Human Immunodeficiency Virus Reactivation by Phorb
http://www.100md.com
病菌学杂志 2005年第15期
Division of Infectious Diseases and Program in Translational Immunovirology and Biodefense
Department of Immunology, Mayo Clinic College of Medicine, Rochester, Minnesota
ABSTRACT
Latently human immunodeficiency virus (HIV)-infected memory CD4+ T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKC, PKCA148E, and PKC, PKCA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+ T cells and that both PKCA148E and PKCA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKC and PKC with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKC or PKC. Since suppression of PKC and PKC together has no greater inhibitory effect on HIV reactivation than inhibition of PKC alone, our data confirm that PKC and PKC act in sequence. The requirement for PKC and PKC for prostratin-induced HIV reactivation and the ability of selective PKC or PKC agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.
INTRODUCTION
The effectiveness of current antiretroviral agents is underscored by their inability to impact transcriptionally inactive viral reservoirs. Accordingly, human immunodeficiency virus (HIV) viral rebound is commonly observed when patients cease taking antiretroviral agents (15, 18, 19, 50, 58). Sequence analysis of rebound viral isolates indicates significant homology with proviral sequences from infected memory CD4+ T cells (61), suggesting that such cell populations act as a principal viral reservoir that is unaffected by antiretroviral therapy. Indeed, memory CD4+ T cells which contain HIV provirus have been defined to be transcriptionally inactive and to have an in vivo half-life of several years (13, 14). Consequently an estimated 60 or more years of fully supportive antiretroviral therapy may be required to fully purge such a reservoir, a time frame which is both impractical and clinically not useful. Three conceptually distinct approaches have been proposed as ways of impacting such viral reservoirs: toxins designed to eliminate virus-containing cells (5, 21, 38), antiviral intensification strategies to prevent repopulation of the latent pools (12, 26, 32, 37, 59), and compounds which stimulate HIV long terminal repeat (LTR) transcription, thereby rendering such cells susceptible to antiretroviral agents and/or the cytotoxic effects of viral replication (30, 49).
The HIV type 1 (HIV-1) LTR is regulated in large part by cellular transcription factors, including NF-B, AP-1, SP-1, and NF-AT (reviewed in reference 28). Consequently, stimuli which activate these transcription factors cause HIV LTR-driven viral replication (17, 20, 41). Antigen presentation or T-cell receptor (TCR) cross-linking can induce HIV reactivation from latently infected CD4+ T cells in vitro (15) through protein kinase C (PKC)-mediated activation of NF-B (40). Direct activation of PKC by phorbol esters reactivates HIV from latency in cell lines (27, 29, 45) and, more importantly, it induces HIV reactivation in peripheral blood mononuclear cells and primary quiescent T cells (1, 9, 10). PKC activation induces HIV from latency by targeting multiple regulatory elements of HIV LTR. In addition to NF-B, PKC induces HIV LTR transcription via activation of the AP-1 transcription factor; however, its action is largely dependent on a cooperation with NF-B (35, 46, 60). PKC also increases HIV-1 gene expression by phosphorylation of the virally encoded TAT transcription factor and cellular TAR-binding factors (25, 27).
Among the PKC isoforms in T cells, the novel PKC isoform PKC plays a crucial role in T-cell activation and proliferation (3, 6, 51). PKC activates NF-B (16, 36, 51) and AP-1 (4, 56) transcription factors, which bind to multiple sites in the enhancer region of the HIV-1 LTR. In addition PKC acts upstream of PKC to induce NF-B following CD3/CD28 ligation (54). Since HIV reactivation following CD3/CD28 ligation is NF-B dependent, together these data suggest that each of these two PKC isoforms may be required to mediate reactivation of latent HIV. Indeed, inhibition of both conventional and novel PKC isoforms blocks HIV reactivation following costimulation with CD3 and CD28 antibodies in the SCID-hu(Thy/Liv) model (9).
While TCR cross-linking can reactivate latent HIV in primary T cells in vitro (9, 11), clinical efforts to reduce the latent pool of HIV by treatment with the anti-CD3 antibody OKT-3 alone or in combination with interleukin-2 fails to meaningfully decrease the viral reservoir (32, 44, 55). The phorbol esters (8, 33) and 1,2-diacylglycerol analogs (24), including the non-tumor-promoting phorbol ester prostratin (23), have been suggested as agents to reactivate HIV and eradicate the pool of latently HIV-infected CD4+ T cells (22, 23). More recently, phorbol esters including prostratin have been directly evaluated for their ability to reactivate latent virus (30, 31). Indeed, prostratin causes significant reactivation of latent HIV from memory T-cell pools (7, 10, 30, 48). The mechanism by which prostratin causes viral reactivation is, therefore, of interest, and two independent studies now suggest that conventional and novel PKC isoforms are required (22, 57), although which specific PKC isoforms are involved and whether they are absolutely required is unknown. The present study was designed to address the specific requirement of different PKC isoforms following TCR- or phorbol ester-mediated HIV LTR reactivation and to determine whether such PKC isoforms are absolutely required.
