burden ranging from seasonal recurrences to the unexpected pandemics. The yearly hospitalizations from seasonal viruses such as influenza can fluctuate greatly depending on the circulating strain(s) and the congruency with the predicted strains used for the yearly vaccine formulation, which often are not predicted accurately. While antiviral agents are available against influenza, efficacy is limited due to a temporal disconnect between the time of infection and symptom development and viral resistance. Uncontrolled, influenza infections can lead to a severe inflammatory response initiated by pathogen-associated molecular patterns (PAMPs) or host-derived danger-associated molecular patterns (DAMPs) that ultimately signal through pattern recognition receptors (PRRs). Overall, these pathogen-host interactions result in a local cytokine storm leading to acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS) with concomitant systemic involvement and more severe, life threatening consequences. In addition to traditional antiviral treatments, blocking the host’s innate immune response may provide a more viable approach to combat these infectious pathogens. The SARS-CoV-2 pandemic illustrates a critical need for novel treatments to counteract the ALI and ARDS that has caused the deaths of millions worldwide. This review will examine how antagonizing TLR4 signaling has been effective experimentally in ameliorating ALI and lethal infection in challenge models triggered not only by influenza, but also by other ALI-inducing viruses.
... Although we and others have shown by ELISA that Tat induces TNFα release, early studies reported that Tat induced TNFα and β production through Tat-mediated activation of the TNF-promoter in T-cells and T-cell lines beginning at 8 h and increasing slightly at 16 h and 24 h [47]. Other studies support these findings in monocytes, macrophages, and T-cells [48][49][50][51], and also suggest a role of calcium in the induction of TNFα [52]. Studies by Leghmari et al. show that in a calcium-free environment, the Tat-mediated TNFα production was inhibited; whereas, the Tat-mediated IL-10 production remained intact and involved downstream activation of p38 MAPK [53], indicating a potential overlap with Tat and p38 in IL-10 production, independent of TNFα. ...
Background:
Diseases and disorders with a chronic neuroinflammatory component are often linked with changes in brain metabolism. Among neurodegenerative disorders, people living with human immunodeficiency virus (HIV) and Alzheimer's disease (AD) are particularly vulnerable to metabolic disturbances, but the mechanistic connections of inflammation, neurodegeneration and bioenergetic deficits in the central nervous system (CNS) are poorly defined. The particularly interesting new cysteine histidine-rich-protein (PINCH) is nearly undetectable in healthy mature neurons, but is robustly expressed in tauopathy-associated neurodegenerative diseases including HIV infection and AD. Although robust PINCH expression has been reported in neurons in the brains of patients with HIV and AD, the molecular mechanisms and cellular consequences of increased PINCH expression in CNS disease remain largely unknown.
Methods:
We investigated the regulatory mechanisms responsible for PINCH protein-mediated changes in bioenergetics, mitochondrial subcellular localization and bioenergetic deficits in neurons exposed to physiological levels of TNFα or the HIV protein Tat. Changes in the PINCH-ILK-Parvin (PIP) complex association with cofilin and TESK1 were assessed to identify factors responsible for actin depolymerization and mitochondrial mislocalization. Lentiviral and pharmacological inhibition experiments were conducted to confirm PINCH specificity and to reinstate proper protein-protein complex communication.
Results:
We identified MEF2A as the PINCH transcription factor in neuroinflammation and determined the biological consequences of increased PINCH in neurons. TNFα-mediated activation of MEF2A via increased cellular calcium induced PINCH, leading to disruption of the PIP ternary complex, cofilin activation by TESK1 inactivation, and actin depolymerization. The disruption of actin led to perinuclear mislocalization of mitochondria by destabilizing the kinesin-dependent mitochondrial transport machinery, resulting in impaired neuronal metabolism. Blocking TNFα-induced PINCH expression preserved mitochondrial localization and maintained metabolic functioning.
Conclusions:
This study reported for the first time the mechanistic and biological consequences of PINCH expression in CNS neurons in diseases with a chronic neuroinflammation component. Our findings point to the maintenance of PINCH at normal physiological levels as a potential new therapeutic target for neurodegenerative diseases with impaired metabolisms.
... This suggests that HIV-1 Tat may have the ability to enhance genes that present a TATA-box promoter element. While the HIV Tat peptide per se has the ability to stimulate a diversity of genes, both in vivo (15,26,27,30), and in vitro (31)(32)(33)(34)(35)(36)(37), the contribution of the TATA-box promoter element to upregulated heterologous transcripts has not been examined. ...
Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.
... In this model, in contrast to the mode of action of gp120, the absence of calcium is found to block TNF-α completely, while IL-10 production in Tat-treated monocytes was partially blocked 86 . In addition, in this model, it has been observed that PKC, but not PKA nor MAP kinases ERK1/2, is required for Tat-induced TNF-α and IL-10 production 18,82,87 . Moreover, an inverted involvement was found, where PKA and MAP kinases ERK1/2, but not PKC, pathways seemed to be essential for the production of TNF-α and IL-10 by gp120-stimulated monocytes. ...
Abstract Human HIV-1 infection leads inevitably to a chronic hyper-immune-activation. However, the nature of the targeted receptors and the pathways involved remain to be fully elucidated. We demonstrate that X4-tropic gp120 induced the production of TNF-α and IL-10 by monocytes through activation of a cell membrane receptor, distinct from the CD4, CXCR4, and MR receptors. Gp120 failed to stimulate IL-10 and TNF-α production by monocytes in Ca2+ free medium. This failure was total for IL-10 and partial for TNF-α. However, IL-10 and TNF-α production was fully restored following the addition of exogenous calcium. Accordingly, addition of BAPTA-AM and cyclosporine-A, fully and partially inhibited IL-10 and TNF-α respectively. The PKA pathway was crucial for IL-10 production but only partially involved in gp120-induced TNF-α. The PLC pathway was partially and equivalently involved in gp120-induced TNF-α and IL-10. Moreover, the inhibition of PI3K, ERK1/2, p38 MAP-kinases and NF-κB pathways totally abolished the production of both cytokines. In conclusion, this study revealed the crucial calcium signaling pathway triggered by HIV-1 gp120 to control the production of these two cytokines: TNF-α and IL-10. The finding could help in the development of a new therapeutic strategy to alleviate the chronic hyper-immune-activation observed in HIV-1 infected patients.
