Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits IκBα and enhances HIV-1 replication in human T lymphocytes
© Coiras et al; licensee BioMed Central Ltd. 2008
Received: 04 July 2008
Accepted: 01 December 2008
Published: 01 December 2008
Degradation of p65/RelA has been involved in both the inhibition of NF-κB-dependent activity and the onset of apoptosis. However, the mechanisms of NF-κB degradation are unclear and can vary depending on the cell type. Cleavage of p65/RelA can produce an amino-terminal fragment that was shown to act as a dominant-negative inhibitor of NF-κB, thereby promoting apoptosis. However, the opposite situation has also been described and the production of a carboxy-terminal fragment that contains two potent transactivation domains has also been related to the onset of apoptosis. In this context, a carboxy-terminal fragment of p65/RelA (ΔNH2p65), detected in non-apoptotic human T lymphocytes upon activation, has been studied. T cells constitute one of the long-lived cellular reservoirs of the human immunodeficiency virus type 1 (HIV-1). Because NF-κB is the most important inducible element involved in initiation of HIV-1 transcription, an adequate control of NF-κB response is of paramount importance for both T cell survival and viral spread. Its major inhibitor IκBα constitutes a master terminator of NF-κB response that is complemented by degradation of p65/RelA.
Results and conclusions
In this study, the function of a caspase-3-mediated carboxy-terminal fragment of p65/RelA, which was detected in activated human peripheral blood lymphocytes (PBLs), was analyzed. Cells producing this truncated p65/RelA did not undergo apoptosis but showed a high viability, in spite of caspase-3 activation. ΔNH2p65 lacked most of DNA-binding domain but retained the dimerization domain, NLS and transactivation domains. Consequently, it could translocate to the nucleus, associate with NF-κB1/p50 and IκBα, but could not bind -κB consensus sites. However, although ΔNH2p65 lacked transcriptional activity by itself, it could increase NF-κB activity in a dose-dependent manner by hijacking IκBα. Thus, its expression resulted in a persistent transactivation activity of wild-type p65/RelA, as well as an improvement of HIV-1 replication in PBLs. Moreover, ΔNH2p65 was increased in the nuclei of PMA-, PHA-, and TNFα-activated T cells, proving this phenomenon was related to cell activation. These data suggest the existence of a novel mechanism for maintaining NF-κB activity in human T cells through the binding of the carboxy-terminal fragment of p65/RelA to IκBα in order to protect wild-type p65/RelA from IκBα inhibition.
The family of transcription factors NF-κB regulates numerous genes controlling immune response, cell growth, and tissue differentiation . These factors exist as dimeric complexes, comprising different proteins: NF-κB1/p50, NF-κB2/p52, p65/RelA, c-Rel, and RelB. The most important active heterodimer of NF-κB is p65/p50. All of these proteins contain a well-conserved amino-terminal region known as the Rel Homology Region (RHR) which is responsible for DNA binding, dimerization and nuclear localization . The activation of NF-κB is inhibited by a variety of mechanisms: first, through the association of the NF-κB dimers with three major inhibitory proteins IκBs (IκBα, IκBβ, IκBε) ; second, through the inhibition of p65/RelA posttranslational modifications such as phosphorylation ; third, via complete or partial degradation of p65/RelA [5–8]; and fourth, by replacement of active NF-κB dimers with dimers showing no transcriptional activity .
The NF-κB pathway also provides an attractive target to viral pathogens. Activation of NF-κB is a rapid, immediate early event that occurs within minutes after exposure to a stimulus, does not require de novo protein synthesis (e.g. the basal pool of p65/RelA is very constant), and produces a strong transcriptional activation of several viral genes . As a result, NF-κB is essential in the regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter . The promoter-proximal (enhancer) region of the HIV-1 LTR contains two adjacent NF-κB binding sites that play a central role in mediating inducible HIV-1 gene expression in blood CD4+ T cells [12, 13].
Besides, NF-κB also acts as a protector against apoptosis or programmed cell death, and is necessary and sufficient for preventing apoptosis induced by tumor necrosis factor alpha (TNF-α), ionizing radiation and chemotherapeutic agents [5, 14]. In fact, the ability to maintain NF-κB activity determines whether the cell survives or undergoes apoptosis [5, 15]. Degradation of p65/RelA is therefore an important mechanism for cell survival in many cell types. Putative recognition sequences for caspase-3 and -6-related proteases are present in the amino acid sequences of p65/RelA . This suggests that certain transduced signals could be responsible for the modulation of NF-κB activity by caspase-mediated cleavage of p65/RelA. The cleavage appears to be cell type- and stimulus-specific and occurs at different sites in the amino- and carboxy-terminus of p65/RelA [5, 6, 16, 17]. As a consequence, it is widely established that truncation of p65/RelA inhibits NF-κB-dependent transactivation and ultimately leads to apoptosis. Therefore, caspase-3-related proteolysis may determine the duration of NF-κB activity in stimulated T cells and may play a critical role in the duration and potency of the immune response .
