Input:
Online supplemental material.
Fig. S1 shows the induction of RUNX1, RUNX3, and FOXP3 mRNA in human CD4+ T cells after anti-CD2/3/28 mAb and TGF-beta stimulation. Fig. S2 shows decreased RUNX1 and RUNX3 mRNA and protein expression after siRNA-mediated knockdown and decreased FOXP3 expression in human CD4+ T cells after RUNX1 and RUNX3 knockdown. Fig. S3 shows the quantification of IL-4, IL-5, IL-10, IL-13, and IFN-gamma levels in control siRNA transfected or RUNX1 and RUNX3 siRNA transfected human CD4+ T cells. Fig. S4 shows the putative RUNX binding sites in the FOXP3 core promoter sequence of human, mouse, and rat. Fig. S5 shows the induction of FOXP3 protein after overexpression of RUNX1 and RUNX3 in human CD4+ T cells. Fig. S6 shows that endogenous IL-4 and IFN-gamma do not effect Foxp3 expression in naive CD4+ T cells of CbfbF/F CD4-cre and CbfbF/F control mice, which were stimulated with anti-CD3 and anti-CD28 mAbs, IL-2 and TGF-beta in the absence or presence of anti-IL-4 and anti-IFN-gamma neutralizing mAbs. Fig. S7 shows similar cell death (A) and proliferation (B) of Foxp3+ and Foxp3- cells in CbfbF/F CD4-cre and CbfbF/F control mice cultures. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090596/DC1.

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Input: RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-beta stimulation in the presence of IL-2. Real-time PCR of human naive CD4+ T cells after 48 h of culture. Bars show the mean +/- SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-beta in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-beta and IL-2. Bars show the mean +/- SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean +/- SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-beta in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

induction

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Input: RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127-, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127-, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-beta, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25- cells. Bars show the mean +/- SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy. Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls. HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining. Data shown are representative from one of the three tissue samples with similar results. Bars, 5 microm. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

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Input: Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide-transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean +/- SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFbeta to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-beta. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean +/- SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P < 0.05

Binding

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Input: Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector. Bars show the mean luciferase activity +/- SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene. Bars show the mean luciferase activity +/- SD measured as arbitrary light units of three independent experiments.

transfected

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Input: Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, +/- 10 nM retinoic acid (RA), and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-gamma expression. One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 x 106 cells per transfer). 6 wk later, TCRbeta+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.

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deficient

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Input: CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8- T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-beta. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown.

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Input:
IKKbeta phosphorylation regulates RPS3 nuclear translocation and NF-kappaB function duringEscherichia coliO157:H7 infection
NF-kappaB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-kappaB subunit that directs specific gene transcription. However, it is unknown how RPS3 nuclear translocation is regulated. Here we report that IKKbeta phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli. Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model. Thus, the IKKbeta-dependent modification of a specific amino acid in RPS3 promotes specific NF-kappaB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.

translocation

nuclear

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nuclear

localization

nuclear

inhibited

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blocked

inhibited

phosphorylation

IKKbeta

phosphorylation

IKKbeta

serine 209

phosphorylation

crucial

phosphorylation

in response to

in response to

localization

activating

activating

modification

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Input: Nuclear Factor-kappa B (NF-kappaB) regulates crucial cellular functions and diverse stimuli activate this pleiotropic transcription factor, which in turn regulates a vast array of genetic targets1-3. The best-known mammalian NF-kappaB subunits are Rel proteins, including RelA (p65), RelB, c-Rel, p50, and p52 (refs. 4,5). However, we recently demonstrated that ribosomal protein S3 (RPS3) is a key non-Rel subunit of certain native NF-kappaB complexes6. RPS3 is defined as a "specifier" subunit of NF-kappaB, because it facilitates high affinity DNA binding thus determining the regulatory specificity of NF-kappaB for selected target genes7. RPS3 regulation of NF-kappaB governs key physiological processes, including immunoglobulin kappa light chain gene expression and receptor editing in B cells6,8, cytokine production in T cells6, and in host defense against enterohemorrhagicEscherichia coli(EHEC)9. In particular, theE. coliO157:H7 type III secretion system (T3SS) effector protein NleH1 selectively blocks NF-kappaB target gene transcription by attenuating RPS3 nuclear translocation, without affecting p65 localization9. Nonetheless, how specific NF-kappaB activating signals induce RPS3 nuclear translocation is unknown.Extra-ribosomal functions have been ascribed to ribosomal proteins10. Besides binding RNA within the 40S ribosomal subunit, RPS3 participates in transcription6, DNA repair11,12, and apoptosis13. Whether or not RPS3 is phosphorylated had been controversial14-18. Since kinase cascades play a critical role in NF-kappaB regulation, we tested whether RPS3 is phosphorylated in the context of NF-kappaB activation and sought to identify the responsible kinase19. Additionally, we aimed to define a regulatory role for the C-terminal tail of RPS3 whose function was unknown.Here we show that the Inhibitor of kappaB (IkappaB) kinase beta (IKKbeta) phosphorylated RPS3 at serine 209 (S209). RPS3 S209 phosphorylation enhanced its association with importin-alpha, mediating RPS3 entry into the karyopherin pathway for nuclear translocation. Furthermore, theE. coliNleH1 effector specifically inhibited RPS3 S209 revealing howE. coliO157:H7 inhibits this important innate immune response mechanism.

binding

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Input:
RPS3 phosphorylation in response to NF-kappaB activation
To test whether RPS3 is phosphorylated during NF-kappaB activation, we performed 32P-labeling experiments in tumor necrosis factor (TNF)-stimulated HEK 293T cells. While RPS3 was scarcely phosphorylated in unstimulated cells, we observed a marked increase in 32P-incorporation after TNF stimulation despite no increase in RPS3 protein (Fig. 1a). To determine which RPS3 residues were phosphorylated, we immunoprecipitated RPS3 from either resting or stimulated cells and performed immunoblotting with phosphorylation-specific antibodies. Both TNF and phorbol myristate acetate/ionomycin (PMA+I) stimulated rapid phosphorylation and degradaion of IkappaBalpha within 5 min which was accompanied by RPS3 phosphorylation on serine residues (Fig. 1b and data not shown), similar to the in vivo labeling. We did not detect tyrosine- or threonine-phosphorylation of RPS3 (Fig. 1b).

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Input:
RPS3 and IKKbeta interaction
The activation of the inhibitor of kappaB kinase (IKK), consisting of a regulatory subunit IKKgamma and two catalytic subunits, IKKalpha and IKKbeta, is critical for the phosphorylation and dispatch of the inhibitory IkappaBs and the liberation of NF-kappaB20-22. Given that RPS3 can be found in the cytoplasmic p65-p50-IkappaBalpha inhibitory complex in resting cells6, we hypothesized that activated IKKbeta might also bind to and phosphorylate RPS3. First, we found that ectopically expressed IKKbeta and RPS3 interacted (Fig. 1c). We next examined resting Jurkat cells and detected a modest endogenous IKKbeta-RPS3 interaction (Fig. 1d), potentially accounting for the basal NF-kappaB transcription required for cell proliferation and survival. RPS3-IKKbeta association was clearly augmented upon TNF stimulation, peaking at 10 min. (Fig. 1d), following similar kinetics to RPS3 serine phosphorylation (Fig. 1b). By contrast, there was no detectable interaction between RPS3 and IKKalpha (Fig. 1d).