... As previously demonstrated by our group and others (4,21,22,(33)(34)(35)(36)(37), we showed that the HIV-1 Tat protein activates the pro-duction of TNF-␣ and IL-10 by human monocytes (Fig. 1A). This action is specific to the Tat protein and is not due to possible contaminants in HIV-1 Tat protein preparations. ...
Importance:
In this study, we demonstrated that by recruiting TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both MyD88 and TRIF pathways and to the activation of PKC-βII, MAP kinases and NF-κB signalling, to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it could be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.
... Thus, TNF production by infected cells might be triggered by a Vpr-independent TNF pathway involving TAK1 and DDB1, a phenomenon that would be enhanced by the presence of Vpr. TNF production is known to be additionally triggered by other viral proteins such as Tat or gp41 (110)(111)(112). Nef has also been reported to increase exosomal release by infected cells, leading to a processing of pro-TNF to TNF (97,98). ...
Importance:
The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or on the contrary prevents pro-inflammatory and antiviral immune responses. In this study, we show that Vpr promotes the release of TNF, a pro-inflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This study provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.
... We also confirmed that TNF-a is significantly increased during HIV-1 infection in primary CD4+ T-lymphocytes ( Fig. S7 in File S1). Interestingly, it has been reported that HIV-1 Tat protein induces the release of TNF-a in different types of cells [60,61]. In this report, we found that TNF-a induced during HIV-1 infection play a key role in HIV-1-induced necroptosis. ...
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by progressive depletion of CD4+ T lymphocytes and dysfunction of the immune system. The numbers of CD4+ T lymphocytes in the human body are maintained constantly by homeostatic mechanisms that failed during HIV-1 infection, resulting in progressive loss of CD4+ T cells mainly via apoptosis. Recently, a non-apoptotic form of necrotic programmed cell death, named necroptosis, has been investigated in many biological and pathological processes. We then determine whether HIV-1-infected cells also undergo necroptosis. In this report, we demonstrate that HIV-1 not only induces apoptosis, but also mediates necroptosis in the infected primary CD4+ T lymphocytes and CD4+ T-cell lines. Necroptosis-dependent cytopathic effects are significantly increased in HIV-1-infected Jurkat cells that is lack of Fas-associated protein-containing death domain (FADD), indicating that necroptosis occurs as an alternative cell death mechanism in the absence of apoptosis. Unlike apoptosis, necroptosis mainly occurs in HIV-infected cells and spares bystander damage. Treatment with necrostatin-1(Nec-1), a RIP1 inhibitor that specifically blocks the necroptosis pathway, potently restrains HIV-1-induced cytopathic effect and interestingly, inhibits the formation of HIV-induced syncytia in CD4+ T-cell lines. This suggests that syncytia formation is mediated, at least partially, by necroptosis-related processes. Furthermore, we also found that the HIV-1 infection-augmented tumor necrosis factor-alpha (TNF-α) plays a key role in inducing necroptosis and HIV-1 Envelope and Tat proteins function as its co-factors. Taken together,necroptosis can function as an alternative cell death pathway in lieu of apoptosis during HIV-1 infection, thereby also contributing to HIV-1-induced cytopathic effects. Our results reveal that in addition to apoptosis, necroptosis also plays an important role in HIV-1-induced pathogenesis.
... HIV-1 infection is associated with an increase of immunosuppressive factors such as IL-10 (64), PD-1/ PD-L1 T-cell coinhibitory pathway (65), and IDO (66), which in turn leads to establishment of an immunosuppressive state. Since HIV-1 Tat protein, used in either recombinant or synthetic form, has been (i) portrayed as an immunosuppressive factor by several reports, including ours (33,40,(67)(68)(69)(70), and (ii) shown to affect DC maturation (37,71), it was of interest to determine whether HIV-1 Tat would also lead to induction of the maturation marker (CD83) or costimulatory (CD80 and CD86) and coinhibitory (PD-1/PD-L1/PD-L2) molecules on DCs. ...
Unlabelled:
Chronic human immunodeficiency virus type 1 (HIV-1) infection is associated with induction of T-cell coinhibitory pathways. However, the mechanisms by which HIV-1 induces upregulation of coinhibitory molecules remain to be fully elucidated. The aim of the present study was to determine whether and how HIV-1 Tat protein, an immunosuppressive viral factor, induces the PD-1/PD-L1 coinhibitory pathway on human dendritic cells (DCs). We found that treatment of DCs with whole HIV-1 Tat protein significantly upregulated the level of expression of PD-L1. This PD-L1 upregulation was observed in monocyte-derived dendritic cells (MoDCs) obtained from either uninfected or HIV-1-infected patients as well as in primary myeloid DCs from HIV-negative donors. In contrast, no effect on the expression of PD-L2 or PD-1 molecules was detected. The induction of PD-L1 on MoDCs by HIV-1 Tat (i) occurred in dose- and time-dependent manners, (ii) was mediated by the N-terminal 1-45 fragment of Tat, (iii) did not require direct cell-cell contact but appeared rather to be mediated by soluble factor(s), (iv) was abrogated following neutralization of tumor necrosis factor alpha (TNF-α) or blocking of Toll-like receptor 4 (TLR4), (v) was absent in TLR4-knockoout (KO) mice but could be restored following incubation with Tat-conditioned medium from wild-type DCs, (vi) impaired the capacity of MoDCs to functionally stimulate T cells, and (vii) was not reversed functionally following PD-1/PD-L1 pathway blockade, suggesting the implication of other Tat-mediated coinhibitory pathways. Our results demonstrate that HIV-1 Tat protein upregulates PD-L1 expression on MoDCs through TNF-α- and TLR4-mediated mechanisms, functionally compromising the ability of DCs to stimulate T cells. The findings offer a novel potential molecular target for the development of an anti-HIV-1 treatment.
Importance:
The objective of this study was to investigate the effect of human immunodeficiency virus type 1 (HIV-1) Tat on the PD-1/PD-L1 coinhibitory pathway on human monocyte-derived dendritic cells (MoDCs). We found that treatment of MoDCs from either healthy or HIV-1-infected patients with HIV-1 Tat protein stimulated the expression of PD-L1. We demonstrate that this stimulation was mediated through an indirect mechanism, involving tumor necrosis factor alpha (TNF-α) and Toll-like receptor 4 (TLR4) pathways, and resulted in compromised ability of Tat-treated MoDCs to functionally stimulate T-cell proliferation.