In this study, a carboxy-terminal fragment of p65/RelA that can be detected in activated human blood T lymphocytes is analyzed. Amino-cleavage of p65/RelA was increased after treatment with stimuli as phytohemagglutinin (PHA), 5-phorbol 12-myristate 13-acetate (PMA) or TNFα, thereby proving this phenomenon is related to T-cell activation. However, despite previous studies [5, 6, 16], this amino-truncated p65/RelA was produced in T cells (PBLs and Jurkat) that did not undergo apoptosis. On the contrary, they showed a high viability and an increased NF-κB-dependent activation. This carboxy-terminal fragment of p65/RelA lacked most of the DNA-binding domains but retained the dimerization domain, the nuclear localization signal (NLS) and the transactivation domains. Consequently, it was able to translocate to the nucleus, associate with NF-κB1/p50 and IκBα, but could not bind DNA. In spite of this, amino-truncated p65/RelA was able to increase NF-κB-dependent transactivation, as well as HIV-1 replication in a dose-dependent manner.
p65/RelA is truncated in PHA-treated human blood T lymphocytes
Caspase-mediated cleavage of p65/RelA is produced in T cells upon activation
To further determine the functionality of the tagged p65/RelA, analysis of subcellular distribution was also determined by confocal microscopy after staining with the monoclonal antibody (mAb) against FLAG tag M2 and a secondary antibody conjugated with TexasRed (Fig. 2d). Tagged p65/RelA could shuttle between the cytosol and the nucleus and it mainly increased inside the nucleus after PMA or PHA activation. To prove that the subcellular distribution of the tagged p65/RelA proteins in T cells after activation with PMA or PHA was similar to the usual pattern described for endogenous p65wt, Jurkat cells transfected with the control plasmid pCMV-Tag1 and stained with an antibody against p65/RelA and a secondary antibody conjugated to Alexa 488 were analyzed by confocal microscopy (Figure 2e). As expected, both p65wt-tag (Figure 2d) and p65wt (Figure 2e) showed a similar distribution pattern after activation with PMA or PHA.
Identification of cleavage site at Asp97through generation of uncleavable N-terminal p65/RelA mutants
As protein p65/RelA was truncated at the amino-terminus and produced a fragment of approximately 55 kDa, the cleavage site was supposed to be at the adjacent putative recognition sites for caspase-6 91VGKD94 or caspase-3 94DCRD97 at the amino terminus of the protein (Fig. 3a). With the aim of determining whether the correct cleavage site responsible for producing ΔNH2p65 in human blood T cells was the putative recognition site for caspase-3 at position 94DCRD97 or the putative recognition site for caspase-6 at position 91VGKD94, the following amino-acid-substitution mutants were obtained from pCMV-p65wt-tag expression vector by site-directed mutagenesis: a double amino-acid-substitution mutant in which the aspartates at the putative P1 positions were exchanged for glutamates (94DCRD97 to 94ECRE97) (p65 D94E;D97E-tag mutant); another double amino-acid-substitution mutant in which 91VGKD94 site was exchanged for 91LGKE94 (p65 V91L;D94E-tag mutant); finally, two single amino-acid-substitution mutants in which 91VGKD94 site was exchanged for 91LGKD94 (p65 V91L-tag mutant) and 94DCRD97 site was exchanged for 94DCRE97 (p65 D97E-tag mutant). Consequently, mutants p65 D94E;D97E-tag and p65 V91L;D94E-tag were resistant to cleavage by both caspase-3 and caspase-6, whereas mutant p65 V91L-tag was resistant to cleavage by caspase-6 and p65 D97E-tag mutant was resistant to cleavage by caspase-3. All of these p65/RelA mutants were transiently transfected in Jurkat cells and incubated for 18 hours in the absence of a stimulus. Cells were then treated with PMA for 2 hours and protein extracts were obtained. Analysis by immunoblotting with an antibody against the carboxy-terminus of p65/RelA (Fig. 3c) or by using an anti-FLAG tag M2 mAb (data not shown) revealed that ΔNH2p65-tag was produced only when p65wt-tag or the mutant p65 V91L-tag (resistant to cleavage by caspase-6) were over-expressed but not when amino-acids at position 94 and/or 97 were mutated. Accordingly, ΔNH2p65 was produced in human T cells as a result of p65/RelA cleavage at 94DCRD97 after caspase-3 activation. Moreover, cleavage of p65/RelA was produced promptly after induction of T-cell activation, because PMA had been added for 2 hours before analyzing the protein extracts.
