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Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

['Uri Mbonye', 'Department Of Molecular Biology', 'Microbiology', 'Case Western Reserve University School Of Medicine', 'Cleveland', 'Ohio', 'United States Of America', 'Konstantin Leskov', 'Meenakshi Shukla', 'Saba Valadkhan']

Date: 2021-10

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4 + T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4 + T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4 + T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

Dissecting the cellular pathways through which HIV emerges from latency is a key step in the development of therapeutically viable approaches for latency reversal and eventual clearance of persistent HIV in infected individuals. The essential host transcription elongation factor P-TEFb, a heterodimer of CDK9 kinase and a regulatory cyclin T subunit, is a critical mediator of the trans-activation of latent HIV. Availability of P-TEFb for proviral transcription is highly limited due to a posttranscriptional restriction in cyclin T1 expression and dephosphorylation of CDK9 on its activation loop. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4 + T cells, we have now defined the signaling pathways that are essential for the generation of transcriptionally active P-TEFb and, consequently, proviral reactivation. Crucial among these findings is the demonstration that protein kinase C (PKC) agonists signal through activation of RasGRP1-Ras-Raf-MEK-ERK1/2 rather than PKC enzymes to effect the reactivation of both P-TEFb and proviral HIV. Understanding these pathways should lead to the discovery of novel highly selective activators of P-TEFb to improve the efficiency of HIV reactivation in the memory T-cell population of virally suppressed individuals.

Funding: This work was supported by grants from the National Institutes of Health, R01 AI148083 (JK and UM), R01 DE025464 (JK), R21 AI127252 (SV) and P30 A136219, CWRU/UH Center for AIDS Research (JK). UM and SV were also supported by developmental awards from CWRU/UH Center for AIDS Research (P30 A136219). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Our current study focuses on defining signaling pathways and T-cell factors that are responsible for regulating proviral reactivation in memory T cells, with a specific focus on identifying the pathways that are essential for the generation of transcriptionally active P-TEFb. PKC agonists are well known and highly potent HIV latency reversing agents, but although progress has been made with development of novel synthetic agonists, they have had limited clinical applications due to concerns about their side effects and cytotoxicity [ 42 , 43 ]. Using a well-characterized and highly reproducible primary QUECEL (quiescent effector cell latency) model of HIV latency [ 44 ], alongside healthy donor memory CD4 + T cells, we found that diacylglycerol-mimicking PKC agonists can stimulate active P-TEFb expression with minimal cytotoxicity in the absence of co-treatment with a calcium ionophore. Unexpectedly, PKC agonists induce P-TEFb via activation of RasGRP1-Ras-Raf-MEK-ERK1/2 rather than through stimulation of PKC enzymatic activity. P-TEFb induction after TCR co-stimulation is further regulated by the PI3K-mTORC2-AKT-mTORC1 pathway. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation completely abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Thus, reactivation of P-TEFb in primary T cells in response to either TCR co-stimulation or PKC agonist challenge is largely independent of PKC suggesting novel opportunities to develop effective latency reversing strategies.

An additional CDK9 T-loop phospho-modification occurs at a conserved residue Ser175 (pSer175 CDK9) which we recently demonstrated is due to CDK7 activity in both Jurkat and primary CD4 + T cells [ 13 ]. pSer175 CDK9 is only found on a subset of P-TEFb that is transcriptionally active (ie. P-TEFb dissociated from 7SK snRNP) and is functionally important in being coopted by Tat to enhance Tat’s interaction with P-TEFb in order to outcompete BRD4 [ 13 , 39 ], a bromodomain-containing protein that is considered to be a major recruiter of P-TEFb to cellular genes [ 40 , 41 ].