MATERIALS AND METHODS
Plasmids. Generation and characterization of isoform-specific PKC suppression vectors were previously described (54). The HIV LTR and HIV LTR (B)-dependent firefly luciferase reporter expression vector (B-luc) has been previously described (17). The pRL-TK expression vector, which provides constitutive expression of Renilla luciferase, is commercially available (Promega, Madison, WI). GFP-PCI was a gift from D. McKean (Mayo Clinic, Rochester, MN). The Sp1-luciferase reporter was kindly provided by D. Billadeau (Mayo Clinic, Rochester, MN). The mammalian expression vector, pEF1/Myc-His, was purchased from Invitrogen (Carlsbad, CA). Constitutively active forms, PKCA25E and PKCA148E, were previously described (54).
Constitutively active IKK?S177E/S181E was generated by site-directed mutagenesis using mutagenic primers (sense, 5'-GAACTTTGCACAGAATTCGTGGGGA; antisense, 5'-TTCTGTGCAAAGTTCGCCCTGATCC; sense IKK? with BamHI site [underlined], GGGATCCATCCATGAGCTGGTCACCT; antisense IKK? with XbaI site [underlined], CGTCTAGATTATGAGGCCTGCTCCAG) and cloned into pGEM-T Easy. The cDNA coding for IKK?S177E/S181E was excised and subcloned into HA-pcDNA3 I at the BamHI XbaI site. The sequences of all generated constructs were verified by sequencing.
Cell culture and reagents. Jurkat T cells and latently HIV-1-infected J1.1 T cells were obtained from the American Type Culture Collection, Rockville, MD and maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine. Cells were grown to a density of 3 x 105 to 5 x 105/ml at the time of the different experiments. Ionomycin was purchased from Calbiochem (La Jolla, CA), and tumor necrosis factor alpha was purchased from R&D Systems (Minneapolis, MN). Leupeptin, aprotinin, and pepstatin A were obtained from Boehringer Mannheim (Indianapolis, IN). Antihemagglutinin (anti-HA) high-affinity antibodies were purchased from Boehringer Mannheim (Indianapolis, IN). OKT3 antibodies were obtained from Ortho Biotech (Raritan, NJ), and anti-CD28 was purchased from BD Biosciences (San Jose, CA). Anti-PKC (C-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC?1 and anti-PKC were purchased from BD Transduction Laboratories (San Diego, CA).
To isolate CD4+ T cells, peripheral blood mononuclear cells from healthy volunteer blood donors were obtained from buffy coats by using RossetteSep cocktail CD4+ antibodies in accordance with the manufacturer's protocol (StemCell Technologies, Vancouver, British Columbia, Canada). The remaining cell population was repeatedly found to be 98% CD4+ T cells as determined by flow cytometry. CD4+ T cells used in the various experiments were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mM L-glutamine, and antibiotics (penicillin [100 U/ml] and streptomycin [100 μg/ml]) at 0.5 x 106 cells/ml. For Jurkat T cells, ionomycin was used at 3.5 μg/ml. Jurkat and J1.1 T cells were cross-linked with 10 μg/ml of anti-CD3 and anti-CD28 antibodies or isotype control antibodies as previously described (53).
Cell extract preparation and immunoblotting. To obtain total cellular proteins, cells were washed with cold phosphate-buffered saline, resuspended in a modified whole-cell extract PD buffer (53) (40 mM Tris-HCl [pH 8], 0.3 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM NaF, 10 mM P-nitrophenyl phosphate 10 mM ?-glycerophosphate, 300 μM sodium orthovanadate, 1 mM dithiothreitol, 2 μM phenylmethylsulfonyl fluoride, aprotinin at 10 μg/ml, leupeptin at 1 μg/ml, pepstatin 1 μg/ml) and centrifuged at 12,000 x g for 15 min at 4°C. The resulting supernatant contained total cellular protein. The amount of cellular protein present in the clarified supernatant was calculated by using the Bio-Rad (Hercules, CA) protein assay. For Western immunoblot assays, equal amounts of whole-cell extract were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Immunoblotting was performed with specific antibodies and visualized by using the ECL Western blotting detection kit (Amersham, Buckinghamshire, England).