Caspase-3-mediated cleavage of p65/RelA was produced in non-apoptotic human blood T cells upon activation
On the other hand, despite the activation of caspase-3, there was no significant decrease in the viability of PBLs treated with PMA or PHA for 4 days in comparison with treatment of PBLs with diethylmaleate (DEM), which has been described as an inductor of apoptosis in Jurkat cells by activation of caspase-3  (Fig. 4d). Jurkat cells were then transiently transfected with either pCMV-p65wt-tag or pCMV-p65 D94E;D97E-tag expression vectors, incubated for 18 hours without stimulus and then treated with PMA for 1, 4, or 18 hours. It was observed that only when pCMV-p65wt-tag was transfected, ΔNH2p65-tag progressively accumulated in both nucleus and cytosol according to increasing PMA time exposure (Fig. 4e, Cytosol and Nucleus, lanes 1–4). However, cleavage of p65/RelA was not detected in Jurkat cells transfected with p65 D94E;D97E-tag mutant, even after activation with PMA for 18 hours (Fig. 4e, Cytosol and Nucleus, lanes 5–8). Cleavage did not occur although a weak band corresponding to endogenous ΔNH2p65 could be observed in the cytosol of Jurkat cells after treatment for 18 hours (Fig. 4e, Cytosol, lane 8), as was assessed by immunoblotting with anti-FLAG tag M2 mAb (data not shown). Densitometric analysis was carried out to determine that there was no linear correlation between the increment of p65wt-tag or p65wt (endogenous) and ΔNH2p65-tag.
Truncated ΔNH2p65 was able to bind both IκBα and NF-κB1/p50 proteins
Truncated ΔNH2p65 lacked of both DNA binding capacity and NF-κB-dependent transcriptional activity
In order to demonstrate that ΔNH2p65 was transcriptionally inactive, Jurkat cells were transiently transfected with a luciferase (LUC) reporter expression vector under the control of three -κB consensus sites (plasmid pκB-conA-LUC) together with pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag, or pCMV-ΔNH2p65-tag expression vectors. These cells were maintained in the absence of activation and analysed 18 hours after transfection to measure the luciferase activity due to the transfected tagged proteins. It was observed that although both the p65wt-tag and the p65 D94E;D97E-tag were able to induce more than 3-fold the NF-κB-dependent transcriptional activity in comparison with basal activity, ΔNH2p65 did not induce significant transcriptional activation (Fig. 6c).
Increasing doses of ΔNH2p65 permitted a persistent NF-κB activity in T cells by sequestering IκBα
In vitro binding affinity of translated proteins p65wt-tag and ΔNH2p65-tag to IκBα
Truncated ΔNH2p65 enhanced HIV-1 replication in human blood T cells
Because the PBLs used for this transfection were in a resting state, it was necessary to ensure that the HIV-1 replication detected in Figure 9a was dependent on the NF-κB transcriptional activity induced by the over-expression of p65wt-tag and/or ΔNH2p65-tag. For this purpose, the same experiment was performed by using a plasmid pNL4.3-wt where the -κB consensus sites had been removed (plasmid pdI-NF), as a control of the NF-κB-dependent HIV-1 expression . It was determined that the production of HIV-1 p24-gag antigen was under the threshold limit of detection even when pCMV-p65wt-tag and/or pCMV-ΔNH2p65-tag expression vectors were co-transfected, thereby proving that this phenomenon is exclusively related to NF-κB-dependent activity (data not shown).
HIV-1 infection is characterized by continuous viral replication throughout the illness  – mainly in T lymphocytes and macrophages – that ultimately leads to the acquired immunodeficiency syndrome (AIDS). NF-κB is essential for the activation of HIV-1 in T cells . In fact, although the HIV-1 LTR contains several additional DNA binding domains that bind other cellular transcriptional factors, only NF-κB and Sp1 binding sites are really indispensable for initiation of HIV-1 replication [13, 28]. Therefore, control of NF-κB activation is essential to impede HIV-1 LTR transcriptional activation as well as viral replication. The activation of NF-κB can be inhibited by a variety of mechanisms, especially the synergistic combination of cytosolic sequestering by the inhibitor IκBα and degradation of p65/RelA.