We and others have previously shown that resting CD4 + T cells are highly deficient in the expression of the CycT1 subunit of P-TEFb while the CDK9 kinase subunit is sequestered in an inactive form in the cytoplasm bound to the kinase-specific chaperone complex Hsp90/Cdc37 [ 12 , 13 ]. Activation of resting T cells stimulates a rapid posttranscriptional synthesis of CycT1, which is tightly coupled with Thr186 phosphorylation of the CDK9 subunit (pThr186 CDK9), a posttranslational modification that is critical for the stable heterodimeric assembly of P-TEFb [ 13 ]. Once P-TEFb is assembled it is incorporated into 7SK snRNP, a nuclear regulatory ribonucleoprotein complex that serves to inhibit its kinase activity and sequester the enzyme in nucleoplasmic substructures called speckles found in close proximity to genomic sites of active transcription [ 29 – 32 ]. Recruitment of P-TEFb from 7SK snRNP by Tat and their cooperative binding to a nascently synthesized RNA hairpin called TAR puts the enzyme in close proximity to the negative elongation factors NELF and DSIF, and promoter-proximally paused RNAP II. CDK9 then acts to phosphorylate the E subunit of the repressive NELF complex at multiple serine residues which causes NELF to dissociate from TAR [ 33 – 35 ]. CDK9 also extensively phosphorylates the CTD of RNAP II mainly at Ser2 residues of the heptad repeats Y-S-P-T-S-P-S and C-terminal repeats (G-S-Q/R-T-P) of the hSpt5 subunit of DSIF at Thr4 residues [ 36 – 38 ]. The overall effect of these phospho-modifications by P-TEFb is to remove the blocks to elongation imposed by NELF and DSIF and to stimulate efficient elongation and co-transcriptional processing of proviral transcripts.

Physiological TCR signaling in CD4 + T cells rapidly induces a tyrosine phosphorylation cascade at the cell membrane that leads to the activation of the lipid metabolizing enzymes phospholipase C-γ (PLC-γ) and phosphoinositide-3-kinase (PI3K) [ 24 ]. In turn, PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to generate the second messengers inositol-1,4,5-triphosphate (IP 3 ), that mobilizes calcium release into the cytoplasm, and diacylglycerol (DAG), which directly contributes to the activation of a number of C1 domain-containing intracellular signaling proteins including protein kinase C (PKC) enzymes and the Ras guanine nucleotide exchange factor RasGRP [ 25 – 27 ]. Upon their activation, PI3Ks act to phosphorylate PIP 2 to form phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ), a lipid resistant to PLC-γ hydrolysis, that serves to anchor a wide range of signaling proteins through their pleckstrin homology (PH) domains [ 28 ].

Maintenance of a transcriptionally inactive state in resting memory CD4 + T cells, which engenders the persistence of proviral HIV in latently infected cells, is associated with a tight restriction imposed on P-TEFb activity [ 12 , 13 ]. T-cell receptor (TCR) activation of primary CD4 + T cells potently provides the intracellular signals that are necessary to efficiently stimulate processive HIV transcription by reversing the epigenetic silencing at the proviral promoter, inducing the assembly of P-TEFb and permitting nuclear entry of transcription initiation factors [ 14 , 15 ]. Therefore, the development of more efficient latency reversal strategies will likely require a combination of agents that mimic the effects of TCR signaling. Thus far, histone deacetylase inhibitors are the only class of latency reversal agents (LRAs) to achieve clinical proof-of-concept although they have been found to be marginally effective at activating latent proviruses in both primary cell models of HIV latency and patient-derived CD4 + T cells [ 16 – 22 ]. In a recent clinical study of the HDACi romidepsin we found that poor HIV reactivation was associated with its limited ability to reactivate P-TEFb and transcription initiation factors [ 23 ].

Comprehensively understanding the host mechanisms that permit HIV to emerge from transcriptional latency is crucial for the development of safe pharmacological strategies to eliminate persistent viral reservoirs that are primarily found within memory CD4 + T cells of infected individuals [ 1 – 5 ]. The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator protein Tat, which functions to recruit the essential host transcription elongation factor P-TEFb along with the super elongation complex (SEC) to proviral HIV [ 6 – 9 ]. P-TEFb is a heterodimer of CDK9 serine/threonine kinase and one of three cyclin T regulatory subunits. However, cyclin T1 (CycT1) is the only cyclin capable of interacting with HIV Tat [ 7 , 10 , 11 ].