... Protein kinase 3, or Protein kinase C (PKC) is a member of the family of serine/threonine kinases that are integrally involved in key cellular signaling pathways and can phosphorylate a wide variety of substrates. Not surprisingly, HIV-1 infection alters the PKC phosphorylation pathway to stimulate TNF-α production by monocytes as well as other cytokines and growth factors such as IL-6, IL-10, and MCP-13536373839 . PKC has also been shown to be necessary for HIV-1 Tat-mediated transactivation as well as directly phosphorylating Tat at serine 46 [40,41] and plays an integral role in the signaling and secretion of cytokines in response to HIV-1 envelope proteins gp120, gp160, and gp41 [42,43]. ...
The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum.
Here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs). Here we have identified unique serum proteins that are differentially expressed in LTNP HIV-1 patients and may contribute to the ability of these patients to combat HIV-1 infection in the absence of HAART. We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.
Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.
... (Albini et al., 1998;Badou et al., 2000;Conaldi et al., 2002;Vellutini et al., 1995) although BIV Tat has been reported to have an apoptotic effect in vitro (Xuan et al., 2008). Since the CA/Tat polyprotein was used as a vaccine (Chapters 5 and 6), the possible proliferative effect of the Tat component needs further investigation to determine if the polyprotein would need to be further modified by oxidization or mutated to remove all Tat activity whilst retaining immunogenicity, as has been attempted with HIV Tat (Bennasser et al., 2002;Caselli et al., 1999;Noonan et al., 2003). ...
embrana disease virus (JDV) is a lentivirus associated with an acute disease syndrome of Bali cattle (Bos javanicus) in Indonesia. Control of Jembrana disease currently involves serological monitoring of infection in endemic areas, restriction of movement of cattle from these areas and ring-vaccination around outbreaks of the disease with a tissue-derived inactivated-virus vaccine.
Earlier investigations at Murdoch University resulted in a vaccine combining a mix of recombinant capsid (CA) and Tat proteins that ameliorated the clinical signs of JDV infection in vaccinated cattle. This thesis reports the development of an alternative novel fused CA and Tat polyprotein antigen emulsified in incomplete Freund’s adjuvant (IFA) as a vaccine. This polyprotein could be produced in a single operation at a lower cost than the mix of the 2 individually produced recombinant proteins. The effect of vaccination with this CA and Tat polyprotein vaccine was compared to vaccination with a mixture of individual CA and Tat proteins in small groups of cattle that were subsequently challenged with virulent JDV. It was found that the fused polyprotein vaccine elicited a greater antibody response against both the CA and Tat antigens than a vaccine containing the mix of individual recombinant proteins. In cattle vaccinated with the fused recombinant polyprotein the viral load and lymphocyte response to infection was similar to that in cattle vaccinated with the mix of individual proteins. When the CA and Tat polyprotein vaccine was administered to cattle under field conditions, the vaccine induced minimal side effects and an antibody response to both CA and Tat proteins that persisted for 12 months.
A recombinant protein antigen was produced for ELISA and Western immunoblotting assays that enabled serological detection. This antigen provided an alternative to the whole virus antigen that is currently used in Indonesia for these serological assays. The whole virus antigen is prepared from plasma of JDV-infected cattle using a differential centrifugation technique and is difficult to produce in Indonesia. It was found that a recombinant full length CA (p26) protein antigen used in an ELISA provided sensitivity and specificity equivalent to that obtained by Western immunoblotting with the whole virus antigen. This recombinant CA protein antigen is now used in routine serological assays for detection of JDV in Indonesia.
... HIV TAT is known to accumulate within the nuclei of infected cells and is also secreted into the plasma of HIV-infected patients where it can exert its effects on uninfected bystander cells. Secreted TAT can act in an autocrine or paracrine manner on neighboring cells, altering their normal functionality directly through its activity on cells (Zauli, 1995), or indirectly through regulating the expression of proinflammatory mediators like TNF-α and IL-1β (Bennasser, 2002;Kinga, 2006). The molecular mechanism underlying TAT-mediated biological effects remains a major subject in AIDS pathogenesis research. ...
Human immunodeficiency virus TAT plays an important role in the disregulation of cytokine production associated with the neurological disorders that follow HIV infection. IL-1beta is one of the important inflammatory cytokines secreted by immune-activated monocytes/macrophages. Previous reports have shown that extracellular TAT stimulates IL-1beta expression in monocytes/macrophages. However, little is known about the mechanisms and possible TAT-responsive elements within the IL-1beta promoter. The present study shows that TAT increases the production of IL-1beta in human monocytes; PLC-PKC pathway-dependent phosphorylation of p44/42 and JNK MAP kinases participates partially in IL-1beta induction by TAT; specific C/EBP and NF-kappaB transcription factor binding elements within the IL-1beta promoter are involved in TAT regulation of IL-1beta production. This study identifies a signaling mechanism for HIV-1-induced IL-1beta production in human monocytes that may be involved in the neuropathogenesis of HIV-associated dementia.
... Viral impact on signaling is frequently studied by investigating discrete pathways in cells treated with an isolated viral component, for example Env gp120 [9,11,56], Tat [57,58], Nef [34,59], Vpr [60], or synthetic RNA corresponding to subgenomic regions of HIV-1 [18]. While these studies have provided a framework for network analysis, our global strategy builds insight through the use of whole virus replication to assess the coordinate impact on multiple cell signaling pathways by viral gene expression during spreading infection. ...
Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1). Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection.
To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK), and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR) pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1beta, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication.
HIV-1 induced a primed, proinflammatory state, M1(HIV), which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic approaches.
... The human immunodeficiency virus type 1 (HIV-1) genome encodes several small regulatory proteins during the course of infection whose functions are beneficial for the overall replication of the viral genome and progression of the acquired immune deficiency syndrome (AIDS) (Douek et al., 2003; Lambotte et al., 2003; Persaud et al., 2003; Phimister, 2003 ). One of the moststudied members of this family of proteins is Tat, which plays a pivotal role in stimulating transcription of the viral genome through a mechanism that includes its association with the viral transcript and several host basal and inducible transcription factors (Bennasser et al., 2002; Berkhout et al., 2002; Esposito et al., 2002; Reynolds et al., 2003). In addition, Tat has been shown to alter several key host regulatory events by affecting the expression of various cytokines and immunomodulators such as TNFa, IL-2, and TGFb, which accelerate the viral infection cycle (Buonaguro et al., 1992; Chen et al., 1997; Sawaya et al., 1998; Rappaport et al., 1999; Gonzalez et al., 2001). ...