Degradation of p65/RelA can occur through different pathways that vary depending on the cell type. This mechanism has been involved not only in the inhibition of NF-κB-dependent activity but also in the onset of apoptosis [5–8, 16, 17, 29]. Protein p65/RelA is a potential target for specific cleavage by caspases , but viruses such as picornaviruses can also promote a rapid and efficient proteolytic cleavage of the carboxy-terminus of p65/RelA by the viral protease 3C . The resultant amino-terminal fragment has also been detected in HUVEC cells and acts as a dominant-negative inhibitor of NF-κB, finally promoting apoptosis . This effect is caused because the elimination of the carboxy-terminus leaves an amino-terminal fragment that retains the ability to bind DNA but lacks the ability to initiate transcription. Similar cleavage can be performed by the human neutrophil elastase (HNE) that removes the carboxy-terminus of p65/RelA near a site predominantly cleaved by caspase-6 . However, the opposite situation has also been described. The neutrophilic and monocytic proteinase 3 (PR3) removes the DNA-binding domain in the amino-terminus of p65/RelA by cleavage at a sequence near a caspase-3 cleavage site, leaving a carboxy-terminal fragment that contains two potent transactivation domains and the nuclear localization signal (NLS) [22, 23, 29]. Furthermore, it has been described that caspase-mediated cleavage of p65/RelA at Asp97 in HeLa cells induced an amino-cleaved fragment of p65/RelA that was responsible for inducing apoptosis in the presence of 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (NA) . On the contrary, Qin Z et al.  suggested that a caspase-3-like protease contributed to NF-κB activation through IκBα degradation, which finally caused apoptosis in rat striatal neurons through the activation of the N-methyl-D-aspartate (NMDA) receptor. Consequently, association of p65/RelA cleavage with the onset of apoptosis or with modulation of the NF-κB-dependent transactivation is not widely understood and it appears to be dependent on the cell type. In this context, the amino-terminal cleavage of p65/RelA (ΔNH2p65) detected in PHA-, PMA-, or TNFα-stimulated PBLs has been investigated.
The Jurkat cell line does not show significant levels of endogenous ΔNH2p65 even after PMA activation; hence it has been used as a recipient for this study because it is a lymphoblast-like cell line and can reproduce the environment of human PBLs. When p65/RelA was over-expressed in Jurkat cells by transfection of vector pCMV-p65wt-tag protein, a weak ΔNH2p65-tag form – coming from cleavage of p65wt-tag protein – could be detected in the absence of activation. This cleavage was greatly enhanced when cells were also activated with PMA and measurement of caspase-3 activity showed that it increased in T cells more than 3-fold after treatment with PMA for 18 hours. However, ΔNH2p65-tag was not detected when Jurkat cells were also treated with caspase inhibitors even upon PMA activation. Consequently, this specific degradation was produced by caspase activation. According to the observed molecular weight of ΔNH2p65 (~55 kDa), the potential site of cleavage was supposed to be at position 94DCRD97 – a putative recognition site DXXD for caspase-3 – or at position 91VGKD94 – a putative recognition site V/I/LXXD for caspase-6. The correct cleavage site was identified at 94DCRD97 because when amino acids at position 94 and/or 97 in p65wt-tag protein were changed, ΔNH2p65-tag was not produced even after activation with PHA or PMA. This major cleavage site of p65/RelA has been previously reported in HeLa  and SK-Hep1 hepatoma cells  but not in human PBLs. Besides, Kang et al.  reported that ΔNH2p65 induced a slight decreasing of the transcriptional activity mediated by NF-κB in HeLa cells. But the transfection of different concentrations of the plasmid pCMV-ΔNH2p65-tag in 3T3-p65ko cells, Jurkat or PBLs did not induce significant variations in the NF-κB-dependent transcriptional activity. Moreover, T-cell viability was not diminished after over-expression of ΔNH2p65-tag protein in these cells. These contradictory data could be due to the cell type used for studying the cleavage of p65/RelA. Activation of caspase-3 is very complex and can be promoted through different pathways in different cells : e.g., deprivation of growth factors in endothelial HUVEC cells produced a caspase-mediated carboxy-terminal cleavage of p65/RelA that acted as a dominant-negative inhibitor of NF-κB, finally causing cell death ; but deprivation of serum in the culture medium of Jurkat cells did not induce detectable apoptosis (data not shown).
On the other hand, PMA has been currently described as a potential inhibitor of apoptosis in human T cells [32, 33]; PHA is a mitogen usually used to induce HIV-1 replication and significant apoptosis has not been reported under this stimulus; and TNFα treatment does not result in the death of Jurkat cells . However, when PBLs were exposed to PMA or PHA for 4 days the amount of procaspase-3 decreased in the cytosol as long as the active nuclear caspase-3-p17 subunit increased up to 4-fold, although these cells were largely viable. Moreover, an increase in caspase-3 activity correlated with the increasing cleavage of p65/RelA, although NF-κB1/p50 remained stable. Accordingly, if these cells were apoptotic, p65/RelA and NF-κB1/p50 protein levels would decrease, as occurs in both Jurkat and PBLs with the onset of apoptosis [16, 17]. Consequently, activation of T cells did induce caspase-3-mediated cleavage of p65/RelA in a process unrelated to apoptosis.
