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Keywords:

  • Tat;
  • c-fos;
  • HIV-1;
  • Jurkat;
  • MAPKinase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

The human immunodeficiency virus (HIV) encodes the highly conserved transcriptional transactivator Tat, which is expressed early in the viral life cycle and plays a pivotal role in viral replication as well as in the progression to overt acquired immune deficiciency syndrome (AIDS) (Cullen, 1993; Jones & Peterlin, 1994). Tat, endogenously expressed by HIV-1-infected cells, exerts its function after binding to a specific RNA target (Dingwall et al, 1989). In particular, Tat protein binds to the transactivation response (TAR) RNA stem-loop located from +1 to +60 in the 5′ of the long-terminal repeat (LTR) viral promoter inducing a massive viral RNA transcription (Feng & Holland, 1988; Marciniak & Sharp, 1991; Cullen, 1993). In addition, endogenous Tat is able to elicit an effective increase of transcriptional RNA viral elongation (Feng & Holland, 1988; Marciniak & Sharp, 1991; Cullen, 1993).

Interestingly, Tat can also induce biological effects in various cell types in a cytokine-like fashion. In fact, it has been shown that Tat can be actively released and detected in the culture supernatants of both acutely HIV-1-infected and tat-transfected cells (Frankel & Pabo, 1988; Ensoli et al, 1993). Moreover, soluble Tat can be rapidly taken up by uninfected cells in which it localizes in both the cytoplasm and nuclear compartments (Frankel & Pabo, 1988; Mann & Frankel, 1991). We, and other authors, have previously demonstrated that, in its extracellular form, Tat protein affects the survival/proliferation of CD4+ T lymphocytes by a paracrine/autocrine loop (Zauli et al, 1995; Zauli & Gibellini, 1996). While Tat inhibits cell proliferation at high (μmol/l-nmol/l) concentrations (Li et al, 1995; Westendorp et al, 1995; Zauli et al, 1996; McCloskey et al, 1997), at lower (pmol/l) physiological concentrations Tat promotes the survival (Gibellini et al, 1995; Zauli et al, 1995) and the proliferation of quiescent CD4+ T cells, in cooperation with anti-CD3 and anti-CD28 monoclonal antibodies (Zauli et al, 1996; Secchiero et al, 2000).

Additional studies established that extracellular Tat elicits an array of intracellular signal transduction pathways in lymphoid T cells, including the p125FAK/PI3-K/Akt, the JNK and the ERK/MAPK pathways (Borgatti et al, 1997; Li et al, 1997; Gibellini et al, 1998; Kumar et al, 1998). Activation of one or more of these pathways ultimately leads to the recruitment of activated nuclear transcription factors, such as CREB (Gibellini et al, 1998) and NF-κB (Demarchi et al, 1996).

In this context, we have previously demonstrated that tat gene endogenously expressed in both lymphoid and monocytic cells upregulates the expression of c-fos (Gibellini et al, 1997), a proto-oncogene, which constitutes the transcription factor AP−1 in association with c-jun. It should be noticed, however, that endogenous tat increased the expression of c-fos only when cells were cultured in the presence of high [15% fetal calf serum (FCS)] serum concentrations and were stimulated with phytohaemagglutinin or phorbol esters.

The aim of this study was to ascertain whether low physiological concentrations of extracellular Tat protein, which have been detected in the body fluids of HIV-1-seropositive individuals (Westendorp et al, 1995), were able to modulate c-fos expression. We also investigated which region of the c-fos promoter was responsive to extracellular Tat and whether the Tat-mediated induction of c-fos led to the formation of the AP-1 complex. This is particularly relevant as AP-1 plays a crucial role in the activation/proliferation of various cell types, including T lymphocytes (Sassone-Corsi et al, 1988; Angel & Karin, 1991; Karin et al, 1997).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Reagents and plasmids Synthetic (Technogen, Caserta, Italy) and recombinant (Intracell, Cambridge, MA, USA) HIV-1 Tat protein and recombinant HIV-1 p24 protein (Intracell) were dissolved in phosphate-buffered saline (PBS) containing 0·1% bovine serum albumin (BSA) and aliquoted at −70°C before use. Stock solutions (10 mmol/l) of PD98059 (Calbiochem, La Jolla, CA, USA) were prepared in dimethyl sulphoxide (DMSO) and stored at −20°C.