Tat is an early regulatory protein of human immunodeficiency virus type 1, which plays a central role in the pathogenesis of AIDS by stimulating transcription of the viral genome and impairing several important cellular pathways during the progression of the disease. Here, we investigated the effect of Tat on cell response to DNA damage. Our results indicate that Tat production causes a noticeable increase in the survival rate of PC12 cells upon their treatment with genotoxic agents. Single-cell gel electrophoresis studies revealed reduced DNA breakage in PC12-Tat cells upon cisplatin treatment relative to the control cells. Furthermore, cytogenetic data exhibited less chromosomal damage in Tat-producing cells after recovery from cisplatin treatment, corroborating electrophoretic data. Examination of several proteins involved in the control of DNA repair showed elevated levels of Rad51, a key regulator of homologous recombination in cells expressing Tat. On the other hand, the level of Ku70, one of the components of the nonhomologous end-joining repair pathway, was slightly decreased in cells expressing Tat. Using a fluorescence-based assay, we demonstrated that repair of DNA double-strand breaks via homologous recombination is increased in Tat-producing cells. The results from in vitro nonhomologous end-joining assay revealed a reduced ability of protein extract from PC12-Tat cells compared to PC12 cells in rejoining linearized DNA. These observations ascribe a new role for Tat in host genomic integrity, perhaps by affecting the expression of genes involved in DNA repair.
... Transient exposure of monocytes or astrocytes to Tat for periods as brief as 5 min is sufficient to induce secretion of cytokines from these cells for extended periods. Involvement of two different signaling pathways, the PKC pathway and the calcium pathway, could stimulate cytokine secretion from cells when exposed to Tat [69]. Additionally, exposure of Tat in the order of only milliseconds is sufficient to induce prolonged depolarization in neurons leading to neurotoxicity [70]. ...
Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.
Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers.
Our report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.
... Tat upregulates the expression of IL-2, IL-8, and IL-10 in T-lymphoblastic cell lines, 59-62 IL-6 in HeLa and B-lymphoblastoid cells, 63,64 and tumor necrosis factor-a (TNFa) in monocytes. 65 Tat also upregulates the IL-4R on Raji cells, a B-lymphoblastoid cell line. 66 Although Tat has been shown to suppress IL-2R a-chain (CD25) expression on the H9 T-lymphoid cell line, 67 it seems to have no effect on the expression of this receptor on primary T-cells isolated from healthy donors. ...
We have previously shown decreased expression of the interleukin (IL)-7 receptor alpha-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.
... HIV-Tat mediates its biological effects by activating a multitude of signaling pathways and transcription factors. For example, Tat induces the activation of MAPK, including JNK, p38, and ERK, PI3K, and calcium signaling pathways (36,(41)(42)(43)(44). These signaling cascades are believed to be activated following interaction of Tat with various cell surface receptors including integrin receptors, members of the vascular endothelial growth factor receptor family (45,46), the -chemokine receptors (CCR2 and CXCR4 (47,48)), as well as with integrins ␣ 5  1 and ␣ v  3 (49,50). ...
The anti-inflammatory cytokine, IL-10 plays an important role in HIV immunopathogenesis. The HIV accessory protein, Tat is not only critical for viral replication, but affects the host immune system by influencing cytokine production including IL-10. During HIV infection, IL-10 production by monocytic cells is up-regulated, representing a critical pathway by which HIV may induce immunodeficiency. Herein, we show that extracellular Tat-induced IL-10 expression in normal human monocytes. To understand the signaling pathways underlying HIV-Tat induced IL-10 transcription, we investigated the involvement of MAPK as well as calcium signaling and the downstream transcription factor(s). Our results suggest that Tat-induced calcium influx regulated IL-10 transcription in monocytic cells. The experiments designed to further understand the molecules involved in the calcium signaling suggested that calmodulin and calmodulin-dependent protein kinase-II (CaMK-II)-activated p38 MAPK played a role in extracellular Tat-induced IL-10 expression in primary human monocytes. Furthermore, Tat-induced IL-10 expression was regulated by p38 MAPK- and CaMK II-activated CREB-1 as well as Sp-1 transcription factors. Taken together, our results suggest that extracellular HIV-Tat induced IL-10 transcription in primary human monocytes is regulated by CREB-1 and Sp-1 transcription factors through the activation of calmodulin/CaMK-II-dependent p38 MAPK.
... For instance, the SupT1 cell line represents an early stage in T cell development and these CD4 + CD8 + cells are relative rare in PBMC, and even absent in our CD8 + -depleted CD4 + preparations . Alternatively, a high level of endogenous NF-kB or activation of the NF-kB pathway by endogenous TNFα production by monocytes, which are still present in the T cells preparations [49], could explain the lack of a TNFα effect in these assays. In case of the HTLV-1 transformed cell lines, the absence of a TNFα response could also be the result of expression of the viral Tax trans-activator protein , which blocks TNFα activation505152535455. ...
Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication.
We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFalpha treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings.
We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.
In HIV positive patients, a dysfunction of the immune system, linked to the progressive disturbance of the cytokine network, is observed well before the decrease in TCD4+ lymphocyte number. This dysfunction is mainly due to the action of viral proteins such as Tat, a protein necessary for viral genome transcription. Secreted by infected cells, Tat protein can be captured and internalized by other cells, whether they are infected or not, in which it can exert many functions including apoptosis or modulation of the expression of numerous cytokines and membrane receptor genes. Thus, for example, in HIV positive patients, an increase in the level of IL-10, an immunosuppressive Th2 cytokine, and TNF-α, a proinflammatory cytokine involved in HIV associated dementia, during the progression of HIV infection towards AIDS. By acting at membrane receptor, Tat protein induces TNF-α and IL-10. This activation is mediated by the N-Terminal domain 1-45 of Tat protein. Signal transduction analysis showed that the protein kinase C is crucial for this induction. Among the 8 PKC isoforms present in human monocyte/macrophages Tat activates PKC-β and δ Downstream from PKC, Tat activates ERK1/2 and P38 MAPkinases and NF-kB whose activation is necessary for IL-10 and TNF-α activation. In addition to these pathways, Tat protein induces in monocytes/macrophages a calcium signal by mobilizing calcium from extracellular stores. This stimulation is mediated via the 20-45 domain of Tat and is totally blocked in the presence of DHP receptor inhibitors such as nimodipine or calcicludine. Interestingly we showed that Tat-induced calcium mobilization is tightly linked to TNF-α production, thus indicating that Tat induced mobilization and TNF-α production are entirely mediated by DHP receptors. Thus, Tat by acting on infected and non infected cells activates multiple pathways leading to both AIDS and neurological disease progression.