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Input:
IKKbeta is required for RPS3 nuclear translocation
To examine whether the RPS3-IKKbeta interaction is required for RPS3 nuclear translocation, we knocked down IKKalpha or IKKbeta expression with siRNAs (Supplementary Fig. 1) and then observed stimulation-induced RPS3 nuclear migration by confocal microscopy. Both TNF and PMA+I triggered RPS3 nuclear translocation in Jurkat cells transfected with a scrambled nonspecific (NS) siRNA (Fig. 2a)6. RPS3 nuclear translocation was only slightly, if at all, impaired by IKKalpha-silencing. Conversely, knockdown of IKKbeta attenuated 60-70% of RPS3 nuclear accumulation following stimulation (Fig. 2a). Immunoblotting of nuclear fractions confirmed that full expression of IKKbeta, but not IKKalpha, was necessary for activation-induced RPS3 nuclear translocation (Fig. 2b). Control immunoblots revealed that p65 nuclear translocation was blocked under the same conditions (Fig. 2b).
We next examined the nuclear translocation of RPS3 in cells ectopically expressing either kinase-dead (SSAA) or constitutively-active (SSEE) mutant IKKbeta proteins. As expected, the SSEE, but not SSAA, mutant of IKKbeta induced NF-kappaB-dependent luciferase reporter activity (Fig. 2c, left). Whereas RPS3 remained cytosolic in IKKbeta (SSAA)-expressing cells (Fig. 2c, right), a substantial proportion of RPS3 translocated to the nucleus in cells expressing IKKbeta (SSEE) (Fig. 2c, right). The percentage of cells containing detectable nuclear RPS3 increased 5-fold in IKKbeta (SSEE)-expressing cells, but not in IKKbeta (SSAA)-expressing ones (Fig. 2d and Supplementary Fig. 2). Thus, IKKbeta activity is necessary and sufficient for RPS3 nuclear translocation in response to NF-kappaB activating stimuli.

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Input:
IkappaBalpha degradation and RPS3 nuclear translocation
Importin-alpha regulates the nuclear import of NF-kappaB Rel subunits23, 24. RPS3 harbors a nuclear localization signal (NLS) sequence and its nuclear translocation occurs in parallel to, but independently of, p65 translocation6. We envisioned that RPS3 could also utilize the importin-alpha/beta pathway. Consistent with this notion, RPS3 association with importin-alpha, but not importin-beta, was enhanced in TNF-stimulated cells (Fig. 3a). Therefore, we examined whether RPS3 binding to importin-alpha is essential for nuclear translocation during NF-kappaB activation.
Since IkappaBalpha degradation is a prerequisite to unmask the NLS of p65, and both RPS3 and IkappaBalpha bind to p65 in the cytoplasmic inhibitory complex, we tested whether IkappaBalpha degradation is required for the liberation of RPS3. We measured the association of RPS3 with importin-alpha in 293T cells overexpressing wild-type IkappaBalpha or an IkappaBalpha mutant (SSAA) resistant to IKKbeta-induced phosphorylation and degradation. In cells transfected with wild-type IkappaBalpha, TNF stimulation augmented the interaction of RPS3 and importin-alpha to a similar degree as in non-transfected cells. By contrast, we observed that the RPS3-importin-alpha association was abolished by the presence of non-degradable IkappaBalpha (Fig. 3b).
To examine whether IkappaBalpha is the only cytoplasmic barrier precluding RPS3 nuclear translocation, we measured both RPS3-importin-alpha association and nuclear RPS3 after reducing IkappaBalpha expression. Compared with nonspecific siRNA, siRNA targeting of IkappaBalpha completely depleted IkappaBalpha in Jurkat cells (Fig. 3c, input). Nevertheless, the RPS3-importin-alpha association was not augmented (Fig. 3c), nor was significant nuclear RPS3 detected (Fig. 3d). Moreover, cells treated with sodium pervanadate (Pv) to induce IkappaBalpha degradation through an IKK-independent mechanism25-27 did not show increased association between RPS3 and importin-alpha (Fig. 3e and Supplementary Fig. 3b) or nuclear accumulation of RPS3, despite complete IkappaBalpha degradation (Supplementary Fig. 3c). We further examined whether a subsequent NF-kappaB activation signal independently promotes the importin-alpha association and nuclear transport of RPS3 after IkappaBalpha degradation. We found that TNF stimulation following Pv treatment was required for the RPS3-importin-alpha association, comparable to TNF stimulation alone (Fig. 3e). Thus, IkappaBalpha phosphorylation and degradation itself is required but not sufficient to cause RPS3 association with importin-alpha followed by nuclear translocation. Rather, an additional signal, potentially IKKbeta phosphorylation of RPS3, is required.

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siRNA

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Input:
IKKbeta phosphorylates RPS3 at serine 209
Although originally defined as the kinase that phosphorylates IkappaB19, IKKbeta also phosphorylates unrelated substrates including 14-3-3beta and Bcl10, which lack the IKK consensus motif (DpSGYXpS/T)28. We therefore hypothesized that IKKbeta could directly phosphorylate RPS3. By in vitro kinase assays using recombinant IKK and RPS3 proteins, we observed strong incorporation of 32P in autophosphorylatd IKKalpha and IKKbeta (Fig. 4a, lanes 2-7) as well as phosporylated GST-IkappaBalpha (1-54) (Supplementary Fig. 4), but not the GST protein alone (Fig. 4a, lanes 3 and 6), when either IKKalpha or IKKbeta was used. We discovered that GST-RPS3 could be phosphorylated by IKKbeta, but not IKKalpha, in vitro (Fig. 4a, compare lanes 4 and 7).
To identify the RPS3 amino acid residue(s) phosphorylated by IKKbeta, we performed liquid chromatography-tandem mass spectrometry analyses using in vitro phosphorylated RPS3. The results indicated that IKKbeta phosphorylated S209, located in the RPS3 C-terminus (Fig. 4b). RPS3 amino acid sequence alignment revealed that S209 is conserved in many species throughout phylogeny with the exception of Caenorhabditis elegans and Schizosaccharomyces pombe, two organisms that do not possess the NF-kappaB signal pathway (Supplementary Fig. 5).
To verify biochemically that S209 is an IKKbeta substrate, we performed 32P-labeling in vitro kinase assays with recombinant wild-type or S209A mutant RPS3 proteins. Compared with the wild-type protein, the S209A mutation reduced IKKbeta-mediated RPS3 phosphorylation (Fig. 4c). There might be alternative phosphorylation site(s) under these conditions given modest residual phosphorylated RPS3 (Fig. 4c). RPS3 S209 does not fall within a conventional IKK recognition motif, but rather resides in a sequence motif (XXXpS/TXXE), potentially recognized by casein kinase II (CK2). Although IKKbeta kinase can display a CK2-like phosphorylation specificity29, no CK2 protein was detectable in our recombinant IKK proteins (Supplementary Fig. 6). Thus, RPS3 S209 phosphorylation was due to the alternate specificity of the IKKbeta kinase rather than any trace amount of CK2 bound to IKKs. To determine whether S209 is the critical site at which IKKbeta phosphorylates RPS3 in living cells, we transfected the wild-type or S209A mutant Flag-RPS3 alone, or together with IKKbeta into cells. Indeed, we observed that overexpressing IKKbeta enhanced Flag-RPS3 phosphorylation, but phosphorylation was effectively eliminated by alanine substitution indicating that S209 is the predominant target site for IKKbeta phosphorylation (Fig. 4d). We next generated a phospho-S209 RPS3 antibody and confirmed that endogenous RPS3 was phosphorylated at S209 in a time-dependent manner upon TNF stimulation (Fig. 4e). Thus, the RPS3 C-terminal tail potentially contains an important regulatory site.