The c-fos-promoter–chloramphenicol acetyltransferase (CAT) plasmid constructs (FC2, FC3, FC4, FC8) containing the bacterial CAT gene under the control of various c-fos promoter sequences from position −1450 (FC2), −711 (FC3), −404 (FC4), −220 (FC8) to position +42 have been previously described (Deschamps et al, 1985). These plasmids were a generous gift from Dr Paolo Sassone-Corsi and Dr Enzo Lalli (IGBMC, Illkirch, France). The wild-type c-fos promoter fosCAT, the wild-type c-fos promoter mutated in −60 CRE (M1-fos) or in all three CRE elements (M2-fos) have been described previously (Ginty et al, 1994). These plasmids were a generous gift of Dr Ginty (Department of Microbiology and Molecular Genetics, Harvard, MA, USA). The backbone control vector was represented by the empty pCAT plasmid.

The TRE-CAT plasmid, represented by two TRE sequences cloned in front of CAT in the backbone pBLCAT2 and the pBLCAT2 plasmid itself were a generous gift of Dr Enzo Lalli.

Cell cultures and treatment The human Jurkat CD4+ lymphoblastoid T-cell line was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% FCS (Gibco) at an optimal cell density of 0·5–1 × 106 cells/ml.

In all experiments described below, Jurkat cells were kept in RPMI supplemented with low (2%) FCS for 24 h (serum starvation) before the addition of 10 ng/ml Tat or 10 ng/ml p24 protein for up to 6 h. In control experiments, Tat was pretreated for 2 h at 37°C with 20 μg/ml of a rabbit polyclonal neutralizing anti-Tat antibody (Ab) (Intracell).

In some experiments, Jurkat cells were pretreated for 60 min at 37°C with 10 μmol/l PD98059, a specific ERK/MAPK pharmacological inhibitor, or with equimolar concentration of DMSO diluted in RPMI-1640, before adding 10 ng/ml of Tat or p24 proteins.

Western blot analysis Samples derived from 2 × 106 Jurkat cell lysates, containing approximately 100 μg of proteins, were migrated in 10% acrylamide gels and blotted onto nitrocellulose filters. Blotted filters were blocked for 30 min in a 3% suspension of dried skimmed milk in PBS and incubated overnight at 4°C with anti-c-fos polyclonal rabbit Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:1000 in PBS plus 1% BSA, or with mouse anti-β-tubulin monoclonal Ab diluted 1:1000 plus 1% BSA (Sigma St.Louis, MO, USA). Filters were washed and further incubated for 60 min at room temperature with peroxidase-conjugated goat anti-rabbit immunoglobulins diluted 1:2000 in PBS plus 1% BSA (Sigma) or with peroxidase-conjugated goat anti-mouse immunoglobulins diluted 1:2000 in PBS plus 1% BSA (Sigma). Specific reactions were revealed with the ECL Western blotting detection reagent following manufacturer's instruction (Amersham, Arlington Heights, IL, USA).

Analysis of c-Fos protein by flow cytometry Cells were fixed and permeabilized with cold 70% ethanol for 30 min at 4°C, washed twice with PBS and treated for 90 min at 37°C with anti-c-Fos protein rabbit polyclonal IgG (Santa Cruz, dilution 1:100 in PBS) plus 1% BSA. Non-specific blocking guinea pig serum (2·5%) was present during each Ab incubation step. Samples were extensively washed and incubated for 30 min at 37°C with goat anti-rabbit serum conjugated to fluorescein (GAR-FITC, Sigma) diluted 1:100 in PBS plus 1% BSA. Negative controls consisted of normal rabbit serum followed by an identical second layer labelled as above. The presence of c-Fos protein was evaluated using the FACScan flow cytometer using the Lysis II software (Becton-Dickinson, Palo Alto, CA, USA).