Retroviruses are RNA viruses that infect birds and mammals. They can be divided into two categories: simple, which contain three main reading frames (gag, pol, env), and complex, which also code for regulatory and accessory proteins essential in viral replication, e.g., Tax and Rex in human T-cell leukemia virus (HTLV), or Tat and Rev in human immunodeficiency virus (HIV). Two terminal non-coding sequences at both ends of the genome, which contain consensus sites specific to cellular and viral transcription factors, act as promoter regions. Retroviruses, as other viruses, lack an independent metabolism and are unable to replicate outside living cells. As a result, their gene expression is regulated by both viral and cellular factors. However, the key feature that differentiates retroviruses from other viruses is that they encode the enzyme reverse transcriptase, which synthesizes a double-stranded DNA copy of the viral genome. Viral DNA is integrated into the host genome as a provirus and can induce an active viral production or a post-integration latency. The provirus acts as a host gene and can be transmitted to the progeny cells. The interplay of the viral genome with the host metabolic machinery involves modifications in both gene expression and regulation. In fact, retroviruses have adapted themselves to use this machinery while maintaining cell integrity, which is essential to preserve their survival. Consequently, there can be variable host pathogenicity associated with several diseases-such as malignancies, immunodeficiencies, and neurological disorders-due to the down- or up-regulation of different cellular genes. For example, the HTLV-1 protein Tax modifies cell proliferation by activating the expression of interleukin receptors and cytokines. Moreover, retroviruses also isolate the infected cell by modifying the expression of surface receptor or molecules essential for cell communication. For example, the HIV-1 protein Nef down-regulates the expression of CD4 receptors in T cells, and therefore contributes to the overall immunodeficiency and the onset of superinfections caused by opportunistic pathogens. Retroviruses are also able to modulate gene expression through direct regulation of the transcriptional machinery. For example, in human acute promyelocytic leukemia (APL), chromosomal translocations and mutations in nuclear hormone receptors yield oncoproteins that alter chromatic structure and deregulate transcription. A better understanding of retroviral gene expression regulation is essential to develop prevention and therapeutic strategies. However, the variability of the viral targets and the strong dependence of viral replication on the host factors are major concerns.
HIV type 1 (HIV-1) is a lentivirus that causes the global acquired immunodeficiency disease pandemic. HIV-1 infection of CD4+ lymphocytes and macrophages modulates signal transduction pathways and immune response. Although rarely oncogenic itself, HIV-1 exacerbates clinical disease course in individuals dually infected by HIV-1 and hepatitis C virus, human papilloma virus, Epstein-Barr virus, or human herpesvirus-8. No vaccines or treatments to prevent or cure HIV-1 infection are available. Combinations of antiretroviral drugs reduce perinatal infection and HIV-1 associated morbidity and mortality. Increased survival among HIV-1 infected adults and children could lead to an increase in malignancies owing to chronic immune suppression, coinfection by oncogenic viruses, and/or persistent HIV-1 infection.
The lives of individuals infected with HIV who have access to combination antiretroviral therapy (cART) are substantially prolonged, which increases the risk of developing non-AIDS comorbidities, including coronary heart disease (CHD). In Europe and the USA, individuals with HIV infection have a ∼1.5-fold increased risk of myocardial infarction relative to uninfected individuals. In Africa, the relative risk of myocardial infarction is unknown, but broadened access to life-extending cART suggests that rates of CHD will rise in this and other resource-constrained regions. Atherogenesis in HIV is affected by complex interactions between traditional and immune risk factors. cART has varied, regimen-specific effects on metabolic risk factors. Overall, cART seems to lessen proatherogenic immune activation, but does not eliminate it even in patients in whom viraemia is suppressed. Current strategies to decrease the risk of CHD in individuals infected with HIV include early initiation of cART regimens with the fewest metabolic adverse effects, and careful management of traditional CHD risk factors throughout treatment. Future strategies to prevent CHD in patients with HIV infection might involve the use of HIV-tailored CHD risk-prediction paradigms and the administration of therapies alongside cART that will further decrease proatherogenic HIV-specific immune activation.
The deaths of neurons, astrocytes and endothelial cells have been described in patients with HIV (human immunodeficiency virus)
dementia. HIV-1 does not infect neurons; instead, neurotoxic substances shed by infected glia and macrophages can induce a
form of programmed cell death called apoptosis in neurons. These neurotoxins include the HIV-1 proteins Tat and gp120, as
well as proinflammatory cytokines, chemokines, excitotoxins and proteases. In this article we review the evidence for apoptosis
of various cell types within the brain of HIV-infected patients, and describein vitro andin vivo experimental studies that have elucidated the mechanisms by which HIV causes apoptosis of brain cells.
The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-κB-dependent pathways. The trans-dominant negative mutants of NF-κB-inducing kinase (NIK), IKKα and IKKβ expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-κB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-κB pathways. In inactivated cells, IKKα is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKα translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKα and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-κB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKα nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKα pathways, to promote production of IL-10.
The dsRNA-dependent protein kinase, PKR, is a central component in antiviral defense. The biological importance of PKR is further remarked by its critical role in apoptosis induced by a variety of stresses. Here, we analyzed the implication of oxidative stress in the induction of PKR-dependent apoptosis in Jurkat cells. Our results revealed that reactive oxygen species (ROS) induced endogenous pkr gene expression at the transcriptional level by activating the interferon (IFN)-gamma gene. However, IFN-gamma siRNA expression abrogated the H(2)O(2)-mediated pkr induction. The radical scavenger N-acetyl-l-cysteine profoundly inhibited pkr induction via the reduction of IFN-gamma expression. The treatment of cells with the specific JAK-STAT inhibitor, AG490, reduced the PKR expression, and suppressed PKR-dependent cell death. Finally, siRNA-mediated depletion of IFN-gamma or pkr efficiently downregulated H(2)O(2)-mediated apoptotic cell death. These results indicated that oxidative stress induces PKR expression essentially via the IFN-gamma activation signal, and causes apoptosis in Jurkat T cells.