recognized

sequence motif

transfected

transfected

IKKbeta

reduced

mutation

mediated

eliminated

phosphorylation

alanine substitution

phosphorylates

IKKbeta

serine 209

phosphorylates

IKKbeta

phosphorylates

IKKbeta

phosphorylate

IKKbeta

incorporation of 32P

IKKalpha

incorporation of 32P

IKKbeta

autophosphorylatd

IKKalpha

autophosphorylatd

IKKbeta

phosporylated

GST-IkappaBalpha

phosporylated

GST

phosphorylated

IKKbeta

phosphorylated

IKKalpha

phosphorylated

amino acid residue(s)

IKKbeta

phosphorylated

phosphorylated

IKKbeta

phosphorylation

phosphorylated

phosphorylation

phosphorylates

IKKbeta

phosphorylation

phosphorylation

phosphorylated

mediated

IKKbeta

phosphorylation

due

phosphorylation

IKKbeta

critical

phosphorylates

overexpressing

IKKbeta

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overexpressing

phosphorylation

upon

dependent

mutant

mutation

alanine substitution

dependent

phosphorylated


Input:
Phosphorylation of RPS3 and its NF-kappaB function
We next examined whether S209 phosphorylation plays a role in the nuclear translocation of RPS3 during NF-kappaB activation. Subcellular fractions from either wild-type or S209A mutant RPS3-transfected cells were prepared and blotted for heat-shock protein 90 (hsp90), a cytoplasmic protein, and poly (ADP-ribose) polymerase (PARP), a nuclear protein, confirming a clean separation (Fig. 5a). As expected, PMA+I stimulation triggered wild-type Flag-RPS3 nuclear translocation (Fig. 5a). However, RPS3 (S209A) nuclear translocation was attenuated (Fig. 5a). We also tested the impact of activating NF-kappaB by overexpressing IKKbeta on RPS3 nuclear translocation. IKKbeta overexpression activated NF-kappaB measured by luciferase assays (Supplementary Fig. 7), and also induced the nuclear translocation of wild-type, but not S209A, RPS3 (Fig. 5b). These data suggest that S209 phosphorylation is critical for the NF-kappaB activation-induced RPS3 nuclear translocation.
To examine the role of S209 phosphorylation of RPS3 to its NF-kappaB function6, 7, 30, we silenced endogenous RPS3 expression using an siRNA that targets the 3' untranslated region (3' UTR) of RPS3 mRNA, followed by complementation with either wild-type or S209A mutant RPS3 via transfection. As expected, RPS3 siRNA severely reduced endogenous RPS3 abundance compared to NS siRNA, but did not affect the robust expression of Flag-tagged RPS3 from a transfected construct lacking the 3' UTR (Fig. 5c). We also found that RPS3 knockdown reduced TNF-induced expression of an Ig kappaB-driven luciferase construct6 (Fig. 5d). The impaired luciferase signal caused by RPS3 deficiency was completely restored by transfecting wild-type, but not by S209A RPS3 (Fig. 5d), despite equivalent expression (Fig. 5c). Moreover, the failure of S209A RPS3 to restore luciferase activity did not result from defective translation because the transient overexpression of green fluorescent protein (GFP) was comparable in cells complemented with wild-type or S029A RPS3 (Supplementary Fig. 8). Taken together, these data suggest that RPS3 S209 phosphorylation is critical for NF-kappaB activity involving the canonical Ig kappaB site.
We next used chromatin immunoprecipitation to determine whether S209 phosphorylation affects RPS3 and p65 recruitment to specific kappaB sites in intact chromatin during NF-kappaB activation. In RPS3 knockdown cells, PMA+I stimulated the recruitment of ectopically expressed, Flag-tagged wild-type, but not S209A RPS3 to the kappaB sites of the NFKBIA and IL8 promoters (Fig. 5e). While expressing RPS3 S209A had no impact on p65 nuclear translocation, it substantially attenuated p65 recruitment (Fig. 5e). Additional experiments revealed that p65 attraction to RPS3-independent NF-kappaB target gene promoters such as CD25 was increased (Supplementary Fig. 9), consistent with our previous observations6. There was no significant Flag-RPS3 or p65 recruitment to ACTB promoter lacking kappaB sites (Fig. 5e), suggesting the recruitment was kappaB site-specific. Thus, the recruitment of RPS3 as well as the contingent recruitment of p65 to key promoters depended on S209.
Interleukin 8 (IL-8) secretion induced by either T cell receptor (TCR) agonist stimulation or PMA+I was decreased as a consequence of reduced RPS3/p65 recruitment to the IL8 kappaB sites in the presence of S209A mutant compared to wild-type RPS3 (Supplementary Fig. 10). However, cell surface CD25 expression was comparable between the wild-type and S209A RPS3 transfected cells (Supplementary Fig. 11). Therefore, RPS3 S209 phosphorylation by IKKbeta is apparently required for RPS3 in directing NF-kappaB to a specific subset of target genes.

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NleH1 inhibits RPS3 phosphorylation in vitro
EHEC pathogens are important causative agents of both foodborne disease and pediatric renal failure31. EHEC utilize T3SS to inject effector proteins directly into intestinal epithelial cells32, a subset of which inhibit NF-kappaB-dependent innate responses9, 33-38. The E. coli O157:H7 EDL933 effector protein NleH1 binds to and attenuates RPS3 nuclear translocation, thus impairing RPS3-dependent NF-kappaB signaling9. We therefore hypothesized that NleH1 may function by inhibiting RPS3 S209 phosphorylation. As expected, transfecting increasing amounts of NleH1-HA plasmid blocked TNFalpha-induced NF-kappaB activation in a dose-dependent manner (Fig. 6a-b)9. Remarkably, NleH1 reduced both TNF-induced, as well as basal RPS3 phosphorylation to roughly 20% of vehicle control (Fig. 6c). Expressing NleH1 does not interfere with either TNF-induced IKK activation or IkappaBalpha degradation, consistent with the lack of NleH1 impact on p65 nuclear translocation9 (Fig. 6c).
To determine if NleH1 inhibits RPS3 phosphorylation, we infected HeLa cells with E. coli O157:H7 strains possessing or lacking either nleH1 (deltanleH1) or with a strain lacking a functional T3SS unable to inject NleH1 into mammalian cells (deltaescN). In uninfected cells, TNF-treatment stimulated a ~7-fold increase in RPS3 S209 phosphorylation, peaking at 30 minutes (Fig. 6d). By contrast, RPS3 S209 phosphorylation was substantially impaired in cells infected with wild-type E. coli O157:H7 (Fig. 6d). However, TNF-induced RPS3 S209 phosphorylation was unimpaired in cells infected with either deltanleH1 or deltaescN (Fig. 6d). We showed previously that wild-type, but not deltanleH1 or deltaescN E. coli O157:H7 significantly attenuated TNF-induced RPS3 nuclear translocation9. The parallel between RPS3 phosphorylation and its nuclear translocation during E. coli infection provides evidence in the context of an NF-kappaB-dependent disease process that RPS3 S209 phosphorylation is important for nuclear translocation.
Our discovery that NleH1 inhibits RPS3 S209 phosphorylation suggested that it should also blocks RPS3-dependent NF-kappaB target gene transcription (e.g. IL8, NFKBIA, and TNFAIP3). Indeed, these genes were only modestly upregulated in cells infected with wild-type E. coli O157:H7, but significantly induced in cells infected with either deltanleH1 or deltaescN strains (Fig. 6e). In contrast, deleting nleH1 had no impact on the expression of RPS3-independent genes, including CD25 and TNFSF13B (Supplementary Fig. 12). Together these results demonstrate that NleH1 specifically inhibits the protective immune response by directly blocking RPS3 S209 phosphorylation and thereby impairing critical RPS3-dependent NF-kappaB target genes.

