Induction of latent provirus by T-cell receptor activation
The 2D10 cell line is a latently infected Jurkat T-cell line carrying a lentiviral vector that expresses the regulatory proteins Tat and Rev in cis
and a short-lived green fluorescent protein (d2EGFP) in place of Nef25
. The provirus in 2D10 cells also carries the H13L mutation in Tat which effectively supports HIV transcription elongation but is attenuated and therefore helps to promote proviral entry into latency24; 25; 51
. Extensive characterization of the 2D10 cell line has shown that the provirus is inserted into the first exon of the SEPX1 gene in the opposite orientation from the SEPX1 promoter 25
An important advantage of 2D10 cells is that although HIV gene expression is highly suppressed (more than 98% of the uninduced cells are d2EGFP negative) the cells are readily induced. For example, over 99% of the cell population becomes d2EGFP positive (mfi > 2 ×101) after exposure to 10 ng/ml TNF-α for 18 hr (). The provirus in 2D10 cells can also be efficiently reactivated by stimulation of the T-cell receptor using a combination of α-CD3 mAb and α-CD28 mAb. As shown in mAb stimulation of the TCR resulted in 62.4% reactivation of the latent proviruses ().
Induction of latently infected 2D10 cells by stimulation of the TCR
In addition to activating NF-κB, TCR stimulation induces a complex cascade of pathways, including activation of NFAT through the Ca+2
-calcineurin pathway. This raises the intriguing possibility that HIV proviral reactivation following TCR stimulation involves multiple transcription factors in addition to NF-κB. In order to monitor the kinetics of activation of transcription factors following TCR stimulation we compared the nuclear levels of NF-κB (p65, p50) and NFAT (NFATc1 and NFATc2) following either TNF-α treatment or stimulation of the TCR with α-CD3/α-CD28 mAb by Western blotting (). During a 6 hr time course TNF-α induced cycling of the NF-κB p65 and p50 subunits between the cytoplasm and nucleus 25; 33; 50
. After an initial peak of nuclear accumulation at 30 min, both NF-κB p65 and p50 levels oscillated with a period of approximately 2.5 hr. As expected, there was no detectable increase in the nuclear levels of either NFATc1 or NFATc2 following TNF-α treatment. NFATc2 was undetectable in the nucleus prior to TNF-α exposure, whereas NFATc1 showed moderate nuclear levels in unstimulated cells which slowly fell during the 8 hr time course.
Stimulation of the TCR with α-CD3/α-CD28 mAbs activated both NF-κB and NFAT with unique kinetics (). Initially, entry of NF-κB p65 into the nucleus followed kinetics that were nearly identical to those observed following TNF-α stimulation and nuclear p65 levels reached a maximum at 30 min. However, in contrast to activation by TNF-α, p65 levels then declined rapidly and showed only slight peaks of activation at 2.5 and 5.5 hr. NF-κB p50 also showed a distinct initial peak at 30 min followed by more sustained activation during the next 8 hr. NFATc2 reached maximal nuclear levels at 30 min before declining to basal levels by 2.5 hr, whereas NFATc1 levels gradually increased before reaching maximal levels between 2.5 and 8 hr after TCR stimulation.
NFAT is not required for HIV transcription in Jurkat T-cells following TCR activation
Since NFATc1 is induced in response to TCR signaling, it is possible that it contributes to HIV transcription under these conditions. To test this possibility, we used cyclosporine A (CsA) to block the Ca2+-calcinurein activation pathway. Control experiments demonstrated that CsA was able to completely block the induction of both NFATc1 and NFATc2 but did not prevent NF-κB p65 or p50 induction (data not shown). Flow cytometry () demonstrated that CsA did not block proviral reactivation following either TNF-α, or TCR stimulation, suggesting that not only is NFAT dispensable for proviral reactivation, but sufficient levels of NF-κB p65 were induced under both conditions to drive HIV transcription.