Results

Regulation of 7SK snRNP and super elongation complex factors in primary T cells Efficient stimulation of proviral HIV transcription elongation by P-TEFb is regulated by 7SK snRNP and facilitated by the super elongation complex (SEC) (Fig 1A) [8,9,29,30]. We therefore examined the expression profiles of 7SK snRNP and SEC components in primary CD4+ T cells before and after activation. Western blotting of resting and TCR-activated Th17 cells demonstrated notable restrictions in protein expression of not just CycT1 but also HEXIM1 and the SEC components AFF1 and ELL2 during quiescence (S6A Fig). Transcriptome analysis (bulk RNA-seq) of primary CD4+ T cells that had been polarized into the four major T-cell subsets (Th1, Th2, Th17, and Treg) [44] was also conducted to provide an additional measure of the differential expression of transcripts belonging to P-TEFb subunits (CDK9, CycT1 and CycT2), 7SK snRNP components (HEXIM1, HEXIM2, LARP7 and MEPCE) and SEC proteins (AFF1, AFF2, ELL, ELL2 and ENL). By including in our analysis a publicly available bulk RNA-seq dataset generated by Zhao et al. using unstimulated and TCR-activated memory CD4+ T cells [48] (SRA accession SRP026389) (Extended data shown in S6B–S6D Fig), we were able to demonstrate that each of these in vitro polarized T-cell subsets can faithfully recapitulate the phenotypic characteristics of memory CD4+ T cells in maintaining the pattern of expression of these factors (Fig 3A–3C). Most notably, the high-throughput sequencing studies demonstrated that there was an overall little change in CycT1 mRNA levels over a 24-h period of TCR co-stimulation (Figs 3A and S6B), consistent with the prevailing idea that this P-TEFb subunit is post-transcriptionally regulated. By contrast, CDK9 transcripts appeared to decrease in all cell types with the most significant decrease observed in the QUECEL subsets (Fig 3A). Therefore, there was an apparent inconsistency between the observed drop in CDK9 transcript levels and the modest increase in protein expression following TCR activation seen in Figs 1C and S6. This discrepancy could be an indication that any elevation in CDK9 protein in the activated cells may emanate from posttranscriptional regulation rather than new mRNA synthesis. Also noteworthy was the observation that HEXIM2 mRNA was very low in resting primary CD4+ T cells (~6 transcripts per million) and was further slightly reduced following TCR co-stimulation (~5 transcripts per million) (Fig 3B). By contrast, HEXIM1 was found to be expressed at approximately 4-fold and 7-fold higher levels compared to HEXIM2 in unstimulated and 24-h TCR-activated primary CD4+ T cells, respectively (Fig 3B). These findings suggest that HEXIM1 could be the major isoform in CD4+ T cells and therefore the more probable regulatory partner for P-TEFb within 7SK snRNP. Consistent with our immunoblotting analysis (S6A Fig) and previous findings that ELL2 is tightly regulated and therefore an important limiting factor for SEC formation [49,50], we observed that ELL2 mRNA is minimal in resting CD4+ T cells and highly inducible in response to TCR activation (Figs 3C and S6C). Single-cell RNA-seq (scRNA-seq) that were carried out using both resting and TCR-activated memory CD4+ T cells also supported the findings from the bulk RNA-seq analysis in showing a restriction of ELL2 in resting T cells and a predominance of HEXIM1 over HEXIM2 (S7A and S7B Fig). Thus, these overall results suggest that the posttranscriptional restriction in CycT1 and repression of the P-TEFb-associated factors HEXIM1, AFF1 and ELL2 may need to be overcome in resting CD4+ T cells in order to achieve a robust reactivation of HIV from latency. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Transcriptome analysis of P-TEFb, 7SK snRNP and SEC expression in primary QUECEL subsets and memory CD4+ T cells. Quiescent CD4+ T cells that had been polarized into Th1, Th2, Treg and Th17 subsets using the QUECEL procedure shown in S4 Fig were activated or not for 24 h with anti-CD3/anti-CD28 Dynabeads. Bulk RNA-seq datasets obtained using these cells were analyzed to examine the expression of P-TEFb subunits (A), 7SK snRNP components (B), and factors belonging to the super elongation complex (C). Transcripts per million (TPM) values were used to evaluate the relative abundance of transcripts under resting and activated conditions. In A, the expression of CD25 was examined as a positive control. A publicly available bulk RNA-seq dataset of primary human memory CD4+ T cells that had been activated or not through TCR co-stimulation (SRA accession SRP026389) with anti-CD3/anti-CD28 coated beads was also analyzed for these factors (Tm Expt 1 and Tm Expt 2). Statistical significance (p values) was calculated using a two-tailed Student’s t test. https://doi.org/10.1371/journal.ppat.1009581.g003