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NleH1 inhibits RPS3 S209 phosphorylation in vivo
We previously utilized a gnotobiotic piglet infection model to determine that piglets infected with deltanleH1 mutant died more rapidly than those infected with wild-type E. coli O157:H7. Piglets infected with deltanleH1 displayed clinical disease consistent with a robust inflammatory response, but with reduced bacterial colonization and little diarrhea9. While seemingly paradoxical, based on our cell culture data (Fig. 6d), we hypothesized that NleH1 blocked RPS3 S209 phosphorylation in vivo, thereby preventing RPS3 nuclear translocation in infected piglets. We isolated piglet colons at necropsy, and subjected them to immunohistochemistry using our phospho-RPS3 antibody. Consistent with in vitro data, piglets infected with wild-type E. coli O157:H7 exhibited diffuse and low intensity phospho-RPS3 staining, whereas in piglets infected with deltanleH1 mutant, phospho-RPS3 expression was florid and intense (Fig. 6f). These data demonstrate that NleH1 inhibits RPS3 S209 phosphorylation both in vitro and in vivo, which might benefit the bacterium in colonization and transmission.

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NleH1 steers the IKKbeta substrate specificities
NleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. To explore the mechanism by which NleH1 inhibits RPS3 S209 phosphorylation, we first performed an in vitro kinase assay with purified wild-type His-NleH1 protein and a mutant His-NleH1 (K159A) protein, confirming that NleH1 is autophosphorylated and the K159A is an NleH1 kinase-dead mutant (Fig. 7a). To examine whether the kinase activity is required for NleH1 to inhibit IKKbeta phosphorlyation of RPS3 on S209, we ectopically expressing either wild-type or K159A NleH1 in 293T cells. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKbeta-mediated phosphorylation.
Citrobacter rodentium is a mouse pathogen that shares pathogenic strategies with E. coli 39, most notably for our investigation, C. rodentium NleH inhibited RPS3 nuclear translocation and RPS3-dependent NF-kappaB luciferase activity to an extent equivalent to E. coli NleH1 (ref. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ~3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, deltanleH). We further examined the role of NleH1 kinase activity using the C. rodentium deltanleH strain as a background on which to express either wild-type or K159A E. coli NleH1. Complementing deltanleH mutant with wild-type NleH1 almost abolished TNF-induced RPS3 S209 phosphorylation whereas complementing with K159A failed to inhibit RPS3 phosphorylation (Fig. 7c). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.
We next examined whether the inhibitory activity of NleH1 is sufficiently robust to impair the strong nuclear translocation of RPS3 trigged by the constitutively-active IKKbeta (IKKbeta [SSEE]) (Fig. 2d). We found that ectopically expressing either wild-type or SSEE IKKbeta proteins, triggered more RPS3 nuclear translocation than the kinase-dead IKKbeta (SSAA) protein (Fig. 7d). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). In contrast, infecting with either deltanleH1 or deltaescN strains only slightly impaired RPS3 nuclear translocation in either IKKbeta- or IKKbeta (SSEE)-expressing cells (Fig. 7d). As expected, E. coli infections did not affect the RPS3 nuclear translocation in IKKbeta (SSAA)-expressing cells, where NF-kappaB signaling was low (Fig. 7d). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKbeta.
We examined whether NleH1 could directly phosphorylate IKKbeta thus inhibiting IKKbeta-mediated RPS3 S209 phosphorylation. We performed in vitro kinase assays using immunoprecipitated Flag-IKKbeta (K44A) as substrate and recombinant His-NleH1 as kinase, so that IKKbeta autophosphorylation would not obscure NleH1-induced phosphorylation. However, we did not observe any detectable 32P incorporation in IKKbeta (Supplementary Fig. 13), thus ruling out this possibility.
We then tested the hypothesis that NleH1 could alter the IKKbeta substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKbeta. As expected, IKKbeta phosphorylated RPS3 (Fig. 7e, lane 7) and GST-IkappaBalpha (1-54) protein (Fig. 7e, lane 9), demonstrating it harbors either CK2 or IKK substrate specificity. Preincubation of IKKbeta with NleH1 reduced IKKbeta-mediated RPS3 phosphorylation, i.e. the CK2 kinase specificity, but not IKKbeta-mediated GST-IkappaBalpha phosphorylation, i.e. the IKK kinase specificity (Fig. 7e). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IkappaBalpha (Fig. 7e). Taken together, NleH1 blocks the CK2 substrate specificity of IKKbeta thus inhibiting the IKKbeta-mediated RPS3 S209 phosphorylation thus representing a novel strategy by E. coli O157:H7 to alter the host innate immune response.

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RPS3 was previously demonstrated to function as an integral subunit conferring NF-kappaB regulatory specificity6. Here we sought to elucidate how NF-kappaB activation signaling triggers RPS3 to translocate and participate in NF-kappaB function in the nucleus. We demonstrate that IKKbeta-mediated RPS3 S209 phosphorylation represents a critical determinant in governing its nuclear import thus unveiling a novel mechanism behind NF-kappaB regulatory specificity. IKKbeta is the major kinase that phosphorylates IkappaBs in the classical NF-kappaB pathway, leading to their subsequent degradation40. Strikingly, RPS3 possesses not any consensus IKK motif; instead, S209 is centered in a consensus CK2 motif. The recent observation that human IKKbeta displayed CK2-like phosphorylation specificity29 coincides with our evidence that recombinant IKKbeta, but not IKKalpha, phosphorylated RPS3. We found this phosphorylation is a critical modulation for RPS3 nuclear translocation (via importin-alpha) and engagement in specific NF-kappaB transcription. CK2 was previously shown to phosphorylate p65 and to bind to and phosphorylate IKKbeta41-43, however, we ruled out the possibility that the IKKbeta-bound CK2 could account for the observed RPS3 phosphorylation because no CK2 was detected in the IKKbeta preparations used for the in vitro kinase assay. Because RPS3 only harbors the CK2 motif and not a traditional IKK motif, this RPS3 regulatory function could explain why IKK harbors the alternative substrate phosphorylation capability.
More importantly, our study has elucidated how RPS3 is biochemically integrated into NF-kappaB activation signaling in a manner that is pivotal for the pathogenesis of foodborne pathogen E. coli O157:H7. IKKbeta-mediated RPS3 S209 phosphorylation is a critical target modulated by this pathogen to subvert host NF-kappaB signaling. The bacterial effector NleH1 specifically binds to RPS3 once injected into host cells and profoundly suppresses NF-kappaB and its attendant protective immune responses9. Our data now show that NleH1 selectively inhibits RPS3 phosphorylation, thus retarding its nuclear translocation and subsequent NF-kappaB function, without altering other NF-kappaB signaling. Although NleH1 did not directly phosphorylate IKKbeta, its kinase activity was required to inhibit IKKbeta-mediated RPS3 S209 phosphorylation. Many bacteria pathogens have products that target key kinases to inactivate them in host cells, whereas E. coli O157:H7 employed NleH1 to steer the substrate specificity of IKKbeta thus specifically fine-tuning host NF-kappaB signaling. This could represent a novel strategy to fine-tune host NF-kappaB signaling that could be shared by other pathogens. These data provide new insights into the poorly understood action mechanism for most T3SS effectors.
NleH1 attenuates the transcription of RPS3-dependent, but not all, NF-kappaB target genes, in particular those genes associated with acute proinflammatory responses, including IL8 and TNF. In contrast, NleH1 does not block NF-kappaB p65 nuclear translocation, which suggests that certain p65-dependent but RPS3-independent NF-kappaB target genes might thus be beneficial for E. coli O157:H7 to replicate and disseminate in the host. By selectively inhibiting RPS3 and its attendant NF-kappaB function with NleH1, the pathogen achieves the ability to increase colonization and diarrhea yet limiting the mortality of the host. This seemingly paradoxical combination of effects make sense when one considers that increased bacterial load and diarrhea together with survival of the infected host would promote the spreading of the bacteria among a population of susceptible individuals. Such complex and paradoxical pathological effects that influence the spread of disease are often poorly understood at the molecular level. Our data elucidate how alterations in selective NF-kappaB function, achieved by impeding RPS3, but not altering p65 nuclear translocation, can influence specific cytokines that affect bacterial colonization, diarrhea diseases and mortality. It may be fruitful in attempting to understand other infectious and autoimmune diseases involving NF-kappaB to consider selective effects of subunits such as RPS3 in addition to global NF-kappaB inhibition.