Kinetics of HIV transcription in response to TCR signaling
In order to study early events during the induction of transcription from the latent HIV proviruses we performed quantitative ChIP assays using an RNAP II antibody (). The ChIP assays were used to measure the accumulation of RNAP II at the TAR region near the promoter (+30 to +134), which provides an accurate estimate of transcription initiation rates. In addition, RNAP II levels at a downstream region (+2593 to + 2691) were measured to provide an assessment of transcription elongation efficiencies.
Stimulation of HIV transcriptional elongation following TNF-α stimulation and T cell receptor-mediated activation of latent proviruses
The data shown in is a subset of an extensive data set used to simultaneously monitor HIV transcription and gene expression from control cellular genes that are responsive to NF-κB and NFAT. The ChIP data were analyzed by fitting a series of Gaussian distributions. This procedure effectively allows for the objective identification of the main peaks of transcription and the relative contribution of successive waves of transcription factor activation to overall transcription. The full data set is provided as supplementary material
along with details of the curve fitting.
As we have previously reported, a low level of RNAP II transcription complexes accumulate near the start site of transcription of latent HIV proviruses25; 50; 51
. Both TNF-α stimulation and α-CD3/α-CD28 mAb co-stimulation led to the rapid recruitment of RNAP II to the HIV promoter concomitantly with the initial rise of NF-κB levels in the nucleus. Under both stimulation conditions, RNAP II accumulation at the promoter reached maximal levels at 30 min and was nearly identical during the first 90 min.
Following the initial cycle of transcription initiation, the kinetics of HIV proviral reactivation after TNF-α treatment and TCR stimulation diverged significantly. In the case of TNF-α activation, transcription initiation rebounded by 3.5 hr and increased throughout the subsequent cycles of NF-κB p65 entry into the nucleus during the next 8 hr. By contrast, following TCR activation, maximal HIV transcription initiation was achieved during the first cycle and lower levels of transcription initiation were observed during each of the later time points.
The IκBα gene, which is highly dependent on NF-κB for transcription initiation, but does not require Tat for transcription elongation, was analyzed in parallel to the HIV provirus in order to provide an internal control for NF-κB activity (). Accumulation of RNAP II near the promoter of the IκBα gene (+155 to +237) was used as a measurement of transcription initiation, while accumulation of RNAP II in a downstream region (+2038 to +2159) was used to evaluate transcription elongation. As in the case of the HIV provirus, there was a rapid increase in transcription initiation of the IκBα gene during the first cycle of NF-κB p65 entry into the nucleus and the first peak of transcription initiation was nearly identical following either TNF-α activation or TCR stimulation. However, comparing the patterns of IκBα gene transcription and HIV proviral transcription at later times demonstrates that the HIV promoter is both more strongly stimulated and sustains a higher level of transcription than the IκBα gene between 2 and 8 hr. Similarly, following TCR activation, IκBα gene responds poorly after the initial cycle of activation whereas there is substantial HIV transcription under these conditions. The simplest explanation for these kinetic differences is that NF-κB p65 is the primary factor driving IκBα gene transcription throughout the time course, whereas in response to TCR signaling additional factors help to sustain HIV transcription following the initial phase of NF-κB p65 activation.
TCR activation stimulates HIV transcriptional elongation
Although the induction of HIV transcription initiation by TNF-α was very efficient during the first cycle of NF-κB p65 entry into the nucleus, downstream RNAP II levels remain at extremely low levels at this time and only began to rise after 2 hours of stimulation when Tat protein begins to accumulate25; 33
. In contrast, stimulation of 2D10 cells by α-CD3/α-CD28 antibodies strongly induced elongation during the first 90 min, a time prior to the synthesis of new Tat protein. Integration under the fitted curves demonstrated that although initiation during the first cycle of transcription is nearly identical when cells are stimulated either by TNF-α, or through the TCR, elongation is approximately twice as efficient following TCR activation.