Gene transfection, p24 enzyme-linked immunosorbent assay (ELISA), and reporter assays. Jurkat T and J1.1 T cells were electroporated using a BTX Electro Square Porator T820 (BTX Corporation, San Diego, CA) at 325 V, 10 ms, and 350 V, 10 ms, correspondingly. Primary CD4+ T cells were electroporated by using Nucleofector (U-14 program; Amaxa Inc., Gaithersburg, MD) in accordance with a protocol for unstimulated human T cells. Where indicated, transfection efficiency was monitored by green fluorescent protein (GFP) expression.
Jurkat T cells were transfected with the indicated plasmids and grown for 18 to 24 h. Cells were stimulated for 12 h with ionomycin (3.5 μg/ml), prostratin (10 μM), or phorbol myristate 13-acetate (PMA; 10 ng/ml) or cross-linked with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies as previously described (54). Thereafter, cells were washed twice in cold phosphate-buffered saline and lysed with 100 μl lysis buffer (Dual-Luciferase reporter assay system; Promega, Madison, WI). Firefly and Renilla luciferase activities from 20 μl of extract were assayed using the Promega Dual-Luciferase reporter assay system reagents and a Berthold Lumat following the manufacturer's recommendations. B-Luciferase activity was normalized to Renilla expression. All transfection experiments were performed in duplicate.
Concentrations of p24 Gag in supernatants of J1.1 T cells were measured in duplicate by using a p24 ELISA kit (ZeptoMetrix Corporation, Buffalo, NY).
RESULTS
Constitutively active forms of both PKC and PKC induce HIV LTR activation via NF-B in Jurkat and human primary CD4+ T cells. Recently we demonstrated that PKC acts upstream of PKC to activate NF-B following CD3/CD28 stimulation in uninfected T cells (54). Since antigen presentation, TCR ligation, and phorbol esters all lead to NF-B activation and subsequent HIV LTR transcription, we first examined the role of both PKC and PKC in induction of HIV-1 LTR transcriptional activity in T lymphocytes mediated by these stimuli.
First, we studied whether CA forms of PKC, PKCA25E, and PKC, PKCA148E, can induce HIV-1 LTR transcriptional activity. Jurkat T cells were transfected with either PKCA25E or PKCA148E, together with luciferase reporter plasmids containing the wild-type HIV-1 LTR or the LTR lacking the two B sites, HIV-LTR (B). As shown in Fig. 1A, expression of either PKCA25E or PKCA148E induced HIV LTR transcriptional activity preferentially via NF-B, since deletion of B sites strongly impaired HIV LTR activation (Fig. 1A). As previously shown (54), ionomycin treatment is required for efficient NF-B activation by PKCA25E, since expression of PKCA25E resulted in only a moderate increase of HIV LTR transcriptional activity (Fig. 1A) which was further augmented by ionomycin treatment. Similar to PKCA148E, PKCA25E induced HIV LTR transcription via B sites (Fig. 1A). While PKCA25E and PKCA148E strongly activated NF-B-dependent transcription (25- to 200-fold) of HIV LTR, both only modestly induced (1.2- to 2-fold) transcriptional activity of the transcription factor, Sp1 (Fig. 1B).
To further confirm the relevance of these findings, we studied whether PKCA25E and PKCA148E can induce HIV-1 LTR transcriptional activity in human primary CD4+ T cells. Purified human CD4+ T cells were transfected with either PKCA25E or PKCA148E, together with the luciferase reporter plasmids mentioned above. Similar to our results with Jurkat T cells, expression of either PKCA25E or PKCA148E in human primary CD4+ T cells induced HIV-LTR transcriptional activity via B sites (Fig. 1C and D).
Taken together these results demonstrate that both PKC and PKC can induce HIV LTR transcription preferentially through B sites in Jurkat and primary CD4+ T cells.
Suppression of either PKC or PKC inhibits HIV LTR activation by CD3/CD28 stimulation in Jurkat T cells. To identify which of the two PKC isoforms mediates CD3/CD28-induced HIV LTR activation, we utilized isoform-specific PKC suppression vectors (54), which direct synthesis of short hairpinned (sh) RNA molecules specific for targeting of either human PKC or human PKC. Jurkat T cells were transfected with either pFRT-PKC or pFRT-PKC together with luciferase reporter plasmids as mentioned above. The levels of PKC and PKC expression in transfected cells were analyzed 48 h later by immunoblotting with anti-PKC and anti-PKC antibodies. As shown in Fig. 2C, transfection of Jurkat T cells with either pFRT-PKC or pFRT-PKC resulted in selective and significant reduction of PKC and PKC expression. Neither of the shRNA constructs affected the levels of PKC?1. Cross-linking of transfected Jurkat T cells with CD3 and CD28 antibodies induced HIV LTR-dependent luciferase activity preferentially via B sites of LTR (Fig. 2A and B). Suppression of either PKC or PKC significantly impaired CD3/CD28-induced HIV LTR-dependent luciferase activity. In contrast, suppression of either PKC or PKC had no effect on a basal or prostratin-induced Sp1-dependent luciferase activity (Fig. 2D), suggesting that both PKCs are dispensable for Sp1 transcription. Altogether, these results indicate that both PKC isoforms are required for CD3/CD28-induced HIV LTR-dependent transcription via B sites in Jurkat T cells.