Reactivation of P-TEFb in primary T cells is not mediated by protein kinase C Treatment with a variety of protein kinase C (PKC) agonists (ingenol, prostratin or PMA) stimulated the dual expression of CycT1 and pSer175 CDK9 in both primary Th17 and memory CD4+ T cells albeit at lower mean levels compared to that observed with TCR co-stimulation (Fig 1D). On average, primary Th17 cells appeared to be more responsive to challenge with PKC agonists compared to memory T cells as we observed a 1.2-, 1.9- and 2.7-fold higher level of P-TEFb expression in the former cell type upon stimulation with ingenol, prostratin and PMA, respectively (Figs 1D and S4B). Also noteworthy was the observation that while P-TEFb expression was barely induced in memory T cells following ionomycin treatment, primary Th17 cells responded significantly better with a 4.9-fold higher expression of P-TEFb (Figs 1D and S4B). Overall, there was a much higher expression of P-TEFb in both cell types following challenge with the PKC agonists than with ionomycin treatment. Co-treatment of memory T cells with ionomycin and each of the three PKC agonists did not further elevate P-TEFb expression in memory T cells but substantially enhanced formation of both the transcriptionally active form of NF-κB (pSer529 p65) and the P-TEFb-modified pSer2 CTD RNAP II (Fig 4A). While treatment with PKC agonists elicited minimal cytotoxicity to memory T cells (relative to unstimulated cells viability was found to be 93%, 91% and 87% for ingenol, prostratin and PMA, respectively), co-treatment with ionomycin resulted in a significant loss of cell viability (35–40% cell death compared to unstimulated cells) (Fig 4A). These findings suggest that while intracellular calcium mobilization is not essential for enabling P-TEFb biogenesis in memory T cells, it significantly potentiates the efficacy of PKC agonists at generating the transcription elongation competent form of RNAP II albeit with significant cell cytotoxicity. Exposure of polarized primary Th17 cells to ingenol or prostratin was sufficient to stimulate the formation of P-TEFb and resulted in a comparable extent of proviral reactivation in latently infected Th17 cells as TCR co-stimulation (Figs 4B and S5D). By comparison, TNF-α and SAHA which are ineffective at generating transcriptionally active P-TEFb in both memory and Th17 cells (Fig 1D), were found to be poor reactivators of proviral HIV in the QUECEL Th17 latency model (S5C Fig). Interestingly, ionomycin was able to reactivate latent HIV in QUECEL Th17 cells but to a lower and much more variable extent compared to ingenol and prostratin (Fig 4B). The ability of ionomycin to potently activate the transcription initiation factor NFAT [44], and the slight induction in P-TEFb levels observed upon treatment of primary Th17 cells (Figs 1D, 4B and S4B) are likely to account for this observation. Thus, the reactivation of HIV from latency appears to be tightly coupled to the biogenesis of P-TEFb in primary T cells and both of these processes can be elicited upon cellular treatment with PKC agonists in the absence of intracellular calcium mobilization. PPT PowerPoint slide