Changes in elongation efficiency of the HIV provirus and the IκBα gene following activation by TNF-α or TCR stimulation were estimated by taking the ratio of RNAP II at the promoter and the downstream region for the HIV provirus () and the IκBα gene (). Following stimulation by TNF-α, there is a progressive increase in elongation efficiency between 2 and 8 hr following an initial lag period of 2 hr. Although initiation rates oscillate throughout this time course the elongation ratios increase monotonically, suggesting that this is due to accumulation of Tat in the system. During TCR activation changes in the elongation efficiency are apparent at the earliest time points and maximal transcriptional elongation efficiencies are reached sooner than during TNF-α activation. Compared to the IκBα gene (), the elongation efficiency of the latent HIV provirus is initially significantly more restricted (i.e. 10% versus 20% to 30%) but becomes dramatically more efficient (i.e. 75% compared to 30%) once Tat is induced. In contrast to the results observed with the HIV provirus, the elongation efficiency from the IκBα gene remains constant during the activation time course since it is not activated by Tat.
TCR stimulation induces P-TEFb recruitment to the HIV provirus
In order to examine if the strong activation of elongation we observed at early times after TCR stimulation was associated with enhanced P-TEFb recruitment to the HIV provirus, we measured the levels of RNAP II and CDK9 at various regions of HIV provirus by ChIP assays. shows that although the amounts of RNAP II near the transcription start site are similar after stimulation for 30 min by either TNF-α, or through the TCR, the amount of RNAP II found downstream of the TAR sequence was significantly higher in the TCR activated samples. This enhanced RNAP II processivity was associated with a 3- to 6-fold increase in CDK9 levels in downstream regions of the provirus (). Since, as described above, there is no significant increase in newly synthesized Tat until at least 120 min following stimulation by TNF-α, these results imply that TCR-mediated signaling uniquely increased the availability of either P-TEFb or Tat.
Enhanced recruitment of P-TEFb to HIV proviruses following TCR activation
Enhanced HIV transcriptional elongation is mediated through an ERK-dependent signaling pathway
To define the molecular mechanisms responsible for increasing P-TEFb pool sizes, 2D10 cells were treated with a range of activators and inhibitors of pathways known to be induced by TCR-mediated signaling (). As shown in , activation of the TCR led to a rapid increase in the amount of phosphorylated ERK1/2 and phosphorylated AKT in both the nucleus and the cytoplasm. Treatment of cells with U0126, an inhibitor of the kinase activity of MAP kinase kinase (MAPKK), selectively blocked ERK1/2 phosphorylation but did not affect AKT phosphorylation. Conversely, treatment of cells with LY294002, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked AKT phosphorylation, and increased ERK1/2 phosphorylation, since blocking of the AKT pathway removes an inhibitor of the ERK pathway52; 53
. For example, in the experiment shown in , at 15 min after TCR stimulation phospho-ERK-2 levels were 2.9-fold higher in cells treated with LY294002 than in control cells and phosho-ERK-1 levels were 1.7-fold higher than in control cells. Treating cells with a combination of LY294002 and U0126 reduced ERK2 levels to 60% of the control values.
ERK kinase pathway mediates TCR activation of HIV transcriptional elongation
ChIP assays were performed to study the effects of these inhibitors and a range of transcription activation conditions on transcription elongation. In the experiments shown in , RNAP II levels were measured at both an upstream (+30 to +134) and a downstream (+2593 to +2691) site and the ratio used to estimate the elongation efficiency, as described above. Exposure of 2D10 cells to TNF-α, PHA, TSA or HMBA did not cause any significant changes in elongation efficiency at 30 min following treatment. However, activation by PMA and a combination of PMA and PHA strongly induced elongation at 30 min post stimulation.
In cells activated either through the TCR or by PMA, exposure to U0126 reduced elongation efficiency by approximately 50%, but had no significant effect on initiation (). By contrast, the AKT inhibitors LY294002 and AI8 (Akt inhibitor 8) were able to further activate transcriptional elongation following TCR stimulation or PMA activation. Since U0126 is able to partially reverse the effects of LY294002, it seems likely that the enhancement in elongation that we have observed is due to the enhanced activation of ERK-1/2 that is mediated by AKT inhibition.