Constitutively active forms of both PKC and PKC reactivate HIV in latently infected J1.1 T cells. To further define the role of both PKCs in HIV reactivation, we studied HIV LTR-dependent transcription and HIV-1 production in the latently HIV-infected CD3+ J 1.1 T-cell line (42). J1.1 T cells were transfected with HA-PKCA25E or HA-PKCA148E or, as a positive control, with the constitutively active form of IKK?, HA-IKK?S177E,S181E, together with either HIV LTR (Fig. 3A) or HIV LTR (B)- luciferase reporters (Fig. 3B) and the tkRenilla reporter plasmid. Thirty-six hours later, supernatants were collected to measure p24 Gag levels and cells were harvested to assay luciferase activities. Expression of either PKCA148E or PKCA25E induced HIV LTR transcriptional activity in J1.1 T cells through both NF-B-dependent and non-NF-B-dependent pathways (Fig. 3A and B).
To test whether PKCA25E, HA-PKCA148E, or HA-IKK?S177E,S181E can reactivate HIV from latency, we measured p24 Gag levels in supernatants of transfected J1.1 cells. As shown in Fig. 3D, the expression of the constitutively active forms of PKC, PKC, or IKK? induce strong p24 Gag production in J1.1 T cells, which correlates with HIV LTR activation (Fig. 3A and B).
Taken together, these results demonstrate that activation of either PKC or PKC is sufficient for the activation of HIV LTR transcription and for subsequent HIV reactivation in latently infected T cells.
Suppression of either PKC or PKC inhibits HIV reactivation by CD3/CD28 stimulation in J1.1 T cells. To confirm which of the two PKC isoforms is required for the CD3/CD28-induced HIV reactivation, J1.1 T cells were transfected with control vector, pFRT-PKC alone, pFRT-PKC alone, or pFRT-PKC together with pFRT-PKC. HIV LTR or HIV LTR (B) luciferase plasmids and Renilla were cotransfected. Forty-eight hours later cells were cross-linked with anti-CD3 and anti-CD28 antibodies and then cells and supernatants were harvested to assay luciferase activity (Fig. 4A), PKC expression (Fig. 4B), and p24 Gag concentrations (Fig. 4C). As shown in Fig. 4A, stimulation of J1.1 T cells with anti-CD3 anti-CD28 antibodies induced a threefold increase in HIV LTR transcription. Deletion of two B sites in the LTR resulted in a 50% decrease of CD3/CD28-induced HIV LTR transcription. Suppression of PKC inhibited B-specific CD3/CD28-induced HIV LTR transcription and resulted in a twofold decrease of p24 levels in CD3/CD28-treated J1.1 T cells. Conversely, PKC suppression inhibited both B-dependent and to a lesser degree B-independent CD3/CD28-induced HIV LTR transcription, potentially altering AP-1 (4, 56) and NF-AT (2, 43, 56) activation. Simultaneous suppression of both PKCs did not have a greater effect on CD3/CD28-induced HIV LTR transcription and p24 production than suppression of PKC or PKC alone (Fig. 4A and C), confirming that PKC and PKC act in sequence following CD3/CD28 stimulation (54).
Both PKC and PKC are required for HIV reactivation by phorbol esters in J 1.1 T cells. Having shown that both PKC isoforms are required for CD3/CD28-mediated HIV reactivation, we addressed the role of both PKCs in phorbol ester-mediated HIV reactivation from latency. Isoform-specific PKC suppression vectors were expressed in J1.1. T cells that were then stimulated with two different phorbol esters, PMA or prostratin. Stimulation of J1.1 T cells with PMA or prostratin induced 12-fold increases in HIV LTR-dependent luciferase activity that was reduced by deletion of the B sites of HIV LTR [Fig. 5A, HIV-LTR (B)] or suppression of PKC. PKC suppression resulted in a twofold decrease of p24 Gag production induced by PMA or prostratin (Fig. 5B). It also blocked 50% of HIV LTR-dependent transcription induced by either PMA or prostratin. As with CD3/CD28 stimulation, suppression of PKC had no effect on PMA- or prostratin-induced HIV LTR (B) transcription (Fig. 5A). Suppression of PKC blocked almost 50% of prostratin PMA-mediated HIV LTR activation via B sites and resulted in a twofold decrease of prostratin- or PMA-induced HIV LTR (B) transcription. PKC suppression also significantly decreased p24 production induced by prostratin (P < 0.05) (Fig. 5B). Similar to CD3/CD28 stimulation, suppression of both PKC and PKC together was no more effective in blocking PMA- or prostratin-induced HIV LTR transcription and p24 production than suppression of PKC alone.