Northern blot analysis To study the endogenous c-fos mRNA expression, 30 × 106 Jurkat cells were treated as described above. Total RNA was extracted, using the RNAzol kit (Biotecx, Galveston, TX, USA) according to the manufacturer's instructions. Poly(A+) RNA (5 μg) was then purified from each sample using the poly(A+) RNA purification kit (Qiagen, Hilden, Germany) and processed for Northern blot as previously described (Gibellini et al, 1997). Control and c-fos probes were represented by the XbaI/PstI 780 bp fragment of the glyceraldeyde-3-phosphate dehydrogenase gene (GAPDH) (Lalli et al, 1992) and by the XhoI/NcoI 2700 base pairs (bp) c-fos fragment (Van Straaten et al, 1983) respectively. Both probes were digoxigenin-labelled using a random primer labelling kit (Roche, Mannheim, Germany) and revealed with a chemiluminescent substrate according to the manufacturer's instructions.

Transfections Transient transfection experiments were carried out using the DEAE-dextran method as previously described (Gibellini et al, 1997). Briefly, 10 × 106 of exponentially growing Jurkat cells, showing a viability > 95%, were transfected in 3 ml of RPMI-1640 with 10 μg of plasmid DNA and 500 μg of DEAE dextran for 90 min. After transfection, the cells were seeded again in RPMI-1640 plus 2% FCS for 24 h and treated with 10 ng/ml of Tat or p24. After 48 h, cells were lysed and the clarified lysates were assayed for CAT activity using volumes of extract corresponding to equal amounts of proteins, as determined by the Bio-Rad protein assay system (Bio-Rad, Richmond, CA, USA). CAT assays were performed according to the method described by Gorman et al (1982). In blocking experiments, prior to its addition to the culture medium, Tat was preincubated for 2 h at 37°C with 20 μg/ml of an anti-Tat neutralizing polyclonal Ab or with 20 μg/ml of an anti-p24 polyclonal Ab (Intracell).

Statistical analysis Results are expressed as means ± standard deviation (SD) of three or more experiments performed in duplicate. Statistical analyses were performed using the two-tailed Student's T-test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Low concentrations of extracellular Tat induce the production of c-Fos protein and c-fos mRNA in CD4+ Jurkat T cells

In order to evaluate whether extracellular Tat was able to modulate the levels of c-Fos protein in CD4+ T cells, total protein homogenates were obtained from Jurkat cells supplemented with 10 ng/ml synthetic Tat for up to 6 h. The c-Fos protein was undetectable in Jurkat cells, serum-starved (2% FCS) for 24 h, while it was detectable after 30 min of Tat addition and its maximal induction was observed after 45–60 min (Fig 1). Alternatively, equimolar concentrations of recombinant p24 or Tat protein pretreated with polyclonal anti-Tat neutralizing Ab were unable to induce c-Fos protein (Fig 1).

image

Figure 1. Western blot analysis of c-Fos protein performed on 24 h serum-starved (2% FCS) Jurkat cells stimulated for up to 6 h (time shown in min) with 10 ng/ml of synthetic Tat protein (A) or for 45 min with Tat pretreated with a rabbit polyclonal anti-Tat Ab (B). Equal loading of protein in each lane was confirmed by staining with a monoclonal Ab to tubulin. A representative of four separate experiments is shown.