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Cells and Reagents
Jurkat E6.1, HEK293T and HeLa cells were cultured in RPMI 1640 and DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin, respectively. IkappaBalpha (C-21, sc-371), p65 (C-20, sc-372), and phospho-threonine (H-2, sc-5267) antibodies were from Santa Cruz Biotechnology; beta-actin (AC-15, A5441), Flag (M2, F3165), HA (HA-7, H3663), importin-alpha (IM-75, I1784), and importin-beta (31H4, I2534) antibodies were from Sigma; PARP (C2-10, 556362), IKKalpha (B78-1, 556532), and IKKbeta (24, 611254) antibodies were from BD Pharmingen; CK2alpha (31, 611610) and Hsp90 (68, 610418) antibodies were from BD Transduction Laboratories; phospho-IkappaBalpha (5A5, 9246S) and phospho-IKKalpha/beta (16A6, 2697S) antibodies were from Cell Signaling Technology; phospho-serine (AB1603) and phospho-tyrosine (4G10, 05-777) antibodies were from Millipore. The rabbit polyclonal RPS3 antiserum was as described previously6. The rabbit polyclonal antibody specific for S209 phosphorylated RPS3 was generated and affinity purified by Primm Biotech using the peptide NH2-CKPLPDHV(Sp)IVE-COOH.


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Plasmid Constructs
The Flag-IKKbeta (SSEE), Flag-IKKbeta (SSAA), and HA-IkappaBalpha (SSAA) constructs were provided by C. Wu (NCI, Bethesda) and U. Siebenlist (NIAID, Bethesda), respectively. The HA-IkappaBalpha and IKKbeta (K44A)-Flag plasmids were purchased from Addgene44, 45. The Flag-RPS3, GST-RPS3, HA-RPS3, VN-HA, NleH1-HA plasmids were described previously6, 9. The point mutants of RPS3 were generated by site-directed mutagenesis using the Quick Change Kit (Stratagene) with primers forward 5'-CTGCCTGACCACGTGGCCATTGTGGAACCCAAA-3' and reverse 5'-TTTGGGTTCCACAATGGCCACGTGGTCAGGCAG-3' for S209A. All mutants were verified by DNA sequencing.


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32P in vivo Labeling
HEK 293T cells were labeled with 2 mCi/ml 32P-orthophosphate (Perkin Elmer) in phosphate-free medium (Invitrogen) for 2 h. Cells were then left untreated or treated with TNF (50 ng/ml, R&D Systems) for indicated periods. Cell lysates were prepared and used for immunoprecipitations with RPS3 antibody.


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In Vitro Kinase Assay
Kinase-active recombinant IKKbeta and IKKalpha proteins were purchased from Active Motif and Millipore, respectively. Bacterially purified glutathione S-transferase (GST), GST-IkappaBalpha (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates. The in vitro kinase assay was performed as previously described29. Briefly, enzyme (100 ng) and substrate (2 mug) were co-incubated in IKK reaction buffer (25 mM Tris-HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 muCi 32P-gamma-ATP (GE Healthcare) added at 37 degreesC for 30 min. The reactions were resolved by SDS-PAGE and visualized by autoradiography.


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LC-MS/MS Analysis
GST or GST-RPS3 was incubated with recombinant IKKbeta protein as described above in an in vitro kinase assay reaction conducted without 32P-gamma-ATP labeling. The reaction was separated by SDS-PAGE, and the protein gel was stained with Colloidal Blue (Invitrogen). The corresponding protein fragments were excised and subjected to trypsin digestion and LC-MS/MS at the Yale Cancer Center Mass Spectrometry Resource (New Haven, CT).


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RNAi and Transfection
The siRNA (sense-strand sequence) IKKalpha, 5'-AUGACAGAGAAUGAUCAUGUUCUGC -3'; IKKbeta, 5'-GCAGCAAGGAGAACAGAGGUUAAUA -3'; IkappaBalpha, 5'-GAGCUCCGAGACUUUCGAGGAAAUA -3'; RPS3-3' UTR, 5'-GGAUGUUGCUCUCUAAAGACC -3' (Invitrogen). Transient transfection of siRNA and DNA constructs into Jurkat cells and 293T cells was described previously6.


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Subcellular Fractionation
Subcellular fractionation was performed by differential centrifugation as previously described6. Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 degreesC for 5 min. Lysates were centrifuged at 4 degreesC, 500 x g for 3 min, and supernatants were collected as cytosolic fractions. Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 degreesC for 10 min. Supernatants were collected as nuclear fractions following a centrifuge at 4 degreesC, 13,800 x g for 10 min.


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Luciferase Reporter Gene Assays
Luciferase reporter gene assays were performed as previously described6. Briefly, cells were cotransfected at a ratio of 10:1 with various promoter-driven firefly luciferase constructs to the Renilla luciferase pTKRL plasmid, together with indicated plasmids. Cells were cultured for 1-2 days and then stimulated in triplicate before harvest. Lysates were analyzed using the Dual-Luciferase Kit (Promega).


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Chromatin Immunoprecipitation (ChIP)
ChIP assays was performed as previously described6. The primers used to amplify the promoter region adjacent to the kappaB sites of IL8 and NFKBIA, as well as ACTB have been described6.


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Immunofluorescence Microscopy
Confocal microscopy was performed as previously described6. Briefly, cells were fixed with 4 % paraformaldehyde in PBS and then Cellspin mounted onto slides. The fixed cells were then permeabilized with 0.05 % Triton X-100 in PBS and stained with FITC-conjugated rabbit anti-RPS3 antibodies (Primm Biotech), or AlexaFluor 594-conjugated rat anti-Flag antibodies (BD) for 40 min together with 1 mug/ml of Hoechst 33342 (Sigma) for 5 min at 25 degreesC. The slides were then rinsed with PBS three times and cover mounted for fluorescence microscopy.