DISCUSSION
In this study we have demonstrated a specific requirement for both PKC and PKC in HIV reactivation following TCR ligation or stimulation with the non-tumor-promoting ester prostratin in T lymphocytes. We conclude this based on observations that selective suppression of PKC, PKC, or both PKC isoforms inhibits HIV LTR transcription and HIV reactivation following CD3/CD28 cross-linking or prostratin treatment in Jurkat and J1.1 T cells. Several interesting features of HIV reactivation by these stimuli are underscored by our results. First, HIV reactivation by either CD3/CD28 engagement or prostratin stimulation requires both PKC and PKC, demonstrating that both PKC isoforms are necessary. Second, suppression of PKC alone or PKC alone has the same inhibitory effect on HIV LTR transcription as suppression of both PKC isoforms together, suggesting that PKC and PKC act in sequence to activate HIV LTR transcription. These findings are consistent with our previous observation that PKC acts upstream of PKC to activate NF-B following TCR ligation (54). Exactly how PKC upregulates PKC is undefined, yet potential direct phosphorylation of PKC by PKC at the plasma membrane may be required for either full PKC activation or to prolong PKC signaling following either CD3/CD28 engagement or prostratin stimulation. The murine PKC undergoes inducible phosphorylation at the C terminus on two adjacent (Thr-692 and Thr-703) residues following TCR or phorbol ester stimulation, which is required for NF-B activation (52). We observed that the C-terminal phosphorylation of PKC occurs in a PKC-dependent manner (data not shown), suggesting that PKC can directly phosphorylate the two adjacent serine residues, S685 and S703, in human PKC (unpublished data). Therefore, PKC-mediated phosphorylation prolongs PKC activation which, as we have previously demonstrated (53), is required for efficient NF-B transcription. Consequently, persistent PKC activation may be necessary for efficient HIV LTR transcription and subsequent HIV reactivation from latency. This contention is supported by the fact that the constitutively active form of PKC provides prolonged signaling and, therefore, it induces HIV LTR transcription in primary CD4+ T cells (Fig. 1C) and efficiently reactivates HIV from latency (Fig. 3D). Finally, we demonstrated that suppression of either PKC or PKC inhibits prostratin-mediated or CD3/CD28-mediated HIV LTR transcription via B sites. However, suppression of PKC also has a significant effect on prostratin-induced HIV LTR (B) transcription and a smaller effect on CD3/CD28-induced HIV LTR (B) transcription, potentially due to inhibition of other transcription factors, such as AP-1. Previously it was shown that prostratin induces both NF-B- and AP-1-dependent transcription (57). Our data are in agreement, since constitutively active forms of both PKC and PKC can induce transcription of HIV LTR lacking B sites (Fig. 3B) and potently induce AP-1-dependent transcription in J1.1 T cells (data not shown). However, it is noteworthy that the magnitude of AP-1 activation by PKC is much greater than that by PKC. Therefore, selective inhibition of CD3/CD28- and prostratin-induced HIV LTR (B) transcription by suppression of PKC but not PKC indicates that PKC and PKC pathways are divergent. This is in agreement with the previous observation that PKC activates NF-B, but not AP-1, in a PKC-dependent manner (54). It is possible that the divergence in the PKC and PKC signaling pathway occurs on the level of Ste20-related proline- and alanine-rich kinase, which recently has been shown to mediate PKC, but not PKC, signaling to AP-1 (34). Therefore, while both PKC isoforms induce HIV LTR transcription via B sites, PKC also contributes in HIV LTR transcription via AP-1 sites. The conclusion that both PKC and PKC are required for HIV reactivation is in agreement with recently published data demonstrating that CD3/CD28 costimulation and prostratin treatment reactivate HIV from latency in the SCID-HU Thy/Liv model (9) in both a PKC- and NF-B-dependent manner. Similarly, a role for PKC in mediating prostratin-induced HIV reactivation is further supported by observations that prostratin induces translocation of several conventional, novel, and atypical PKC isoforms to the cell membrane, suggesting that all these PKC isoforms can contribute to HIV reactivation (57). That suggestion is supported by observations that simultaneous inhibition of conventional and novel PKC isoforms together completely blocks prostratin-induced HIV reactivation (9, 57). However, inhibition of conventional PKC isoforms or PKC with inhibitor TER14687 which blocks PKC translocation, demonstrated that these drugs failed to prevent prostratin-induced HIV reactivation from latency (47, 57). These data are in sharp contrast to our results obtained with specific suppression constructs (Fig. 5), where we showed that PKC suppression abrogates both prostratin- and CD3/CD28-induced HIV LTR reactivation. Whether the previous report succeeded in achieving complete inhibition of conventional PKC isoforms is untested. Similarly, although TER14687has been shown to prevent PKC translocation to the membrane following OKT3 stimulation by interfering with PKC-Fyn interactions (47), it is not clear whether the PKC-Fyn interaction is required for prostratin-induced PKC translocation and activation. Therefore, direct suppression of PKC with shRNA seems to be a more reliable approach to address the requirement for PKC function in prostratin-induced HIV reactivation. In doing so, we have demonstrated the absolute requirement for PKC in both TCR- and prostratin-induced HIV reactivation.