In this study, we demonstrate that HIV-1 Tat protein is able to induce IL-10 and TNF-alpha in human macrophages. We show that N-terminal Tat 1-45 fragment initiates the PKC pathway by acting at the membrane. Inhibition of PKC pathway, by chemical inhibitors or after PMA treatment, abolishes both IL-10 and TNF-alpha production. Among the eight PKC isoforms present in macrophages, we show that only PKC-betaIotaIota and -delta are activated by Tat or Tat 1-45 in human macrophages. However, their selective inhibition affects only IL-10 production. Downstream of PKC, Tat activates the MAP kinases p38 and ERK1/2 and the transcription factor NF-kappaB. Using chemical inhibitors we show that (i) both ERK1/2 MAP kinase and NF-kappaB transcription factor play an important role in IL-10 and TNF-alpha production, in macrophages stimulated by Tat. However, p38 MAP kinase seems to be involved only in IL-10 and not TNF-alpha production.
The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.
The Tat regulatory protein of human immunodeficiency virus type 1 (HIV-1) is secreted by infected cells and plays a key role in viral pathogenesis and replication. Tat protein has been proposed as a target antigen for vaccine design since anti-Tat antibodies may interfere with virus spread and disease progression. The aim of this study was to analyse the serum antibody response of mice, rabbits, macaques and humans immunized with recombinant Tat, synthetic Tat, Tat toxoid or Tat peptides and to examine the biological properties of these antibodies in terms of Tat-induced transactivation and HIV-1 replication. Only sera with antibody specificity to both N-terminal and basic functional domains were able to inhibit extracellular Tat-dependent transactivation significantly in vitro. Antibodies from a human subject immunized with Tat also reduced HIV-1 replication in acutely infected T cells and blocked reactivation of virus replicating low levels in chronically infected cells by exogenous Tat. These results demonstrate that immunization with Tat protein or a combination of synthetic Tat peptides elicits the production of Tat-neutralizing serum antibodies and suggest that Tat vaccination could be used to block in vivo extracellular Tat autocrine/paracrine transactivation of HIV-1 replication.
The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations.
HIV-1 Tat protein, acting at the cell membrane, stimulates the production by human monocytes of TNF-alpha, a cytokine implicated in both HIV-1 replication and pathogenesis. Here, we analyze, in primary human monocytes, the mechanisms involved in Tat-stimulated calcium mobilization and its relationship with TNF-alpha production. We show that the Tat protein induces a calcium signal by mobilizing calcium from extracellular stores. This calcium signal is totally blocked when cells are stimulated in the presence of DHP receptor inhibitors such as nimodipine or calcicludine, thus suggesting the implication of this L-type calcium channel. By using RT-PCR amplification, Western blot with antibodies directed against the alpha1D subunit, binding assays with specific agonists or antagonists, and inhibition with specific antisense oligonucleotides, we show that DHP receptors are expressed and functional in primary human monocytes. Interestingly, we demonstrate that Tat-induced calcium mobilization is tightly linked to TNF-alpha production, thus indicating that Tat-induced mobilization and TNF-alpha production are entirely mediated by DHP receptors, as shown by their total inhibition by nimodipine, calcicludine, or anti-alpha1D antisense oligonucleotides.
The transactivator of transcription (Tat) protein is essential for efficient HIV type 1 (HIV-1) replication and is involved in the transcriptional regulation of the host immune response gene, TNF. In this study, we demonstrate that Tat proteins from representative HIV-1 subtype E isolates, but not from subtypes B or C, selectively inhibit TNF gene transcription and protein production in CD4(+) Jurkat T cells. Strikingly, we show that this repression is due to a tryptophan at residue 32 of Tat E and is secondary to interference with recruitment of the histone acetyltransferase P/CAF to the TNF promoter and with chromatin remodeling of the TNF locus. This study presents a novel mechanism by which HIV-1 manipulates a host immune response gene that is important in its own replication. Moreover, these results demonstrate a new mechanism by which the TNF gene is regulated via chromatin remodeling secondary to viral infection.
To know why HIV-1-infected persons are particularly susceptible to amebic liver abscess (ALA), we investigated the role of CD4CD25 T cells in the susceptibility of HIV-1-infected persons to this disease. Herein we show, in early stage HIV-1-infected subjects, that CD4 T-cell responses to Entamoeba histolytica antigen (EhAg) were selectively impaired, especially in those with ALA. EhAg-specific CD4 T-cell responses were normalized by depletion of CD4CD25 cells or by addition of anti-cytotoxic T lymphocyte antigen 4 (CTLA4) antibody. Regulatory activity of CD4CD25 T cells to suppress the EhAg-specific CD4 T-cell response could be induced by EhAg-primed dendritic cells (DCs) in HIV-1-infected subjects, especially in those with ALA, but not in healthy controls. Exogenous Tat-incubated DCs derived from HIV-negative subjects also could upregulate CTLA4 expression on autologous CD4CD25 T cells and selectively suppress the EhAg-specific CD4 T-cell response. The results imply an interaction of the two pathogens: HIV-1, perhaps through the effect of Tat on DCs, may upregulate EhAg-specific regulatory T-cell activity to suppress T-cell response to E. histolytica, thus increasing the susceptibility to invasive amebiasis in even early-stage HIV-1-infected persons.
Human immunodeficiency virus-1 (HIV-1) infection is characterized by chronic immune activation and progressive loss of CD4+ T cells, leading to a wide array of immune dysfunction, particularly involving immune response directed against viral antigens. HIV-1 encodes for fifteen proteins, which might serve as a target for immune recognition. Immune response to the envelope proteins have been studied more due to their presence on the surface of the virus. Recent studies on HIV vaccine development have focused on the Gag and Pol proteins. The transactivator Tat and Rev proteins have also been the focus of immunization studies due to their potent regulatory activity. The Tat (transactivator of transcription) protein although being nuclear in localization is also released from infected cells and acts on uninfected cells. Extracellular Tat seems to play an important role in AIDS pathogenesis. Furthermore, a correlation has been found between anti-Tat immune response and slow progression of the disease. Although several studies have shown Tat as a potential vaccine candidate with encouraging results, there are also reports raising doubt about its efficacy in multi-component HIV vaccine strategy. Here, we have addressed the issue of immune response to the most indispensable HIV-1 regulatory protein Tat.