Control experiments confirmed that none of the inhibitors studied affected transcriptional elongation in untreated cells or in cells activated by TNF-α (). Finally, we note that blocking of NFAT activation by CsA had no impact on transcription elongation ().
CycT1 and CDK9 levels are not altered in response to TCR signaling
The signaling events stimulated by the TCR could either result in enhanced synthesis of components of the transcription elongation machinery, such as CycT1 and CDK9, or act to enhance their activity by inducing post-transcriptional modifications. To distinguish between these two possibilities additional Western blotting experiments were undertaken to test whether nuclear ERK, CycT1 and CDK9 levels were altered within the first 2 hr after TCR activation. As shown in , nuclear ERK-1 levels remain constant during the first hr after TCR activation. By contrast, high levels of phosphorylated ERK-1/2 appear in the nucleus within 15 min and then slowly decay. Consistent with the results shown in , the phosphorylation of ERK-1/2 is blocked by U0126.
P-TEFb association with chromatin is enhanced following TCR activation in the absence of new P-TEFb subunit synthesis
There are no detectable changes in the nuclear levels of CycT1, CDK9 or CDK7 during the first 2 hr after TCR stimulation (). The constitutive expression of CycT1 and CDK9 in Jurkat T cells is in contrast to the situation in primary resting memory T-cells and monocytes where CycT1 are low prior to TCR stimulation and new CycT1 is synthesized within 60 min 24; 48; 49
TCR signaling induces global increases of P-TEFb association with chromatin
In order to test whether TCR signaling induced a general increase in transcription associated with P-TEFb recruitment to the chromatin, we performed Western blots on a series of nuclear extracts (). In the Jurkat T-cells there is an excess of HEXIM1 and CDK9 which can be detected at high levels in cytoplasmic extracts prepared in hypotonic buffers. CycT1 is absent from these fractions. By contrast, nuclear extracts prepared using buffers containing 0.36 M NaCl, contain stoichiometric amounts of CycT1 and CDK9 and variable amounts of HEXIM1. The initial nuclear extracts contain high levels of HEXIM1 suggesting that they are enriched in the nucleoplasmic 7SK RNP complex. Subsequent extracts, which represent fractions that are more tightly associated with the chromatin, contain only CycT1 and CDK9. Treatment of cells with either DRB or actinomycin D (Act D), which induce dissociation the 7SK RNP complex37; 38
(see also below) induced increased association of the CycT1 and CDK9 subunits of P-TEFb with the chromatin, but did not alter HEXIM1 levels in the nuclear extracts. Similarly, TCR signaling induced a global increase in P-TEFb association with the chromatin. This is consistent with the ChIP data presented in demonstrating that TCR signaling increased the levels of CDK9 recruited to latent HIV proviruses.
TCR signaling induces the disassembly of the 7SK RNP complex
Thus, the activation of elongation on HIV by TCR signaling that we have observed seems to be associated with an ERK-mediated post-translation modification of presynthesized P-TEFb rather as the result of new synthesis of its subunits.
U0126 blocks TCR activation of HIV transcriptional elongation
To verify that elongation is selectively activated early after TCR activation, we performed time course experiments similar to those shown in in the presence and absence of U0126. U0126 selectively blocked ERK phosphorylation, but it did not interfere with NF-κB p65 and p50 translocation to the nucleus following TCR (). U0126 also had no impact on NFATc1 and NFATc2 activation. In the corresponding ChIP experiments, U0126 treatment did not inhibit RNAP II recruitment to the promoter but did result in a dramatic decrease in HIV elongation during the first wave of transcription (). Similarly, U0126 is able to restrict transcriptional elongation and CDK9 recruitment following PMA stimulation of cells (data not shown). U0126 also strongly inhibits HIV elongation during the 2 to 4 hr period when Tat synthesis has resumed. Complementary studies using PD98059, which inhibits the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and blocks the induction of AP-1 during TCR signaling have shown that AP-1 contributes to HIV transcription during the 2 to 4 hr period but plays no role during the first round of HIV elongation between 0 to 2 hr (Hokello, J., Mbonye, U., & Karn, J., unpublished).