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TIFF original image Download: Fig 4. P-TEFb biogenesis in primary T cells does not require intracellular mobilization of calcium. (A) Left graph; Cross-comparison of the effectiveness of 50 nM ingenol, 1 μM prostratin or 50 ng/ml PMA on their own or combined with 1 μg/ml ionomycin at inducing active P-TEFb in primary T cells measured by co-staining for CycT1 and pSer175 CDK9. Middle graph; Cross-comparison of the effectiveness of 50 nM ingenol, 1 μM prostratin or 50 ng/ml PMA on their own or combined with 1 μg/ml ionomycin at coordinately inducing the C-terminal domain Ser2 phosphorylated form of RNA polymerase II (pSer2 RNAP II CTD) and the transcriptionally active form of NF-κB (pSer529 p65). Right graph; Measurement of the effect of 50 nM ingenol, 1 μM prostratin or 50 ng/ml PMA on their own or combined with 1 μg/ml ionomycin on cell viability as assessed by flow cytometry following staining with the eFluor450 viability dye. (B) Measurement of P-TEFb (left graph) and latent HIV (right graph) reactivation following 24 h treatment of resting and polarized Th17 cells with 50 nM ingenol, 1 μM prostratin or 1 μg/ml ionomycin. The graphs show data generated using polarized Th17 cells prepared using naïve T cells from at least two different donors. (C) Proposed schemes for the promoter recruitment of RNA polymerase II (RNAP II) to proviral HIV and its phosphorylation by active P-TEFb on its C-terminal domain (pSer2 RNAP II CTD). Arrows with red question marks indicate a P-TEFb regulatory signaling pathway(s) intended to be defined by the current study. https://doi.org/10.1371/journal.ppat.1009581.g004 PKC agonists, commonly referred to as phorbol esters, are structural mimics of endogenously generated diacylglycerol (DAG) and bind the C1 domains of both conventional (α, βI, βII, and γ) and novel (δ, θ, ε, and η) PKC isozymes leading to their membrane recruitment and stimulation of their kinase activities [27]. Previously, we had shown that treatment of Jurkat T cells with a pan-PKC inhibitor Ro-31-8220 effectively blocked the inducible phosphorylation of CDK9 at Ser175 that is rapidly stimulated by the phorbol ester PMA [13]. These results suggested that pSer175 CDK9 was likely to be PKC-dependent, although in the same study we did unequivocally demonstrate that this CDK9 T-loop modification was directly due to CDK7 activity. We therefore examined whether DAG-binding PKC enzymes can mediate the expression of CycT1 and/or T-loop phosphorylated CDK9 in primary T cells in response to TCR co-stimulation or phorbol ester treatment (Fig 4C). Healthy donor memory CD4+ T cells and polarized primary Th17 cells were either activated through the TCR or challenged with phorbol ester in the presence or absence of treatment with a conventional pan-PKC inhibitor (Ro-31-8220), an inhibitor that potently inhibits PKC α, β, γ, and δ isoforms (Gö-6983), or one that selectively targets PKC-θ (sotrastaurin). PKC-θ is considered to be the predominantly functional PKC isoform in T cells and it critically mediates both TCR-induced NF-κB and global T-cell activation [51–53]. Surprisingly, all three PKC inhibitors failed to efficiently block either the TCR- or the phorbol ester-mediated inducible expression of CycT1, pSer175 CDK9 and pThr186 CDK9 in both memory CD4+ T cells and polarized primary Th17 cells or had only very modest effects in their inhibition (Figs 5A, 5B, S8 and S9C). Similarly, these inhibitors were ineffective at suppressing the reactivation