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Immunoprecipitation and immunoblot
The cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 x protease inhibitor cocktail (Roche) and 1 x phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min. The lysates were centrifuged at 10,000 x g at 4 degreesC for 10 min to remove insoluble material. After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4degreesC. The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions. Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6. For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min. Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46. Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.


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ELISA
The amount of IL-8 present in supernatants collected from Jurkat cell culture was measured using a Human Interleukin-8 ELISA Ready-SET-Go kit (eBioscience) according to the manufacturer's instructions.


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Cell Infections
HeLa cells were infected with E. coli O157:H7 or C. rodentium strains as described previously9.


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Immunohistochemistry
Gnotobiotic piglets were infected with E. coli O157:H7 strains as described previously9. Spiral colon specimens were collected at necropsy and embedded in paraffin. Paraffin sectioning and immunohistochemical staining using phospho-RPS3 antibody were performed by Histoserv Inc.


Input:
Human immunodeficiency virus-1 Tat activates NF-kappaB via physical interaction with IkappaB-alpha and p65
Nuclear factor (NF)-kappaB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-kappaB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-kappaB by hijacking the inhibitor IkappaB-alpha and by preventing the repressor binding to the NF-kappaB complex. Moreover, Tat associated with the p65 subunit of NF-kappaB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IkappaB-alpha and p65 association, respectively, and for sustaining the NF-kappaB activity. Among an array of NF-kappaB-responsive genes, Tat mostly activated the MIP-1alpha expression in a p65-dependent manner, and bound to the MIP-1alpha NF-kappaB enhancer thus promoting the recruitment of p65 with displacement of IkappaB-alpha; similar findings were obtained for the NF-kappaB-responsive genes CSF3, LTA, NFKBIA and TLR2. Our results support a novel mechanism of NF-kappaB activation via physical interaction of Tat with IkappaB-alpha and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.

interaction

Tat

IkappaB-alpha

hijacking

Tat

IkappaB-alpha

binding

IkappaB-alpha

associated

Tat

binding

association

Tat

IkappaB-alpha

bound

Tat

NF-kappaB enhancer

recruitment

NF-kappaB enhancer

displacement

IkappaB-alpha

interaction

Tat

IkappaB-alpha

expression

preventing

Tat

binding

increased

Tat

binding

required

IkappaB-alpha

required

responsive

activated

Tat

dependent

dependent

expression

promoting

bound

recruitment

promoting

bound

displacement


Input:
Nuclear factor (NF)-kappaB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1). The NF-kappaB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding. The transcriptional activity of the NF-kappaB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).
Inhibitors of NF-kappaB (IkappaB) associate with the NF-kappaB complex and interfere with its binding to DNA (3). In the canonical pathway of NF-kappaB activation, the activated IkappaB kinase (IKK) phosphorylates IkappaB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-kappaB complex in the nucleus. IkappaB-alpha, the most abundant inhibitor of NF-kappaB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6). The NF-kappaB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCzeta (9) and Ser536 by IKKalpha (10,11). Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IkappaB-alpha, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13). Post-activation turn off of NF-kappaB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-kappaB, such as IkappaB-alpha and ubiquitin-editing protein A20 (14-20). Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-kappaB activity (12,21,22).
Persistent activation of NF-kappaB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-kappaB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23-25). Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27). Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29). Thus, understanding the mechanisms of NF-kappaB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.
In HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-kappaB activity by activation of IKK (30) and procaspase 8 (31). Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication. Tat binds to RNA stem-loop structures generated by the 5' end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor beta (TNFbeta) (33) and interleukin-6 (IL-6) (34) to activate gene transcription. Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPbeta (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41-43). When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).
We first reported that NF-kappaB was constitutively active in Jurkat cells that stably expressed the Tat gene (47). Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IkappaB-alpha (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49). These findings suggested that Tat modulates crucial enzymes involved in NF-kappaB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-kappaB, which is mainly dependent on de novo synthesis of IkappaB-alpha (15,17). We previously found that IkappaB-alpha binds to Tat and promotes the nuclear export of the viral transactivator (50,51). In this study, we report that Tat counteracts the post-activation turn off of NF-kappaB through direct interaction with IkappaB-alpha and p65, which enhances the DNA binding and transcriptional activity of the NF-kappaB complex. The new mechanism of NF-kappaB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.

Acetylation

Acetylation

acetylation

dimerization

RelA

dimerization

c-Rel

dimerization

RelB

dimerization

dimerization

binding

RelA

binding

c-Rel

binding

RelB

binding

binding

binding

association

IkappaB-alpha

binding

interacts

Tat

binds

Tat

interacting

Tat

interacting

Tat

TFIID

interacting

Tat

interacting

Tat

C/EBPbeta

interacting

Tat

cyclin T1

interacting

Tat

interacting

Tat

interacting

Tat

CBP

interacting

Tat

P/CAF

binding

Tat

binds

IkappaB-alpha

Tat

interaction

Tat

IkappaB-alpha

Deacetylation

histone deacetylase-3

Deacetylation

acetylation

acetylation

deacetylation

expressed

Tat

synthesis

IkappaB-alpha

released

Tat

impairs

Acetylation

association

inhibiting

Tat

mediated

phosphorylated

IkappaB-alpha

phosphorylated

IkappaB-alpha

phosphorylation

phosphorylation

phosphorylation

for

RelA

Rel homology domain

dimerization

for

RelA

Rel homology domain

binding

for

c-Rel

Rel homology domain

dimerization

for

c-Rel

Rel homology domain

binding

for

RelB

Rel homology domain

dimerization

for

RelB

Rel homology domain

binding

for

Rel homology domain

dimerization

for

Rel homology domain

binding

for

Rel homology domain

dimerization

for

Rel homology domain

binding

by

degraded

by

phosphorylation

by

phosphorylation

PKCzeta

by

phosphorylation

IKKalpha

increases

Acetylation

binding

induces

binding

activation

activation

procaspase 8

activate

binds

transcription

induced

Tat

degradation

mediated

deacetylation

degraded

IkappaB-alpha

degradation

IkappaB-alpha

transcription

tumor necrosis factor beta

transcription

ubiquitylated

IkappaB-alpha

ubiquitylated

IkappaB-alpha


Input:
Plasmids
The plasmids pcDNA-3xHA-IkappaB-alpha, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50). The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pkappaBluc and pSV-beta-Gal were purchased from Promega (Madison, WI, USA). The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65deltaC(1-318), pRc/CMV-3xHA-p65deltaN(122-551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.


Input:
Cells, transfection, treatments and luciferase assay
HeLa, p50-/-p65-/- mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640. PBMCs were isolated as previously described (53). Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany). HeLa, p50-/-p65-/- MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA). For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-alpha (TNF-alpha; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture. For luciferase assays, pSV-beta-Gal was co-transfected with pkappaBluc to monitor the transfection efficiency. Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and beta-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA). The ratio of firefly luciferase activity to beta-galactosidase activity was expressed as relative light units.


Input:
RNA interference
Jurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, aliquots (5 x 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 microF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium. RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU. RNA interference of p65 and IkappaB-alpha was performed with SMART pool siRNA p65 and IkappaB-alpha (Dharmacon, Chicago, IL, USA).

interference

interference

IkappaB-alpha


Input:
Pseudotyped virions and single round infection
293T cells (1 x 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 microg) together with pHXB2 Env (10 microg), and 48-h post-transfection cell supernatant was collected. Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration. PBMCs, Jurkat or U937 cells (5 x 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).