In fact, effector caspase-3 can be processed following T-cell activation in the absence of apoptosis [34–37]. It has been described that caspase-3 translocates from the cytosol to the nucleus after activation in apoptotic cells . However, although human PBLs showed nuclear activity of caspase-3 after treatment with PMA or PHA, these cells were viable and caspase-3 activation did not affect T cell proliferation. Moreover, although PMA activates caspase-3 through the PKC signaling pathway and this leads ultimately to apoptosis in a gastric cancer cell line , caspases also play a central role in T lymphocyte activation as well as in IL-2 release [40–42]. Accordingly, activation of caspase-3 should be considered in the context of the general environment of the cell, where the equilibrium between pro-apoptotic and anti-apoptotic factors will determine if the cell undergoes apoptosis or survives. In this context, Varghese et al.  described that treatment of Jurkat cells with diethylmaleate (DEM) induced apoptosis through activation of caspase-3 but PMA was able to restore the XIAP (X-linked inhibitor of apoptosis protein) levels in DEM-treated cells, which blocks apoptosis by directly binding to caspase-3, -7 and -9. Similar mechanisms should occur in PMA or PHA treated T cells that promote cell survival and proliferation in spite of the activation of caspase-3. Moreover, treatment of Jurkat cells with DEM also induced degradation of p65/RelA to ΔNH2p65 (data not shown), thereby proving that degradation of p65/RelA is not enough to induce apoptosis at least in T cells but other processes such as the decrease in pro-apoptotic factors as XIAP should be involved. Besides, it has been suggested that caspase-1, -3 and -6 could not be the primary caspases required for apoptosis in T cells .
Because ΔNH2p65 was mainly observed after T cell activation, this form could be somehow involved in NF-κB-dependent transcriptional activity. Protein ΔNH2p65-tag retained the dimerization domain, NLS, and both transactivation domains [22, 23, 44] but it lacks the DNA-binding domain. As a result, it could bind IκBα and NF-κB1/p50 but not DNA, neither as a homodimer nor as a heterodimer with NF-κB1/p50. This can be explained because both subsites containing the residues that contact DNA present in each subunit of the dimer are necessary to bind the complex to the DNA backbone . On the other hand, the mutation of NF-κB1/p50 that disrupted DNA binding could not affect the ability of association with other members of the NF-κB family . Accordingly, the deletion of amino acids 1–97 in protein p65/RelA did not interfere with the ability of interaction with NF-κB1/p50 or IκBα, although it impaired the ability to bind to DNA, likely by modification of the structural conformation of the heterodimer. Likewise, in a similar mechanism, other dimers such as p65wt/ΔNH2p65 would also be unable to bind DNA, thereby ruling out the possibility that those dimers could be responsible for the transcriptional activation observed when ΔNH2p65-tag was over-expressed along with p65wt-tag. In fact, over-expression of increasing quantities of ΔNH2p65-tag protein in the 3T3-p65ko cell line – maintaining the p65wt-tag protein concentration as invariable – showed that NF-κB-dependent activation increased in a dose-dependent manner up to 3-fold. On the other hand, ΔNH2p65 was able to bind IκBα and when nuclear export was obstructed with LMB in PHA-treated PBLs, IκBα showed high affinity to ΔNH2p65. This effect suggested the possibility that after ΔNH2p65 was generated – probably in the nucleus by activated caspase-3  –, it would hijack IκBα, being actively exported to the cytosol and thereby permitting a sustained NF-κB-dependent activity by free p65wt. The active shuttling of ΔNH2p65 from the nucleus to the cytosol would explain why it largely accumulated in the nucleus of PHA-activated PBLs after treatment with LMB. But when in vitro translated proteins were mixed at different ratios in the presence of IκBα to evaluate the affinity of both proteins for this inhibitor, data showed that there was similar affinity of IκBα for ΔNH2p65 and p65wt. This could be explained because the translated proteins could present different behavior in vitro and in vivo. However, these results did not rule out the possibility that when ΔNH2p65 was over-expressed, IκBα could bind preferentially to this cleaved form in the nucleus.
NF-κB is essential for triggering HIV-1 LTR-transcription in blood CD4+ T cells  and these cells are one of the long-lived cellular reservoirs of HIV-1 in vivo . In this context, the importance of p65/RelA degradation in HIV-1 infected human blood T cells was also analyzed. Evaluation of the HIV-1 replication in PBLs after the over-expression of pCMV-p65wt-tag and/or pCMV-ΔNH2p65-tag plasmids was performed in resting conditions to evaluate the virus production due exclusively to the transfected tagged p65/RelA proteins and not to the endogenous p65/RelA induced upon activation. Data showed that HIV-1 replication was also enhanced in resting PBLs co-transfected with both p65wt-tag and ΔNH2p65-tag proteins – ratio 1:2 – in comparison with PBLs transfected only with p65wt-tag protein.