of latent HIV in primary Th17 cells that was elicited by either TCR co-stimulation or challenge with phorbol ester (Figs 5B, 5C and S5E). By contrast, sotrastaurin and Ro-31-8220 effectively inhibited the activation of NF-κB in memory T cells in response to phorbol esters as measured by monitoring the formation of pSer529 p65 (Fig 5A). In addition to serving as a positive control for the activity of these PKC inhibitors, this observation is consistent with the prevailing idea that PKC, and more specifically PKC-θ, is a primary mediator of NF-κB activation in T cells. We were also able to demonstrate that Ro-31-8220 can disrupt the degradation of IκB-α due to TCR activation in memory T cells, thereby delaying the peak mobilization of NF-κB subunits p65 and p50 into the nucleus (S9A and S9B Fig). Overall, these results indicate that the coordinate reactivation of P-TEFb and latent HIV in primary T cells in response to either TCR co-stimulation or phorbol ester challenge is largely independent of PKC signaling pathways. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Coordinate reactivation of P-TEFb and latent HIV in primary T cells is largely independent of PKC activity. (A) PKC-θ and pan-PKC inhibition are inefficient at repressing ingenol- or prostratin-induced expression of P-TEFb, but effective at blocking the activating phosphorylation of the p65 subunit of NF-κB at Ser529 in response to these phorbol esters. CD4+ memory T cells from four different donors and in vitro polarized primary Th17 cells were treated for 24 h with the indicated concentrations of either ingenol or prostratin in the presence or absence of either 100 nM sotrastaurin or 100 nM Ro-31-8220 prior to subjecting cells to dual immunofluorescence staining for pSer529 p65 (top graph) or for CycT1 and pSer175 CDK9 (bottom graph). (B) PKC inhibitors modestly repress TCR-mediated expression of P-TEFb in CD4+ memory T cells but are unable to block reactivation of proviral HIV in primary Th17 cells. Left graph; Measurement of P-TEFb expression by dual CycT1 and pSer175 CDK9 immunofluorescence staining of memory T cells that were TCR-activated with anti-CD3/anti-CD28 Dynabeads for 24 h in the presence or absence of treatment with either 100 nM sotrastaurin or 100 nM Ro-31-8220. Right graph; Assessment of the effect of PKC inhibition on proviral reactivation in ex vivo HIV-infected primary Th17 cells prepared from naïve CD4+ T cells belonging to three healthy donors. Latently infected cells were treated or not for 30 min with 100 nM Ro-31-8220 in two experiments or with 100 nM Sotrastaurin in three experiments prior to TCR co-stimulation with anti-CD3/anti-CD28 Dynabeads for 24 h. Thereafter, cells were analyzed by flow cytometry following immunostaining using a fluorophore-conjugated antibody towards HIV Nef. (C) PKC inhibitors are unable to repress the reactivation of latent HIV in primary Th17 cells that is in response to PKC agonists. Latently infected Th17 cells were treated or not for 30 min with either a combination of Ro-31-8220 and Gö 6983 (Donor 14 (Expt 1) and Donor 14 (Expt 2)) at 100 nM each or 100 nM Sotrastaurin (Donor 16 (Expt 1) and Donor 14 (Expt 3)) prior to TCR co-stimulation or challenge with 50 nM ingenol or 1 μM prostratin. Thereafter, cells were immunostained using a fluorophore-conjugated antibody towards HIV Nef and analyzed by flow cytometry. Statistical significance (p values) in A, B and C was calculated using a two-tailed Student’s t test. https://doi.org/10.1371/journal.ppat.1009581.g005