HIV-1 Tat protein stimulates the production of both TNF-alpha and IL-10 in human monocytes. Taking into account the ability of TNF-alpha to induce IL-10 production, we evaluated the link between Tat, TNF-alpha and IL-10 and the implication of PKC and p38 MAP kinase pathways. Our data showed that (i) in the presence of neutralizing anti-TNF-alpha antibodies, IL-10 production is only partially inhibited; (ii) in a calcium-free medium, while TNF-alpha production is totally inhibited, Tat continues to induce IL-10; (iii) under these conditions, Tat-mediated IL-10 production is associated with PKC-delta activation; and (iv) downstream of PKC, p38 MAP kinase is crucial for TNF-alpha independent IL10 production. Overall, our data suggest a new mechanism, implicating Tat protein, by which HIV-1 may maintain a constant production of the immunosuppressive IL-10 cytokine, even in the absence of TNF-alpha production. In consequence, HIV-1 may escape immune surveillance and thus promote the establishment of an immunosuppressive state.
The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 125I-tat38-86 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 microM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.
The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.
Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes
to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma
cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a
corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular
signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic
acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the
addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by
a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.
Temporal cellular events responsible for hormonal activation of responses mediated by the cAMP-dependent protein kinase (PKA) have been studied in living cells. By selectively perturbing molecular function of Gs, the catalytic subunit of PKA (C), or the nuclear factor CREB, in cells through microinjection of inhibitory agents specific for these molecules or activated forms of these molecules, we have obtained evidence for a requirement for the function of each of these molecules in the hormonal stimulation of cAMP-regulated genes. Moreover, by introducing fluorescently labeled PKA subunits into these cells as molecular tracers, or by immunofluorescence of C subunit, we have observed biological translocation of C subunit from the cytoplasm to the nucleus during transcriptional activation and a quenching of this by the inhibitor molecule, PKI. The implications of these cellular and molecular events in the signal transduction of hormonal responses are discussed.
During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.
Human immunodeficiency virus (HIV) infection may cause a dementing illness. HIV-mediated dementia is clinically and pathologically
correlated with the infiltration of activated macrophages and elevated levels of tumor necrosis factor (TNF)-α, both of which
occur in an environment of small numbers of infected cells. We examined the possibility that HIV protein Tat, which is released
extracellularly from infected cells, may induce the production of TNF-α. Tat induced TNF-α mRNA and protein production dose-dependently,
primarily in macrophages but also in astrocytic cells. The TNF-α induction was NF-κB-dependent and could be eliminated by
inhibiting protein kinase A or protein tyrosine kinase activity. In addition, Tat-induced TNF-α release was also linked to
phospholipase C activation. However, Tat effects were independent of protein kinase C. These observations suggest that Tat
may provide an important link between HIV and macrophage/glial cell activation and suggest new therapeutic approaches for
HIV dementia.
We have previously shown that NF-kappaB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559-5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-kappaB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56(lck) as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56(lck), or both molecules, we provide direct evidence that expression of CD4 and p56(lck) is required for HIV-1-induced NF-kappaB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-kappaB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56(lck) indicates the requirement for a functional CD4-p56(lck) complex.
Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.
The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 125I-tat38-86 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 microM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.
The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38–58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.
Almost exactly ten years following the first publication on NF-κB (Sen and Baltimore 1986), researchers working on transcriptional regulation by NF-κB/Rel and IκB proteins gathered for the third time to discuss recent developments in the field (Madrid, July 8-10, 1996). The first meeting of its kind was a Howard Hughes workshop at the NIH in November 1992 and the second one a Banbury Conference held at Cold Spring Harbor in October 1993. This year's meeting was organized by R. Bravo (Bristol-Myers Squibb, Princeton) and P. S. Lazo (Universidad de Oviedo) and held at the Juan March Foundation in Madrid, Spain.
The HIV−1 Tat protein transactivates HIV, viral and some host cell genes1. Tat can be released by infected cells2 and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells3,4. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor−1 and E selectin5−7). Tat induces growth of Kaposi's sarcoma (KS) spindle cells8 and is angiogenic in vivo 7,9 and in transgenic mice10−12. We previously reported that Tat is a direct angiogenic factor7 and noted the Tat arginine− and lysine−rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor−A (VEGF−A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk−1/kinase insert domain receptor (Flk−1/KDR), a VEGF−A tyrosine kinase receptor (for review see ref. 13), and that Tat−induced angiogenesis is blocked by agents blocking the Flk−1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT−1, another VEGF−A tyrosine kinase receptor.
THE depletion of CD4+ T cells in AIDS is correlated with
high turnover of the human immunodeficiency virus HIV-1,2 and
associated with apoptosis3-5. The molecular mechanism of
apoptosis in HIV infection, however, is largely unknown. T-cell apoptosis might
be affected by viral proteins such as HIV-1 Tat6-9 and gp120
(refs 10, 11). T-cell-receptor (TCR)-induced apoptosis was recently shown to
involve the CD95 (APO-1/Fas) receptor12. We show here that HIV-1
Tat strongly sensitizes TCR- and CD4(gpl20)-induced apoptosis by upregulation of
CD95 ligand expression. Concentrations of Tat found to be effective in cultures
of HIV-1-infected cells were also observed in sera from HIV-1-infected
individuals. Taken together, our results indicate that HIV-1 Tat and gp!20
accelerate CD95-mediated, activation-induced T-cell apoptosis, a mechanism that
may contribute to CD4+ T-cell depletion5,13,14 in AIDS.
Human immunodeficiency virus (HIV) infection may cause a dementing illness. HIV-mediated dementia is clinically and pathologically
correlated with the infiltration of activated macrophages and elevated levels of tumor necrosis factor (TNF)-α, both of which
occur in an environment of small numbers of infected cells. We examined the possibility that HIV protein Tat, which is released
extracellularly from infected cells, may induce the production of TNF-α. Tat induced TNF-α mRNA and protein production dose-dependently,
primarily in macrophages but also in astrocytic cells. The TNF-α induction was NF-κB-dependent and could be eliminated by
inhibiting protein kinase A or protein tyrosine kinase activity. In addition, Tat-induced TNF-α release was also linked to
phospholipase C activation. However, Tat effects were independent of protein kinase C. These observations suggest that Tat
may provide an important link between HIV and macrophage/glial cell activation and suggest new therapeutic approaches for
HIV dementia.
A plethora of mechanisms of action have been proposed for exogenous Tat to explain the pleiotropic, and sometimes controversial, extracellular effects reported for this molecule. Anna Rubartelli and colleagues discuss the molecular bases of these multiple functions.