Activation of T cells induced caspase-mediated cleavage of p65/RelA at Asp97. This carboxy-terminal fragment of p65/RelA was observed in the cytosol although it also accumulated in the nucleus when cells were also treated with PMA, PHA, TNFα, or LMB, a specific inhibitor of the nuclear export . Because active forms of caspase-3 accumulated in the nucleus , p65/RelA should be degraded at the nuclear compartment. Then, ΔNH2p65 would bind IκBα and would be rapidly exported to the cytosol, due to the fast nucleocytosolic shuttling of NF-κB in PBLs even in resting conditions . This mechanism would protect the nuclear full-length p65/RelA from IκBα inhibition and would permit a sustained NF-κB-dependent transcriptional activity, thereby increasing HIV-1 replication in human T cells. This function has never been described before for a carboxy-terminal fragment of p65/RelA, which is generally supposed to be a previous form before complete degradation in pro-apoptotic cells. Consequently, these findings describe a novel pathway in the activation and regulation of the NF-κB/IκBα system in human T cells, the best-defined reservoir of HIV-1 latent infection. More studies will be necessary to evaluate the importance of degradation of p65/RelA to ΔNH2p65 and of caspase-3 activity in the mechanisms of HIV-1 latency and replication.
Peripheral blood lymphocytes (PBLs) were isolated from blood of healthy donors by centrifugation through a Ficoll-Hypaque gradient (Pharmacia Corporation, North Peapack, NJ). Cells were collected in RPMI 1640 medium (Biowhitaker, Walkersville, MD) with 10% fetal calf serum (PAN Biotech GmbH, Aidenbach, Germany), 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, and maintained at 2 × 106 cells/ml and at 37°C. PHA-treated T lymphocytes were obtained from PBLs cultured for 3 days in the presence of 5 μg/ml phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO) and for the 9 consecutive days with 300 U/ml interleukin-2 (IL-2) (Chiron, Emeryville, CA). These long-term cultures of PHA-treated T lymphocytes were maintained without supplemental IL-2 18 hours before the experiments. Resting PBLs were maintained in culture at 2 × 106 cells/ml in supplemented RPMI without any stimulus. Jurkat cell line was cultured in supplemented RPMI at 37°C. Mouse 3T3 fibroblast cells lacking p65/RelA (3T3-p65ko) have been previously described  and were kindly provided by Dr Alexander Hoffmann (Department of Chemistry and Biochemistry, University of California, San Diego, CA). 3T3-p65ko cells were plated at 3 × 105 cells/60-mm dish every 3 days in Dulbecco's modified Eagle's medium (Biowhitaker) supplemented with 10% defined calf serum (Hyclone Laboratories, Logan, UT).
Reagents and antibodies
5-phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) was used at 25 ng/ml. PHA (Sigma-Aldrich) was used at 5 μg/ml. Leptomycin B (LMB) was used at 20 nM (Sigma-Aldrich). Anti-FLAG tag M2 monoclonal antibody (mAb) was purchased from Stratagene (La Jolla, CA). Primary antibodies against p65/RelA (clones C-20 and F-6), NF-κB1/p50 (clone H-119), IκBα (clone C-21), and caspase-3 (CPP32) – precursor and p20 and p17 subunits – (clone H-277) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal primary antibody against IκBα (clone 10B) was kindly provided by Dr. Ron T. Hay (College of Life Sciences, University of Dundee, Dundee, UK) . Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare (Uppsala, Sweden). Secondary antibodies conjugated to TexasRed and Alexa 488 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). Generic caspase inhibitor z-VAD-fmk (Calbiochem, Merck Chemicals Ltd, Nottingham, UK) was used at 100 μM and the specific caspase-3 inhibitor Ac-DMQD-CHO (Calbiochem) was used at 10–100 μM. Both inhibitors were dissolved at 10 mM in DMSO and stored at -80°C.
pCMV-Tag1 epitope tagging mammalian expression vector was purchased from Stratagene. The pκB-conA-LUC vector carries a luciferase gene placed under the control of three copies of the -κB consensus . Plasmids pRSV-NF-κB(p105) and pBluescript-RelA(p65) were obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, from Dr Gary Nabel and Dr Neil Perkins [50, 51]. pcDNA3.1(+) plasmid was used as a negative control (Invitrogen). Vector pNL4.3 that contained the HIV-1 complete genome and induced an infectious progeny after transfection was kindly provided by Dr M.A. Martin . Vector pNL4.3-Renilla was obtained by replacing the gene nef of the HIV-1 proviral clone pNL4.3 with the Renilla luciferase gene, as previously described . Vector pdI-NF was a pNL4.3-wt plasmid where the -κB consensus sites had been removed and it was used as a control of the NF-κB-dependent HIV-1 expression . Vector pSV-β-Galactosidase (Promega, Madison, WI) was used as an internal control for transient expression assays. Vector LTR-GFP was generated by replacing the LUC gene from the LTR-LUC vector with the green fluorescent protein (GFP) gene obtained from the pEGFP vector (BD Biosciences Clontech) . All plasmids were purified using Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.