The sequential nature of PKC and PKC activation is probably not limited to TCR or prostratin signaling pathways. Studies with another non-tumor-promoting phorbol ester, 12-deoxyphorbol 13-phenylacetate (DPP), suggest that DPP induces the same set of genes as prostratin and PMA (8), implying a similar PKC- and PKC-dependent signal transduction pathway. In contrast to phorbol esters, synthetic diacylglycerol (DAG) lactones can reactivate HIV with the same efficiency as DPP without causing T-cell proliferation and upregulation of tumor necrosis factor alpha (24). Of note, these DAG lactones have 20- to 100-fold lower PKC binding affinity than DPP, raising the possibility that DAG lactones can preferentially target PKC, thereby avoiding PKC-mediated T-cell activation.
Altogether our data provide novel insights on the role of PKC and PKC in HIV reactivation in T cells. The inability to efficiently reactivate HIV from latency represents a major barrier to HIV eradication. Identification of PKC and PKC as necessary for HIV reactivation following TCR and phorbol ester stimulation indicates that these cellular targets may provide a key for the development of new therapies. Moreover, since PKC activation has been implicated in tumorigenesis (reviewed in reference 39), selection of PKC as a therapeutic target may yield clinically relevant new agents.
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ABSTRACT
Latently human immunodeficiency virus (HIV)-infected memory CD4+ T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKC, PKCA148E, and PKC, PKCA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+ T cells and that both PKCA148E and PKCA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKC and PKC with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKC or PKC. Since suppression of PKC and PKC together has no greater inhibitory effect on HIV reactivation than inhibition of PKC alone, our data confirm that PKC and PKC act in sequence. The requirement for PKC and PKC for prostratin-induced HIV reactivation and the ability of selective PKC or PKC agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.
INTRODUCTION
The effectiveness of current antiretroviral agents is underscored by their inability to impact transcriptionally inactive viral reservoirs. Accordingly, human immunodeficiency virus (HIV) viral rebound is commonly observed when patients cease taking antiretroviral agents (15, 18, 19, 50, 58). Sequence analysis of rebound viral isolates indicates significant homology with proviral sequences from infected memory CD4+ T cells (61), suggesting that such cell populations act as a principal viral reservoir that is unaffected by antiretroviral therapy. Indeed, memory CD4+ T cells which contain HIV provirus have been defined to be transcriptionally inactive and to have an in vivo half-life of several years (13, 14). Consequently an estimated 60 or more years of fully supportive antiretroviral therapy may be required to fully purge such a reservoir, a time frame which is both impractical and clinically not useful. Three conceptually distinct approaches have been proposed as ways of impacting such viral reservoirs: toxins designed to eliminate virus-containing cells (5, 21, 38), antiviral intensification strategies to prevent repopulation of the latent pools (12, 26, 32, 37, 59), and compounds which stimulate HIV long terminal repeat (LTR) transcription, thereby rendering such cells susceptible to antiretroviral agents and/or the cytotoxic effects of viral replication (30, 49).
The HIV type 1 (HIV-1) LTR is regulated in large part by cellular transcription factors, including NF-B, AP-1, SP-1, and NF-AT (reviewed in reference 28). Consequently, stimuli which activate these transcription factors cause HIV LTR-driven viral replication (17, 20, 41). Antigen presentation or T-cell receptor (TCR) cross-linking can induce HIV reactivation from latently infected CD4+ T cells in vitro (15) through protein kinase C (PKC)-mediated activation of NF-B (40). Direct activation of PKC by phorbol esters reactivates HIV from latency in cell lines (27, 29, 45) and, more importantly, it induces HIV reactivation in peripheral blood mononuclear cells and primary quiescent T cells (1, 9, 10). PKC activation induces HIV from latency by targeting multiple regulatory elements of HIV LTR. In addition to NF-B, PKC induces HIV LTR transcription via activation of the AP-1 transcription factor; however, its action is largely dependent on a cooperation with NF-B (35, 46, 60). PKC also increases HIV-1 gene expression by phosphorylation of the virally encoded TAT transcription factor and cellular TAR-binding factors (25, 27).