Input:
Cell extracts, western blotting, IKK activity and NF-kappaB DNA binding
Total, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data. Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris-HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% beta-mercaptoethanol, 25% glycerol), resolved on 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories). Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham). Primary antibodies were purchased from: Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IkappaB-alpha C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-gamma-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65). Densitometry of single bands was analysed by ImageJ software package (NIH, USA). IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA). Binding of p65, p50 and FLAG-Tat to the double-stranded NF-kappaB oligonucleotide was measured using NF-kappaB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.

Binding

Binding

Binding

FLAG-Tat


Input:
In vitro translation
HA-IkappaB-alpha, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50). Details are described in Supplementary Data.

expressed

HA-IkappaB-alpha

expressed

expressed

Tat

under

expressed


Input:
Immunoprecipitation assay and GST-pull down
Immunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50). Details are described in Supplementary Data.


Input:
Real-time PCR
Total RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions: 95degreesC, 1 min; (94degreesC, 10 s; 60degreesC, 30 s) x40. Primers for Tat and MIP-1alpha are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-kappaB signaling pathway (QIAGEN Sciences, MD, USA). Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control. Relative expression was calculated as 2(Ct gene under investigation - Ct GAPDH).

levels

GAPDH


Input:
Chromatin immunoprecipitation assay
Cells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%. After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped. After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1x Complete Protease Inhibitor (Roche Diagnostic GmbH). Nuclei were pelletted (1000 x g, 5 min), and resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA). Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 x g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris-HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA). Samples were pre-cleared by 3-h incubation with 20 microl of protein G agarose beads followed by incubation with antibodies against the analysed proteins. Primary antibodies were: anti-p65 (sc-372), anti-IkappaBalpha (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma-Aldrich. Immunoprecipitations were carried out at 4degreesC overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS. Protein-DNA cross-links were reverted by heating at 65degreesC overnight. DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 microl sterile distilled water. Specific enrichment in NF-kappaB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen). Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions: 95degreesC, 1 min; (94degreesC, 10 s; 60degreesC, 30 s) x40. Primers used for MIP-1alpha, GAPDH and ACTB are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN). For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.


Input:
Statistical analysis
Statistical analysis was performed by two-tail unpaired Student's t-test. Data were reported as means +/- SE. Differences between the means were considered as statistically significant at the 95% level (P < 0.05).


Input:
Tat enhances the NF-kappaB activity by hijacking IkappaB-alpha and inhibiting the post-activation turn off of NF-kappaB
We analysed the kinetic of NF-kappaB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference. Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R-E- virions. By cytofluorimetric analysis, ~40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1). The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A). The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).
The progressive increase in NF-kappaB activity, as measured by p65-p50 binding to an NF-kappaB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C). IkappaB-alpha was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol). Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D). Similar kinetics of NF-kappaB activity, IKK activation and IkappaB-alpha degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2). These results indicated that in single-round HIV-1 infection the NF-kappaB activation initially correlated with the IKK activation and IkappaB-alpha degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IkappaB-alpha.
Next, we tested the single action of Tat on the kinetic of NF-kappaB activity induced by PMA. To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-kappaB activation (17,18). In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IkappaB-alpha content was not significantly affected (Figure 2B, cytosol). Transient stimulation with PMA increased the NF-kappaB DNA binding activity and nuclear p65 at 5 min peaking at 60-120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B). In addition, Tat bound to the NF-kappaB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-kappaB complex bound to DNA. Degradation of IkappaB-alpha was progressively induced at 5-30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol). Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C). These results indicated that Tat enhanced the NF-kappaB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IkappaB-alpha content.
As we previously found that Tat binds to the sixth ankyrin of IkappaB-alpha (50,51), we tested whether Tat counteracted the generation of the IkappaB-alpha/NF-kappaB complex. To this end, the association of IkappaB-alpha with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IkappaB-alpha, respectively. Coimmunoprecipitation of IkappaB-alpha with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IkappaB-alpha (Figure 2D, lanes 2 and 4), indicating that Tat competed the IkappaB-alpha binding to p65.

hijacking

Tat

IkappaB-alpha

binding

binding

binding

binding

binding

bound

Tat

binding

Tat

binds

Tat

sixth ankyrin

IkappaB-alpha

generation

IkappaB-alpha

association

IkappaB-alpha

Coimmunoprecipitation

IkappaB-alpha

coimmunoprecipitated

Tat

IkappaB-alpha

binding

IkappaB-alpha

expression

Tat

expression

Tat

expression

luciferase

synthesized

IkappaB-alpha

re-synthesis

IkappaB-alpha

synthesis

IkappaB-alpha

synthesis

IkappaB-alpha

synthesis

IkappaB-alpha

barely detected

expression

siRNA Tat

dropped

binding

absence

dropped

absence

absence

Tat

dropped

binding

negative

dropped

negative

negative

Tat

counteracted

Tat

generation

halved

Coimmunoprecipitation

Tat

competed

Tat

binding

increased

expression

dependent

expression

induced

dependent

elevated

binding

Tat

elevated

Tat

observed

degradation

observed

re-synthesis

enhanced

binding

Tat

enhanced

Tat

increased

elevated

binding

Tat

elevated

Tat

increased

binding

induced

Degradation

followed

induced

synthesis

degraded

IkappaB-alpha

degradation

IkappaB-alpha

degradation

IkappaB-alpha

Degradation

IkappaB-alpha

modulating

expression

affected

Tat

IkappaB-alpha

independently

increased

Tat

affecting

Tat

IkappaB-alpha


Input:
Tat counteracts the IkappaB-alpha inhibition of p65 by competing the repressor binding
We further investigated the physical interaction of Tat with IkappaB-alpha using in vitro translated proteins in coimmunoprecipitation assays. For mapping the interaction domains, we used the following Tat mutants: Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49-57)A (Figure 3A). IkappaB-alpha coimmunoprecipitated with all Tat proteins, except Tat R(49-57)A (Figure 3B). These results indicated that the arginine-rich domain of Tat was involved in the binding to IkappaB-alpha, which was consistent with previous observations (50,51). Moreover, Tat associating with IkappaB-alpha competed the binding of IkappaB-alpha to p65 in a dose-dependent manner (Figure 3C, lanes 3-5), while Tat R(49-57)A, which lacked the binding site for IkappaB-alpha, did not associate with IkappaB-alpha and did not compete the binding of IkappaB-alpha to p65 (Figure 3C, lanes 6-8).
Next, we evaluated whether Tat counteracted the IkappaB-alpha-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IkappaB-alpha proteins with the NF-kappaB probe in presence or absence of Tat followed by EMSA. The p65 DNA binding activity was inhibited by IkappaB-alpha and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49-57) (Figure 3D). Further, we analysed the Tat effect on the IkappaB-alpha repression of p65 transcriptional activity by transfecting p50-/-p65-/-MEFs with the NF-kappaB-Luc reporter together with expression vectors of p65 and IkappaB-alpha, in presence or absence of Tat. IkappaB-alpha inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49-57)A (Figure 3E). Altogether these results indicated that Tat counteracts the IkappaB-alpha repression of the p65 DNA-binding and transcriptional activity by associating with IkappaB-alpha and competing the repressor binding to p65.