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Parallel experiments were carried out to analyse the levels of c-Fos protein using intracellular staining followed by flow cytometry analysis, which represents a very sensitive and reliable method for the detection of c-Fos protein in haematopoietic cells (Kastan et al, 1989). A representative experiment, illustrated in Fig 2, confirmed that extracellular Tat significantly increases the levels of c-Fos protein in Jurkat cells.

image

Figure 2. Analysis of c-Fos protein using indirect (polyclonal anti-c-Fos Ab plus GAR-FITC) immunofluorescence revealed by flow cytometry. Fluorescence background is represented by Jurkat cells stained with GAR-FITC (A). Jurkat cells were treated for 45 min with 10 ng/ml of p24 (B), 10 ng/ml Tat pretreated with anti-Tat polyclonal Ab (C) or 10 ng/ml of Tat alone (D). Horizontal axis: relative c-Fos expression detected by fluorescence intensity (logarithmic scale). Vertical axis relative number of cells. A representative of three independent experiments is shown.

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To investigate whether extracellular Tat also modulated the c-fos mRNA expression, a Northern blot assay was used. As shown in Fig 3, c-fos mRNA was detected as early as 15 min from Tat addition to serum-starved Jurkat cells, its expression peaked at 30–45 min and declined thereafter. In agreement with the data obtained at the protein level, neither p24 nor Tat preincubated with a polyclonal anti-Tat Ab were able to stimulate c-fos mRNA transcription (Fig 3).

image

Figure 3. Northern blot analysis of c-fos and GAPDH mRNA in Jurkat cells. RNA was extracted from cells serum-starved (2% FCS) for 24 h and then treated for the time-points indicated (in min) with 10 ng/ml synthetic Tat (A) or for 45 min with Tat pretreated with a rabbit polyclonal anti-Tat Ab or p24 (B). A representative of three independent experiments is shown.

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Extracellular Tat activates the c-fos promoter in CD4+ lymphoblastoid T cells

To ascertain whether extracellular Tat promoted c-fos expression in Jurkat cells acting at the promoter level, transient transfection experiments were initially performed using a plasmid (FC2), in which the entire c-fos promoter was cloned in front of CAT. As shown in Fig 4, extracellular Tat protein significantly (P < 0·01) activated the FC2 c-fos promoter activity.

image

Figure 4. Upregulation of the c-fos-CAT promoter activity by extracellular Tat. Serum-starved (2%FCS) Jurkat cells were transfected with FC2 and then treated with synthetic Tat (10 ng/ml), recombinant p24 (10 ng/ml), Tat pretreated with rabbit polyclonal anti-Tat Ab or Tat (10 ng/ml) pretreated with rabbit polyclonal anti-p24 Ab. FC2 promoter activity was measured as folds of activation with respect to Jurkat cells left untreated. Data are reported as means ± SD of three independent transfection experiments performed in duplicate.

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In order to determine the critical domains involved in the extracellular Tat-mediated c-fos activation, the next transfection experiments were performed using several 5′ deletion mutants of the c-fos promoter cloned in front of CAT (Fig 5A). CAT assay analysis showed that the Tat-responsive regions were located in the first 356 bp of the c-fos promoter (fosCAT plasmid), containing several cis-acting regulatory elements (Fig 5B). In fact, the fosCAT plasmid contains several important sequences: the dyad symmetry element that encompasses the serum responsive element (SRE), an AP-1 site, the PDGF responsive sequence (SIE), and three CRE elements of which the best characterized is placed at −60 bp. Moreover, as the FC8 plasmid was much less sensitive than fosCAT to Tat protein, it was suggested that the region between −356 (fosCAT) and −220 (FC8) was the most sensitive to the activation mediated by extracellular Tat.

image

Figure 5. Analysis of the c-fos promoter regions responsive to extracellular Tat. (A) A schematic map of all the c-fos promoter clones employed in the transient transfection experiments. (B) The activity of deletion c-fos promoter mutants was measured as folds of activation with respect to Jurkat cells transfected with pCAT control plasmid. Data are expressed as means ± SD of three independent experiments performed in duplicate.