Generation of p65/RelA mutants and directed mutagenesis
The p65/RelA wild-type (wt) gene was obtained from pBluescript-RelA (p65) and cloned in pcDNA3.1 using HindIII/BamHI cloning sites. The p65wt gene to clone in vector pCMV-Tag1 (Stratagene) was obtained from pcDNA3-p65 plasmid using the following primers: p65s-NotI, 5'-TCGTAACAACTGCGGCCGCTTGACGCAAATGGGCGGT-3' and p65as, 5'-GCTGGATATCTGCAGAATTCCACC-3'. Then, p65wt gene was cloned in pCMV-Tag1 plasmid using NotI/BamHI cloning sites to generate the p65wt-tag mutant. The ΔNH2p65-tag mutant was also obtained from pcDNA3-p65 plasmid using primer p65s-NotI-97A, 5'-AGGAAAGGGCGGCCGCGATGGGCTTCTAT-3', which introduced an ATG codon at position 97, and primer p65as. It was then cloned in pCMV-Tag1 plasmid using NotI/BamHI cloning sites. The substitution mutants were generated from pCMV-p65wt-tag plasmid by site-directed mutagenesis with the Quikchange Site-Directed Mutagenesis kit (Stratagene): in p65 D94E;D97E-tag the sequence 94DCRD97 was converted to 94ECRE97 with the primer 5'-CGAGCTTGTAGGAAAGGAATGCCGGGAAGGCT-3'; in p65 V91L;D94E-tag the sequence 91VGKD94 was converted to 91LGKE94 with the primer 5'-CGAGCTTCTAGGAAAG GAATGCCGGGATGGCT-3'; in p65 V91L-tag the sequence 91VGKD94 was converted to 91LGKD94 with the primer 5'-CGAGCTTCTAGGAAAGGACTGCCGGGATGGCT-3'; in p65 D97E-tag the sequence 94DCRD97 was converted to 94DCRE97 with the primer 5'-CGAGCTTGTAGGAAAGGACTGCCGGGAAGGCT-3' The sequence of the entire p65/RelA coding region was confirmed by DNA-sequence analysis in all substitution mutants. The p50wt gene was obtained from pRSV-NF-κB(p105) and cloned in pcDNA3.1 using HindIII/XbaI cloning sites.
In vitro transcription and translation assays were performed with TNT Couple Wheat Germ Extract Systems (Promega) according to manufacturer's instructions, using unlabeled methionine. Plasmid pcDNA3-p50 was used for co-translation experiments along with vectors pCMV-p65wt-tag, pCMV-ΔNH2p65-tag and pCMV-p65 D94E;D97E-tag.
Transient transfections of Jurkat cells were performed by electroporation with an Easyjet Plus Electroporator (Equibio, Middlesex, UK). In brief, 15 × 106 cells were resuspended in 350 μl of RPMI without supplements and mixed with 1 μg of plasmid DNA per 106 cells in a 4 mm electroporation cuvette (Equibio). Cells were transfected at 280V, 1500 μF and maximum resistance. After transfection, cells were incubated in supplemented RPMI at 37°C for 18 hours before analysis. Luciferase and Renilla activities were assayed using Luciferase Assay System according to manufacture's instructions (Promega). Both total protein concentration and the β-Galactosidase activity were used for the standardization of relative luciferase units (RLU). β-Galactosidase activity was measured in transfected cell lysates using the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer according to manufacturer's instructions (Promega).
Transient transfections of 3T3-p65ko fibroblast cells were performed as previously described with modifications . Briefly, 3T3-p65ko cells were cultivated at 80% of confluence and then split 1:8 in 6-well plates. Cells were cultured in fresh medium 4 hours prior transfection. Reaction mixture containing 200 μl of HBS solution (50 mM HEPES, 1.5 mM Na2HPO4, 140 mM NaCl, pH7.05), 200 μl of 250 mM CaCl2 solution and 5 μg of DNA, was incubated at room temperature for 1 minute and then added to cells drop by drop. Cells were incubated at 37°C, 5%CO2 for 12 hours. Transfection mix was removed and fresh medium was added. After incubation for 12 hours, cells were trypsinized, washed and luciferase expression was analyzed. Although 3T3 cells are supposed to be calcium intolerant cells, no significant cell death was observed after 12 hours in presence of calcium precipitate.
Caspase-3 activity and cell viability
Caspase-3 activity has been measured with the Colorimetric CaspACE™ Assay System (Promega), following the manufacturer's instructions. Briefly, 1 × 106 cells were harvested by centrifugation, washed twice with PBS1x and lysed by freeze-thaw cycles. Cell lysates were centrifuged at 15.000 × g for 20 minutes at 4°C and supernatants were collected and protein concentration was determined by the method of Bradford using a bovine serum albumin (BSA) standard curve . For each experimental point, 25 μg of total protein extracts were analyzed with the colorimetric substrate Ac-DEVD-p-nitroaniline (pNA) 0.1 mM. The assay was incubated over-night at 22°C and the absorbance was measured at 405 nm. Calculation of caspase specific activity was determined by the construction of pNA standard curve.