U0126 blocks TCR mediated HIV transcription elongation
Recruitment of P-TEFb to HIV proviruses following PMA activation is blocked by U0126 and enhanced by LY294002
The inhibitor studies shown in strongly suggested that both TCR activation and PMA stimulation enhanced HIV transcription elongation through a common mechanism involving the ERK kinase pathway. To confirm that PMA stimulation of cells resulted in enhanced P-TEFb recruitment to the HIV provirus additional ChIP experiments were performed ().
Recruitment of P-TEFb to HIV proviruses following PMA activation is blocked by U0126 and enhanced by LY294002
Activation of cells for 30 min with PMA stimulated both RNAP II recruitment to the provirus, transcriptional elongation, and CDK9 recruitment. Both recruitment of RNAP II and CDK9 were strongly inhibited by treatment with U0126. Importantly, treatment of cells with a combination of PMA and LY294002 boosted the levels of both RNAP II and CDK9 associated with the activated HIV provirus and was inhibited by U0126. Thus the amount of RNAP II and CDK9 associated with the HIV provirus varied in proportion to the degree of ERK activation.
Disruption of the 7SK RNP complex is mediated by ERK
The preceding data strongly suggests that restrictions imposed on P-TEFb activity in resting T cells can be relieved through activation of the MAPKK/ERK pathway. We therefore decided to investigate whether TCR activation could alter the levels of the 7SK RNP complex found in T-cells.
As shown in , fractionation of Jurkat T-cell extracts by gel filtration chromatography demonstrates that the majority of P-TEFb is found as part of the large 7SK RNP complex. Integration of the areas under the peaks showed that in the unstimulated cells 95% of the CDK9 and 68% of the HEXIM-1 in the cell is associated with the large complex while 5% of the CDK9 and 32% of the HEXIM-1 were associated with the low molecular weight fraction. Thus, there is a significantly higher proportion of CDK9 associated with the 7SK RNP complex in Jurkat T-cells than has been observed in the more extensively studied HeLa cells where typically less than 50% of the P-TEFb in the cell is in the large complex37; 38; 46; 54
As shown in , stimulation of the cells for 30 min through the TCR or by exposure to PMA results in a significant disruption of the large complex and release of P-TEFb into a low molecular weight fraction. Following TCR treatment 29% of the CDK9 and 35% of the HEXIM-1 is found in the lower molecular weight fractions. The degree of complex dissociation following TCR signaling is comparable to the disruption seen following 30 min exposure to HMBA (low molecular weight fraction: CDK9, 26%; HEXIM1, 47%), but less than that induced by DRB (low molecular weight fraction: CDK9, 60%; HEXIM1, 88%) ().
U0126 effectively blocks the disruption of the 7SK RNP complex by TCR signaling. In the experiment shown in , 12% of the CDK9 and 26% of the HEXIM-1 was found initially in the low molecular weight fraction. After stimulation of the TCR for 30 min, 22% of the CDK9 and 31% of the HEXIM1 was found in the low molecular fraction. Treatment with U0126 not only blocked TCR induced dissociation but resulted in slightly more accumulation of CDK9 and HEXIM1 in the 7SK RNA/P-TEFb/HEXIM1 complex than in unstimulated cells (93% CDK9 and 88% HEXIM1 in the complex). This strongly suggests that the ERK signaling is responsible for the release of functional P-TEFb that we have described above.
The ERK pathway mediates the disassembly of the 7SK RNP complex in response to TCR activation
Although activation of cells through the TCR or by PMA and treatment with HMBA and DRB all resulted in 7SK RNP disruption, only activation by TCR or PMA resulted in enhanced HIV elongation efficiencies. Since DRB is also an inhibitor of CDK9 activity, it would be expected to only release inactive P-TEFb.