In this paper Zeda Rosenberg and Anthony Fauci review the prevailing hypotheses on the mechanisms by which human immunodeficiency virus (HIV) progressively and relentlessly destroys immune function in infected individuals. Although HIV can directly kill CD4+ T cells in vitro, the protracted course of HIV infection in vivo suggests that other pathogenic mechanisms are also involved. As a member of the lentivirus family, HIV can remain latent within the genome of the infected cell. Activation of HIV expression from a latent or low-level state of replication is dependent, in part, on the state of activation of the host cell. As a result, activation of HIV-infected CD4+ T cells or monocyte/macrophages during normal immune responses may ultimately result in the activation of HIV expression and spread of the infection. Thus, HIV may have developed the ability to use normal immune processes to its own reproductive advantage.
Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.
Infection with the human immunodeficiency virus (HIV) results in a profound immunosuppression due predominantly to a selective
depletion of helper/inducer T lymphocytes that express the receptor for the virus (the CD4 molecule). HIV also has tropism
for the brain leading to neuropsychiatric abnormalities. Besides inducing cell death, HIV can interfere with T4 cell function
by various mechanisms. The monocyte serves as a reservoir for HIV and is relatively refractory to its cytopathic effects.
HIV can exist in a latent or chronic form which can be converted to a productive infection by a variety of inductive signals.
The depletion of CD4+ T cells in AIDS is correlated with high turnover of the human immunodeficiency virus HIV-1 and associated with apoptosis. The molecular mechanism of apoptosis in HIV infection, however, is largely unknown. T-cell apoptosis might be affected by viral proteins such as HIV-1 Tat and gp120 (refs 10, 11). T-cell-receptor (TCR)-induced apoptosis was recently shown to involve the CD95 (APO-1/Fas) receptor. We show here that HIV-1 Tat strongly sensitizes TCR- and CD4(gp120)-induced apoptosis by upregulation of CD95 ligand expression. Concentrations of Tat found to be effective in cultures of HIV-1-infected cells were also observed in sera from HIV-1-infected individuals. Taken together, our results indicate that HIV-1 Tat and gp120 accelerate CD95-mediated, activation-induced T-cell apoptosis, a mechanism that may contribute to CD4+ T-cell depletion in AIDS.
The transcription factor NF-ATp is a target in activated T cells for the calcium-regulated phosphatase calcineurin, and is therefore a secondary target for the immunosuppressive drugs cyclosporin A and FK506. Here, Anjana Rao describes how NF-ATp cooperates with Fos- and Jun-family proteins to mediate transcription of the interleukin 2 gene, and how it may participate in the calcium-dependent transcription of other cytokine genes in several types of immune-system cells.
The expression of T-cell derived lymphokines is regulated by signal transduction events initiated by the T-cell antigen receptor and other T-cell surface molecules. Substantial progress has been made in characterizing the signal transduction events initiated at the plasma membrane of the T cell and their targets which control lymphokine gene expression in the nucleus. This review will summarize recent progress in this area of investigation.
Following primary HIV infection, a state of chronic, persistent infection fueled by cellular activation usually ensues and the expression of virus overtime is determined by a number of vira factors as well as by upregulatory and down regulatory host factors. Paramount among these host factors are endogenous cytokines that tightly control HIV expression in an autocnne and paracrine manner For example, TNF-a, IL-10, and IL-6, which are overexpressed in lymphoid tissue of HV-infected individuals potently induce HIV replication, and virus replication can be markedly downregulated by blocking these autocnne pathways. Of notera number of factors including the β-chemokines RAMIES. MIP-1 a, and MIP-βdirectly downmodulate virus expression in certain culture systems. These chemokines are secreted by a variety of cell types including CD4+ T cells, monocytes, B cells, as well as CD8+ T cells. Suppression of HIV replication by the β-chemokines is dependent upon the culture system employed as well as the cellular troprsm of the virus. In ceruin culture systems, CD8+ T cell factors other than the β-chemokines are responsible for suppression of HfV replication. The net expression of virus is determined at least in part by the balance of these opposing factors. The real and potential mechanisms of these regulatory phenomena are complex and diverse, and w'fl be discussed.
Although the involvement of protein kinase C (PKC) in the activation of the mitogen-activated protein (MAP) kinase pathway has been implicated through experiments using 12-O-tetradecanoylphorbol-13-acetate (TPA), there has been no direct demonstration that PKC activates the MAP kinase pathway. A Raf-dependent intact cell assay system for monitoring the activation of MAPK/ERK kinase (MEK) and extracellular signal-related kinase (ERK) permitted us to evaluate the role of PKC isotypes in MAP kinase activation. Treatment of cells with TPA or epidermal growth factor resulted in the activation of MEK and ERK. The activation of the MAP kinase pathway triggered by epidermal growth factor was completely inhibited by dominant-negative Ras (RasN17), whereas the activation triggered by TPA was not, consistent with previous observations. The introduction of an activated point mutant of PKCdelta, but not PKCalpha or PKCepsilon, resulted in the activation of the MAP kinase pathway. The activation of MEK and ERK by an activated form of PKCdelta requires the presence of c-Raf and is independent of RasN17. These results demonstrate that activation of PKCdelta is sufficient for the activation of MEK and ERK and that the pathway operates in a manner dependent on c-Raf and independent of Ras.
The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.
The level of human immunodeficiency virus (HIV) replication in patients reflects a balance between stimulatory and inhibitory host factors (particularly endogenous cytokines). New information concerning the cellular co-receptors for HIV and the cellular tropism of different strains of virus will advance our understanding of HIV-induced pathogenesis and suggests new therapeutic and preventive strategies.
HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.
HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.
In HIV-infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with the disease progression toward AIDS. We have previously shown that HIV-1 Tat induces IL-10 production by human monocytes via a protein kinase C (PKC) -dependent pathway. Here we show that PKC activation by Tat is essential for IL-10 induction. Among the eight PKC isoforms present in human monocytes, we investigated which isoform(s) plays this crucial role in Tat-mediated IL-10 production and show that 1) Tat can activate PKC-alpha, PKC-betaII, PKC-delta, and PKC-epsilon, 2) of these four potential candidates, only PKC-betaII, PKC-delta, and PKC-epsilon are activated by the active domain Tat 1-45, which is responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-alpha and PKC-epsilon by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-betaII or PKC-delta partially inhibits IL-10 production; and 4) the simultaneous inhibition of PKC-betaII and PKC-delta totally inhibits Tat-mediated IL-10. Altogether, these results suggest that the induction of IL-10 by Tat is strictly dependent on the PKC-delta and -betaII isoforms.