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In a recent study (Gibellini et al, 1998), we have demonstrated that extracellular Tat induces CREB phosphorylation on the critical Ser133 residue. Phosphorylation on Ser133 allows the transcriptional activation of this nuclear transcription factor, which, in turn, induces the activation of promoters containing CRE domains. In order to elucidate whether the c-fos promoter was activated by extracellular Tat through its CRE sites, further transient transfection experiments were performed, employing a plasmid containing the c-fos promoter mutated −60 CRE site (M1fos).

As shown in Fig 6, the inactivation of the −60 CRE site significantly (P < 0·05) decreased the c-fos promoter activity in response to extracellular Tat, suggesting that CREB plays an important role in mediating c-fos activation. As two additional CRE sites (−290 near to the SRE motif and −340 in the SIE region) have been characterized in the c-fos promoter, further experiments were performed using a plasmid in which all three CRE sites in the c-fos promoter were mutated (M2fos). The M2fos activity in the presence of extracellular Tat was significantly (P < 0·05) lower with respect to either fosCAT or M1fos plasmids, suggesting that all these regulatory domains contributed to c-fos activation induced by extracellular Tat (Fig 6).

image

Figure 6. Evaluation of the activity of c-fos promoter fosCAT and mutants M1fos and M2fos was measured as folds of activation with respect to Jurkat cells transfected with pCAT control plasmid. Data are expressed as means ± SD of three independent experiments performed in duplicate.

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Extracellular Tat induces AP-1 transcription factor

The proto-oncogene c-Fos exerts its transcriptional activity only when it forms an heterodimer with c-Jun, constituting the AP-1 transcription factor (Sassone-Corsi et al, 1988; Angel & Karin, 1991; Karin et al, 1997), which transactivates TRE-containing promoters. In order to elucidate whether Tat-induced c-Fos protein was functionally active, transient transfection experiments were carried out using the pTRECAT plasmid, in which TRE sequences were cloned in front of CAT. The formation of an active AP-1 complex in Jurkat cells was demonstrated by a significant (P < 0·01) increase of CAT activity when these cells were treated with extracellular Tat (Fig 7).

image

Figure 7. Evaluation of the activity of TRE-CAT promoter measured as folds of activation with respect to Jurkat cells transfected with pBLCAT2. Data are performed as means ± SD of three independent experiments performed in duplicate.

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ERK/MAPK pathway mediates the extracellular Tat-induced c-fos activation

Among various intracellular signal transduction pathways activated by Tat protein in lymphoid cells (Borgatti et al, 1997; Li et al, 1997; Gibellini et al, 1998; Kumar et al, 1998), the ERK/MAPK pathway plays a major role in CREB Ser133 phosphorylation (Gibellini et al, 1998). Moreover, this pathway is implicated in the activation of transcription factors, which bind the SRE region of the c-fos gene (Cahill et al, 1995). To elucidate whether the ERK/MAPK pathway was also involved in the activation of c-fos promoter, Jurkat cells were pretreated with the specific ERK/MAPK kinase inhibitor PD98059 and then transfected with FC8,fosCAT, M1fos and M2fos plasmids. These experiments clearly demonstrated that the extracellular Tat-mediated induction of the SRE and CRE elements of the c-fos promoter was significantly (P < 0·01) decreased in the presence of PD90458 (Fig 8). These results indicate that the ERK/MAPK pathway plays an important role in the Tat-mediated activation of the SRE and CRE domains of the c-fos promoter.

image

Figure 8. Inhibition of the c-fos-CAT promoter activity mediated by extracellular Tat in the presence of 10 μg/ml of PD98059 MAPK kinase inhibitor. Data are performed as mean ± SD of three independent experiments performed in duplicate.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Several studies have convincingly demonstrated that Tat protein has a profound impact on the survival/function of both HIV-1-infected and uninfected lymphoid CD4+ T cells, through its ability to be secreted by HIV-1-infected cells (Ensoli et al, 1993; Zauli et al, 1995). This intriguing cytokine-like accomplishment indicates that Tat can act in an autocrine/paracrine fashion (Ensoli et al, 1993; Zauli et al, 1995).