Among the PKC isoforms in T cells, the novel PKC isoform PKC plays a crucial role in T-cell activation and proliferation (3, 6, 51). PKC activates NF-B (16, 36, 51) and AP-1 (4, 56) transcription factors, which bind to multiple sites in the enhancer region of the HIV-1 LTR. In addition PKC acts upstream of PKC to induce NF-B following CD3/CD28 ligation (54). Since HIV reactivation following CD3/CD28 ligation is NF-B dependent, together these data suggest that each of these two PKC isoforms may be required to mediate reactivation of latent HIV. Indeed, inhibition of both conventional and novel PKC isoforms blocks HIV reactivation following costimulation with CD3 and CD28 antibodies in the SCID-hu(Thy/Liv) model (9).
While TCR cross-linking can reactivate latent HIV in primary T cells in vitro (9, 11), clinical efforts to reduce the latent pool of HIV by treatment with the anti-CD3 antibody OKT-3 alone or in combination with interleukin-2 fails to meaningfully decrease the viral reservoir (32, 44, 55). The phorbol esters (8, 33) and 1,2-diacylglycerol analogs (24), including the non-tumor-promoting phorbol ester prostratin (23), have been suggested as agents to reactivate HIV and eradicate the pool of latently HIV-infected CD4+ T cells (22, 23). More recently, phorbol esters including prostratin have been directly evaluated for their ability to reactivate latent virus (30, 31). Indeed, prostratin causes significant reactivation of latent HIV from memory T-cell pools (7, 10, 30, 48). The mechanism by which prostratin causes viral reactivation is, therefore, of interest, and two independent studies now suggest that conventional and novel PKC isoforms are required (22, 57), although which specific PKC isoforms are involved and whether they are absolutely required is unknown. The present study was designed to address the specific requirement of different PKC isoforms following TCR- or phorbol ester-mediated HIV LTR reactivation and to determine whether such PKC isoforms are absolutely required.
MATERIALS AND METHODS
Plasmids. Generation and characterization of isoform-specific PKC suppression vectors were previously described (54). The HIV LTR and HIV LTR (B)-dependent firefly luciferase reporter expression vector (B-luc) has been previously described (17). The pRL-TK expression vector, which provides constitutive expression of Renilla luciferase, is commercially available (Promega, Madison, WI). GFP-PCI was a gift from D. McKean (Mayo Clinic, Rochester, MN). The Sp1-luciferase reporter was kindly provided by D. Billadeau (Mayo Clinic, Rochester, MN). The mammalian expression vector, pEF1/Myc-His, was purchased from Invitrogen (Carlsbad, CA). Constitutively active forms, PKCA25E and PKCA148E, were previously described (54).
Constitutively active IKK?S177E/S181E was generated by site-directed mutagenesis using mutagenic primers (sense, 5'-GAACTTTGCACAGAATTCGTGGGGA; antisense, 5'-TTCTGTGCAAAGTTCGCCCTGATCC; sense IKK? with BamHI site [underlined], GGGATCCATCCATGAGCTGGTCACCT; antisense IKK? with XbaI site [underlined], CGTCTAGATTATGAGGCCTGCTCCAG) and cloned into pGEM-T Easy. The cDNA coding for IKK?S177E/S181E was excised and subcloned into HA-pcDNA3 I at the BamHI XbaI site. The sequences of all generated constructs were verified by sequencing.
Cell culture and reagents. Jurkat T cells and latently HIV-1-infected J1.1 T cells were obtained from the American Type Culture Collection, Rockville, MD and maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine. Cells were grown to a density of 3 x 105 to 5 x 105/ml at the time of the different experiments. Ionomycin was purchased from Calbiochem (La Jolla, CA), and tumor necrosis factor alpha was purchased from R&D Systems (Minneapolis, MN). Leupeptin, aprotinin, and pepstatin A were obtained from Boehringer Mannheim (Indianapolis, IN). Antihemagglutinin (anti-HA) high-affinity antibodies were purchased from Boehringer Mannheim (Indianapolis, IN). OKT3 antibodies were obtained from Ortho Biotech (Raritan, NJ), and anti-CD28 was purchased from BD Biosciences (San Jose, CA). Anti-PKC (C-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC?1 and anti-PKC were purchased from BD Transduction Laboratories (San Diego, CA).