The failure of HMBA to stimulate HIV transcription elongation appears to be associated with the phosphorylation state of CDK9. Enzymatically active CDK9 must be phosphorylated on Thr186. As shown in , disruption of the 7SK RNP complex by TCR signaling resulted in the release of CDK9 that remains phosphorylated on Thr186. In this experiment 95% of the CDK9 in the unstimulated cells remained associated with the large complex and 5% was found in the small complex. Similarly, the large complex contained 92% of the phospho-Thr-CDK9 while the small complex contained 8%. After TCR stimulation, 21% of the total CDK9 and 11% of the phospho-Thr-CDK9 was released into the small complex. However, as originally reported by Chen et al.54
, although HMBA induced release of 45% of the CDK9 into the small complex, the released P-TEFb was extensively dephosphorylated on Thr-186. For example, the amount of phospho-Thr-CDK9 in the small complex following HMBA treatment corresponded to only 18% of the total CDK9.
TCR-mediated disruption of the 7SK RNP complex does not require Tat
Previous studies have documented that Tat acts to disrupt the 7SK RNP complex by competing with HEXIM for CycT1 binding44; 45; 46
. In order to determine whether residual Tat present in the latently infected 2D10 cells could be contributing to the disruption of the 7SK RNAP complex we measured the dissociation of the 7SK RNP complex in response to TCR signaling and PMA treatment in 2B2D cells25
, which harbor a latent provirus carrying the inactive C22G mutation in Tat, which prevents its interactions with P-TEFb (). As shown in , both TCR and PMA are able to induce 7SK RNP complex disruption in 2B2D cells, at levels comparable to those seen in the 2D10 cells. For example, in the uninduced cells 86.5% of the HEXIM1 is found in the 7SK RNP complex and 13.5% is in the low molecular weight fraction. After induction through the TCR or by PMA treatment, the amount of HEXIM1 in the low molecular weight fraction more than doubles (29.8% TCR; 27.0% PMA).
Disruption of the 7SK RNP complex in response to TCR activation occurs in the absence of HIV Tat
Subthreshold levels of Tat contribute to HIV reactivation in latently infected cells
To evaluate whether P-TEFb released by TCR signaling in 2B2D cells required Tat in order to activate HIV transcription elongation we used ChIP assays to compare the HIV transcription elongation efficiencies in 2D10 and 2B2D cells at 0.5 hr, 2.5 hr and 5 hr after activation (). Consistent with the results described above, stimulation of both cells for 0.5 hr with TNF-α did not increase elongation efficiency above the basal level of 4.2 ± 1.5% in either cell line. However, activation of the TCR for 0.5 hr increased HIV elongation efficiency to 22.3% in the TCR stimulated 2D10 cells, but only to 6.5% in the 2B2D cells. Consistent with the results shown , at HIV elongation efficiency at 2.5 hr (15.0%) and 5.0 hr (48.4%) after activation by TNF-α there was a dramatic increase in due to the resumption of Tat synthesis from the reactivated provirus. There was only a modest further increase in elongation efficiency at 2.5 hr (25.0%) and 5.0 hr (27.7%) after TCR stimulation compared to the already high levels of elongation observed at 0.5 hr (22.3%). By contrast, in the 2B2D cells the elongation efficiency remains only slightly elevated with an average value of 6.2 ± 0.6 % for the 0.5 hr, 2.5 hr and 5.0 hr time points.
Thus, the increase in HIV elongation efficiency that we have observed following TCR stimulation appears to require a combination of P-TEFb activation and the availability of Tat. Since as described above, there is no new synthesis of P-TEFb subunits following TCR activation, these data suggest that there is either activation of residual Tat activity in the 2D10 cells or there is new synthesis of Tat which does not occur after TNF-α stimulation (see Discussion).
In conclusion, our data demonstrates that functional P-TEFb can be released through an ERK-dependent cell signaling pathway. This previously uncharacterized signaling pathway represents one of the first examples of the physiological enhancement of P-TEFb activity and transcriptional elongation.