PKC agonists signal through the MAPK ERK pathway to stimulate P-TEFb expression and reactivate latent HIV in primary T cells TCR-mobilized diacylglycerol and exogenous phorbol esters can also target several other proteins with C1 domains including RasGRP, a guanine nucleotide exchange factor for Ras in T cells [26]. Membrane-anchored RasGRP1 facilitates the activity of SOS, a second Ras GDP-GTP exchange factor, in a positive feedback mechanism that involves the allosteric activation of SOS by RasGRP-generated Ras-GTP [54]. Exchange of GDP for GTP on Ras by both RasGRP and activated SOS causes the formation of Ras–Raf complexes at the plasma membrane that lead to the activation of the MAPK isoforms ERK1 and ERK2 (ERK1/2) (Fig 6A) [55,56]. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Inducible P-TEFb expression and proviral reactivation in primary T cells are mediated by the MAPK ERK pathway. (A) Proposed scheme for the intracellular stimulation of P-TEFb expression in primary T cells by PKC agonists or TCR-generated diacylglycerol (DAG) leading up to the reactivation of HIV from latency. (B) The MEK inhibitor U0126 substantially blocks expression of active P-TEFb in primary CD4+ T cells in response to treatment with PKC agonists but modestly inhibits P-TEFb expression in response to TCR co-stimulation. Memory CD4+ or primary Th17 cells were treated or not for 30 min with U0126 at the indicated concentrations prior to TCR co-stimulation with anti-CD3/anti-CD28 Dynabeads or challenge with either 50 nM ingenol or 1 μM prostratin for 24 h. Cells were then analyzed by flow cytometry for P-TEFb expression following immunostaining using fluorophore-conjugated antibodies towards CycT1 and pSer175 CDK9. (C) U0126 substantially retards proviral reactivation in primary Th17 cells in response to TCR co-stimulation or challenge with PKC agonists. Latently infected Th17 cells prepared using naïve CD4+ T cells from two different donors were pretreated or not with 20 μM U0126 for 30 min prior to TCR co-stimulation with anti-CD3/anti-CD28 Dynabeads or challenge with 50 nM ingenol or 1 μM prostratin for 24 h. Cells were analyzed by flow cytometry following immunostaining using a fluorophore-conjugated antibody towards HIV Nef. All the p values shown in B and C were calculated using a two-tailed Student’s t test. https://doi.org/10.1371/journal.ppat.1009581.g006 We were able to confirm that phorbol ester treatment of T cells rapidly stimulates the activating phosphorylation of ERK1/2 (pThr202/pTyr204) concomitant with the inducible synthesis of c-Fos and its localization to the nucleus (S10A Fig). Therefore, we examined whether the Ras–Raf–MEK–ERK1/2 pathway (Fig 6A) can also mediate the biogenesis of P-TEFb in response to TCR or phorbol ester challenge and if it could be a primary pathway for the reactivation of latent HIV in primary CD4+ T cells. Treatment of Th17 and CD4+ memory T cells with the MEK inhibitor U0126, which effectively blocks the activating phosphorylation of ERK1/2 (S10A and S10B Fig), resulted in a modest inhibition of TCR-induced P-TEFb expression (by an average of 1.5-fold) (Fig 6B). By contrast, U0126 treatment much more effectively inhibited the expression of P-TEFb in both Th17 and memory T cells stimulated by either ingenol or prostratin (by an average of 3.7-fold and 7-fold, respectively) (Fig 6B). Consistent with this observation, U0126 also substantially repressed proviral reactivation in HIV-infected Th17 cells that was induced by ingenol and prostratin (by an average of 2-fold and 3.7-fold, respectively) (Figs 6C and S12). Although U0126 had a modest and variable repressive effect on TCR-induced P-TEFb expression, there was a stronger repressive effect on TCR-induced proviral reactivation in HIV-infected Th17 cells (1.75-fold repression of HIV compared to the 1.5-fold repression observed on P-TEFb) (Figs 6B, 6C and S12). To test the hypothesis that TCR co-stimulation may be exerting effects on the initiation of proviral transcription by generating the activator protein 1 (AP-1) transcription factor complex of c-Fos and c-Jun through a crosstalk between the ERK1/2 and JNK MAPK pathways (S13A Fig), we examined whether inhibiting JNK either individually or in combination with MEK had any significant effects on proviral reactivation as well as P-TEFb expression. The anthrapyrazolone SP600125 is a well characterized inhibitor of JNK that has been shown to effectively block its kinase activity in T cells at a concentration range of 10–25 μM [57]. Inhibition of JNK signaling with SP600125 caused a 1.3-fold reduction in TCR-induced proviral reactivation compared to a 1.7-fold reduction observed with U0126 treatment (S13B Fig). Treatment of latently infected Th17 cells with both inhibitors failed to elicit additional suppression of TCR-induced proviral gene expression beyond that observed with U0126 treatment (S14 Fig). Similarly, inhibition of JNK did not further enhance the modest repression of TCR-induced P-TEFb expression observed upon MEK inhibition (S15 Fig). Moreover, and not surprisingly, inhibition of JNK had a very modest suppressive effect on ingenol- and prostratin-induced proviral and P-TEFb expression (S14 and S15 Figs) clearly indicating that these phorbol esters do not signal through JNK in eliciting their stimulatory effects. By contrast, a well-characterized Raf inhibitor AZ 628 which selectively and potently inhibits Raf1 and B-Raf kinase activities (with IC 50 values of 29 and 105 nM, respectively) was effective at suppressing P-TEFb induction in response to ingenol and prostratin but had no inhibitory effect on TCR-induced P-TEFb expression (S16 Fig). In summary, our results clearly suggest that MEKK1-MKK4/7-JNK-c-Jun is unlikely to be an essential MAPK pathway for regulating the formation of P-TEFb or the reactivation of latent HIV in CD4+ T cells. Instead, our results strongly support the idea that the Raf-MEK-ERK1/2 MAPK pathway is a primary mediator of the expression of active P-TEFb in memory T cells upon challenge with DAG-mimicking phorbol esters and that this signaling pathway is also essential for the reactivation of latent HIV by these candidate LRAs.

[END]

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