Opposite effects of lymphoid cell survival/growth have been demonstrated in the presence of low (pmol/l) (Gibellini et al, 1995; Zauli et al, 1995) and high (μmol/l-nmol/l) (Li et al, 1995; Westendorp et al, 1995; Zauli et al, 1996) concentrations of extracellular Tat, indicating that the vast array of biological responses observed in the presence of this regulatory HIV-1 protein is concentration dependent. In this respect, Tat has been detected in body fluids at subnanomolar concentrations, which suggests that picomolar concentrations of Tat probably reflect the physiopathological concentrations present in vivo at the sites of an active HIV-1 replication (Westendorp et al, 1995). Moreover, tat-transgenic mice studies showed an increased proliferative activity, leading to oncogenic transformation in lymphoid tissues, which further suggests that the main Tat biological effect in vivo is a proliferative one (Vellutini et al, 1995; Altavilla et al, 1999).

To further investigate how low concentrations of extracellular Tat affect lymphoid CD4+ T-cell function, in this study we demonstrated for the first time that synthetic extracellular Tat induces c-fos expression in low serum-starved Jurkat cells. The c-fos proto-oncogene represents a key factor of the AP-1 transcription factor complex, which stimulates several gene promoters by binding to the TRE sequences (Angel & Karin, 1991; Karin et al, 1997). Of note, the Tat-mediated induction of c-Fos protein resulted in the functional activation of AP-1, as demonstrated in CAT assay experiments. The ability of extracellular Tat to activate AP-1 is of primary importance to explain the upregulation of interleukin 2 (IL-2) production observed in lymphoid T cells treated with both intracellular or extracellular Tat (Westendorp et al, 1994; Ott et al, 1997).

By using promoter deletion mutants, we also demonstrated that the two main c-fos promoter elements responsive to extracellular Tat are the CRE and SRE domains located at −60 and −323, respectively, even though the SIE element (located at −340) also contributes to the Tat-mediated activation.

It should be noted that these data obtained with extracellular Tat are significantly different from those obtained with endogenously expressed tat (Gibellini et al, 1997). In fact, we have previously shown that the activation of c-fos promoter in response to endogenous tat gene in the same (Jurkat) cell line (i) requires the presence of high serum concentration (> 10% FCS) and phytohaemagglutinin, and (ii) is mediated by the SRE element, but not the SIE and CRE elements. Our present and previous findings suggest that intracellular and extracellular Tat may select different targets on the c-fos promoter, depending on the activation state of the cells as well as on the presence of additional stimuli. In this respect, it is also noteworthy that the c-fos promoter shows a very complex regulation (Van Straaten et al, 1983), undergoing a post-induction downregulation in response to c-Fos protein itself, which negatively regulates the SRE site of its own promoter. Interestingly, the CRE element (−60) of the c-fos promoter is not affected by c-Fos protein. Taken together, these findings suggest a dual role of endogenous and extracellular Tat in the c-fos promoter regulation in CD4+ T cells.

We have also demonstrated that, among the various intracellular signal transduction pathways activated by Tat protein (Borgatti et al, 1997; Li et al, 1997; Gibellini et al, 1998; Kumar et al, 1998), the ERK/MAPK pathway, which leads to CREB Ser133 phosphorylation (Gibellini et al, 1998), plays an important role in mediating c-fos promoter activity by extracellular Tat. However, it is conceivable that additional pathways are involved in c-fos activation, probably acting at the level of the SIE element.

In conclusion, the ability of extracellular Tat to activate c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to AIDS and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells (Gallo, 1999).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by the ‘AIDS project’ of the Italian Ministry of Health and Funds for Selected Research Topics of the University of Bologna. We are grateful to Dr Paolo Sassone-Corsi, Dr Enzo Lalli and Dr David Ginty for providing all the plasmids.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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