binding

IkappaB-alpha

interaction

Tat

IkappaB-alpha

coimmunoprecipitated

IkappaB-alpha

Tat

coimmunoprecipitated

IkappaB-alpha

Tat R(49-57)A

binding

Tat

IkappaB-alpha

associating

Tat

IkappaB-alpha

binding

IkappaB-alpha

binding

Tat

site

IkappaB-alpha

associate

Tat R(49-57)A

IkappaB-alpha

binding

IkappaB-alpha

binding

binding

binding

associating

Tat

IkappaB-alpha

binding

IkappaB-alpha

expression

luciferase

counteracts

inhibition

competing

inhibition

IkappaB-alpha

competing

Tat

binding

competed

associating

binding

lacked

Tat

site

compete

Tat R(49-57)A

binding

counteracted

Tat

inhibition

inhibition

IkappaB-alpha

binding

inhibited

binding

IkappaB-alpha

restored

inhibited

Tat

restored

inhibited

Tat R(49-57)

inhibited

IkappaB-alpha

dependent

restored

inhibited

Tat

restored

inhibited

Tat R(49-57)A

counteracts

repression

associating

counteracts

repression

competing

repression

IkappaB-alpha

binding

competing

Tat

binding

involved

arginine-rich domain

Tat

binding

dependent

expression


Input:
Tat increases the p65 affinity binding to DNA through association with p65
We tested whether Tat physically interacted with p65. HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts. The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49-57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A). Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49-57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B). These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.
To identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65deltaC (1-318)], or the RHD [p65deltaN (122-551)] (Figure 4C). Tat coimmunoprecipitated with p65deltaC (1-318), and not with p65deltaN (122-551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.
To address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-kappaB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA. Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E). The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-kappaB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).

binding

association

Tat

interacted

Tat

coimmunoprecipitated

Tat

coimmunoprecipitation

Tat C(22,25,27)A

binding

Tat

binding

Tat C(22,25,27)A

associated

Tat

cysteine-rich domain

binding

Tat

coimmunoprecipitated

Tat

p65deltaC (1-318)

interaction

RHD

Tat

binding

binding

binding

binding

Tat

site

binding

binding

immunoprecipitated

Tat

decrease

coimmunoprecipitation

Tat C(22,25,27)A

deleted

transactivation domain

deleted

RHD

lacking

Tat

site

increases

binding

association

mediate

associated

required

domain

binding

increased

Tat

binding

ineffective

binding

Tat C(22,25,27)A

enhanced

Tat

binding

effect

Tat

binding


Input:
Tat activates the p65-dependent expression of NF-kappaB-responsive genes, occupies the NF-kappaB enhancers and promotes the p65 recruitment with IkappaB-alpha displacement
We analysed the action of Tat on the expression of NF-kappaB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49-57)A, and 48 h later the expression of a number of NF-kappaB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1alpha, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49-57)A were ineffective (Figure 5A-E). The Tat-dependent transactivation of the NF-kappaB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A-E). Differently, the expression of the NF-kappaB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-kappaB activity, suggesting that the up-regulation of NF-kappaB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IkappaB-alpha.
MIP-1alpha, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1alpha expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1alpha production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1alpha has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-kappaB enhancers in the proximal promoter region of the MIP-1alpha gene: NF-kappaB1, -1023/-1014 nucleotides; NF-kappaB2, -661/-652 nucleotides; NF-kappaB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-kappaB binding sites of the MIP-1alpha promoter, we stimulated HeLa cells with TNF-alpha, a well-known NF-kappaB inducer, and measured the expression levels of MIP-1alpha by real-time PCR, and the p65 occupancy of the NF-kappaB sites by ChIP. TNF-alpha activated the expression of MIP-1alpha and induced the p65 recruitment to the NF-kappaB1 site of MIP-1alpha without affecting the occupancy of the putative NF-kappaB2 and NF-kappaB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-kappaB1 site of MIP-1alpha, while it did not affect the putative NF-kappaB2 and NF-kappaB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-kappaB1 site was the only effective NF-kappaB enhancer of the MIP-1alpha promoter in response to TNF-alpha and Tat.
Similarly to MIP-1alpha, Tat increased the recruitment of p65 to the NF-kappaB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-kappaB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IkappaB-alpha was chromatin-immunoprecipitated at the NF-kappaB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IkappaB-alpha RNA interference (Figure 5J), indicating that Tat displaced IkappaB-alpha from promoters.

occupies

Tat

recruitment

displacement

IkappaB-alpha

interaction

Tat

occupancy

NF-kappaB sites

recruitment

NF-kappaB1 site

occupancy

recruitment

NF-kappaB1 site

recruitment

NF-kappaB enhancers

recruitment

NF-kappaB enhancers

LTA

recruitment

NF-kappaB enhancers

NFKBIA

recruitment

NF-kappaB enhancers

bound

Tat

GAPDH

promoters

occupancy

Tat

interaction

Tat

chromatin-immunoprecipitated

IkappaB-alpha

from

IkappaB-alpha

expression

expression

expression

LTA

expression

NFKBIA

expression

expression

production

expression

expression

loss

bound

interference

interference

removed

chromatin-immunoprecipitated

Tat

removed

chromatin-immunoprecipitated

interference

interference

IkappaB-alpha

displaced

Tat

from

promotes

Tat

recruitment

promotes

Tat

displacement

increased

Tat

expression

ineffective

expression

Tat C(22,25,27)A

ineffective

expression

Tat R(49-57)A

activated

Tat

increased

expression

induced

production

Tat

activated

TNF-alpha

expression

induced

TNF-alpha

recruitment

affecting

TNF-alpha

occupancy

promoted

Tat

recruitment

increased

Tat

recruitment

ineffective

Tat

promoters

GAPDH

ineffective

Tat

promoters

ACTB

via

occupancy

interaction

regulation

transcriptional

transcriptional


Input:
In HIV-1-infected monocytes Tat sustains the NF-kappaB activity and promotes the transcriptional activation of MIP-1alpha by interacting with IkappaB-alpha and p65
We next analysed whether Tat affected the NF-kappaB activity and the expression of MIP-1alpha in the course of HIV-1 infection. To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R-E- virions, which were used in single-round infection of U937, a human monocytic cell line. Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-kappaB activation following viral infection. The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).
The NF-kappaB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B). Degradation of IkappaB-alpha was observed at 3-h post-infection, and was followed by de novo synthesis of IkappaB-alpha at 12 h independently of the Tat presence (Figure 6C). Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D). These results agreed with the kinetic of NF-kappaB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-kappaB.
In HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IkappaB-alpha and p65 (Figure 6E), and activated the MIP-1alpha expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F). Moreover, Tat and p65 were both recruited to the NF-kappaB enhancer of MIP-1alpha, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H). Altogether these results indicated that in single round HIV-1 infection Tat associated with IkappaB-alpha and p65, and induced the p65-dependent activation of MIP-1alpha expression through occupancy of the MIP-1alpha promoter and increased recruitment of p65.

interacting

Tat

IkappaB-alpha

binding

coimmunoprecipitated

Tat

IkappaB-alpha

recruited

Tat

NF-kappaB enhancer

occupancy

NF-kappaB enhancer

associated

Tat

IkappaB-alpha

occupancy

Tat

promoter

recruitment

promoter

expression

expression

Tat

expression

Tat

expression

synthesis

IkappaB-alpha

expression

expression

detected

expression

siRNA Tat

lack

activated

abolished

occupancy

siRNA Tat

promotes

activation

activation

transcriptional

by

promotes

interacting

induced

binding

increased

binding

Tat

followed

Degradation

synthesis

activated

Tat

dependent

activated

expression

following

lack

induced

activation

activation

expression

increased

Tat

recruitment

Degradation

IkappaB-alpha

affected

Tat

expression

modulate

expression

independently

synthesis

Tat

transcriptional