Cell viability was determined with the CellTiter-Glo® Luminescent Cell Viability Assay (Promega), following the manufacturer's instructions. Briefly, 1 × 105 cells were harvested by centrifugation, washed twice with PBS1x and resuspended in lysis buffer. After incubation for 10 minutes at room temperature to stabilize luminescent signal, cell lysates were deposited in an opaque-walled multiwell plate and analyzed in an Orion Microplate Luminometer with Simplicity software (Berthold Detection Systems, Oak Ridge, TN).
For immunofluorescence assays, cells were immobilized in PolyPrep slides (Sigma-Aldrich) for 15 minutes and then fixed with 2% paraformaldehyde-0.025% glutaraldehyde in PBS for 10 minutes at room temperature. After washing twice with 0.1% glycine/PBS, cells were permeabilized with 0.1% Triton X-100/PBS for 10 minutes. After washing, cells were treated with 1 mg/ml NaBH4 for 10 minutes. Incubation for 1 hour at room temperature with each primary and secondary antibodies and subsequent washes were performed with PBS/2% bovine serum albumin (BSA)/0.05% saponine buffer. Coverslips were immobilized with 70% glycerol/PBS. Images were obtained with a Radiance 2100 confocal microscope (BioRad, Hercules, CA).
Immunoblot and immunoprecipitation assays
Cytosolic and nuclear protein extracts were obtained as described  and protein concentration was determined by the method of Bradford using a BSA standard curve . Control of purity of nuclear and cytosolic extracts was determined by immunoblotting with an antibody against p105 (exclusive cytosolic distribution) and p50 (clone H-119, Santa Cruz Biotechnology). Ten micrograms were fractionated by SDS-PAGE and transferred onto Hybond-ECL nitrocellulose paper (GE Healthcare). After blocking and incubation with primary and secondary antibodies, proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
Cytosolic and nuclear protein extracts were subjected to immunoprecipitation with specific antibodies. In brief, 100 μg of nuclear or cytosolic proteins were incubated overnight at 4°C with 10 μg of an agarose-conjugated antibody against IκBα (Santa Cruz Biotechnology), gently shaking, in RIPA buffer (1 × PBS, 0.1%SDS, 1%NP-40) and 0.5% sodium deoxycholate (DOC). In case of anti-FLAG mAb, 100 μg of protein extracts were incubated with 4 μg of anti-FLAG for 30 minutes at 4°C and then 150 μg of goat anti-mouse IgG agarose-conjugated antibody (Sigma-Aldrich) were added. Protein extracts were then incubated overnight at 4°C with gentle shaking. Immunoprecipitate was collected by centrifugation at 4°C, 2.500 rpm for 5 minutes and washed four times with RIPA/DOC buffer. Finally, the agarose pellet was denatured at 95°C for 2 minutes and analyzed by SDS-PAGE followed by immunoblotting with specific antibodies.
Electrophoretic mobility shift assays (EMSA)
Nuclear protein extracts and TNT co-traduced proteins (3 μg) were analyzed using the [α-32P]-dCTP-labeled double-stranded synthetic wild-type HIV enhancer oligonucleotide 5'-AGCTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA-3' containing both -κB consensus motifs. The nucleoprotein-oligonucleotide complexes were analyzed by electrophoresis on a non-denaturing 6% polyacrylamide gel.
HIV-1 replication assay
Resting PBLs were transfected with pNL4.3-Renilla, pNL4.3-wt or pdI-NF vectors. Viral replication was assessed after 72 hours by quantification of HIV-1 p24 gag antigen in culture supernatants using an enzyme-like immunoassay (Innotest™ HIV Ag mAb, Innogenetics, Barcelona, Spain) or by Renilla quantification. Briefly, cells were resuspended in 100 μl of lysis buffer 1× provided by Renilla Luciferase Assay System Kit (Promega), incubated for 30 minutes at 4°C and centrifuged 5 minutes at 13.000 rpm. RLUs were measured in supernatants with a luminometer Sirius (Berthold Detection Systems, Oak Ridge, TN) after adding the appropriate substrate.
We thank Sanne Spijkers and Sonia Cabezas for technical assistance; Dr. Javier García-Pérez for excellent support in standardization of directed mutagenesis assays and for providing pNL4.3-Renilla vector; and Olga Palao for secretarial assistance. We also acknowledge Dr. Alexander Hoffmann (Department of Chemistry and Biochemistry, University of California, San Diego, CA) for kind gift of mouse 3T3-p65ko fibroblast cells. We thank Centro Nacional de Transfusiones from Comunidad de Madrid, Spain, for providing the buffy coats. This work was supported by the following projects: FIPSE 36633/07; ISCIII-RETIC RD06/0006; FIPSE 36584/06; FIS PI040614; Network of Excellence EUROPRISE; and VIRHOST Network from Comunidad de Madrid, Spain. M.R. López-Huertas is a pre-doctoral fellow funded by FIPSE 36453/03 and "Plan Nacional del SIDA" (MVI 1434/05-5).
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