To isolate CD4+ T cells, peripheral blood mononuclear cells from healthy volunteer blood donors were obtained from buffy coats by using RossetteSep cocktail CD4+ antibodies in accordance with the manufacturer's protocol (StemCell Technologies, Vancouver, British Columbia, Canada). The remaining cell population was repeatedly found to be 98% CD4+ T cells as determined by flow cytometry. CD4+ T cells used in the various experiments were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mM L-glutamine, and antibiotics (penicillin [100 U/ml] and streptomycin [100 μg/ml]) at 0.5 x 106 cells/ml. For Jurkat T cells, ionomycin was used at 3.5 μg/ml. Jurkat and J1.1 T cells were cross-linked with 10 μg/ml of anti-CD3 and anti-CD28 antibodies or isotype control antibodies as previously described (53).
Cell extract preparation and immunoblotting. To obtain total cellular proteins, cells were washed with cold phosphate-buffered saline, resuspended in a modified whole-cell extract PD buffer (53) (40 mM Tris-HCl [pH 8], 0.3 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM NaF, 10 mM P-nitrophenyl phosphate 10 mM ?-glycerophosphate, 300 μM sodium orthovanadate, 1 mM dithiothreitol, 2 μM phenylmethylsulfonyl fluoride, aprotinin at 10 μg/ml, leupeptin at 1 μg/ml, pepstatin 1 μg/ml) and centrifuged at 12,000 x g for 15 min at 4°C. The resulting supernatant contained total cellular protein. The amount of cellular protein present in the clarified supernatant was calculated by using the Bio-Rad (Hercules, CA) protein assay. For Western immunoblot assays, equal amounts of whole-cell extract were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Immunoblotting was performed with specific antibodies and visualized by using the ECL Western blotting detection kit (Amersham, Buckinghamshire, England).
Gene transfection, p24 enzyme-linked immunosorbent assay (ELISA), and reporter assays. Jurkat T and J1.1 T cells were electroporated using a BTX Electro Square Porator T820 (BTX Corporation, San Diego, CA) at 325 V, 10 ms, and 350 V, 10 ms, correspondingly. Primary CD4+ T cells were electroporated by using Nucleofector (U-14 program; Amaxa Inc., Gaithersburg, MD) in accordance with a protocol for unstimulated human T cells. Where indicated, transfection efficiency was monitored by green fluorescent protein (GFP) expression.
Jurkat T cells were transfected with the indicated plasmids and grown for 18 to 24 h. Cells were stimulated for 12 h with ionomycin (3.5 μg/ml), prostratin (10 μM), or phorbol myristate 13-acetate (PMA; 10 ng/ml) or cross-linked with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies as previously described (54). Thereafter, cells were washed twice in cold phosphate-buffered saline and lysed with 100 μl lysis buffer (Dual-Luciferase reporter assay system; Promega, Madison, WI). Firefly and Renilla luciferase activities from 20 μl of extract were assayed using the Promega Dual-Luciferase reporter assay system reagents and a Berthold Lumat following the manufacturer's recommendations. B-Luciferase activity was normalized to Renilla expression. All transfection experiments were performed in duplicate.
Concentrations of p24 Gag in supernatants of J1.1 T cells were measured in duplicate by using a p24 ELISA kit (ZeptoMetrix Corporation, Buffalo, NY).
RESULTS
Constitutively active forms of both PKC and PKC induce HIV LTR activation via NF-B in Jurkat and human primary CD4+ T cells. Recently we demonstrated that PKC acts upstream of PKC to activate NF-B following CD3/CD28 stimulation in uninfected T cells (54). Since antigen presentation, TCR ligation, and phorbol esters all lead to NF-B activation and subsequent HIV LTR transcription, we first examined the role of both PKC and PKC in induction of HIV-1 LTR transcriptional activity in T lymphocytes mediated by these stimuli.
First, we studied whether CA forms of PKC, PKCA25E, and PKC, PKCA148E, can induce HIV-1 LTR transcriptional activity. Jurkat T cells were transfected with either PKCA25E or PKCA148E, together with luciferase reporter plasmids containing the wild-type HIV-1 LTR or the LTR lacking the two B sites, HIV-LTR (B). As shown in Fig. 1A, expression of either PKCA25E or PKCA148E induced HIV LTR transcriptional activity preferentially via NF-B, since deletion of B sites strongly impaired HIV LTR activation (Fig. 1A). As previously shown (54), ionomycin treatment is required for efficient NF-B activation by PKCA25E, since expression of PKCA25E resulted in only a moderate increase of HIV LTR transcriptional activity (Fig. 1A) which was further augmented by ionomycin treatment. Similar to PKCA148E, PKCA25E induced HIV LTR transcription via B sites (Fig. 1A). While PKCA25E and PKCA148E strongly activated NF-B-dependent transcription (25- to 200-fold) of HIV LTR, both only modestly induced (1.2- to 2-fold) transcriptional activity of the transcription factor, Sp1 (Fig. 1B).
To further confirm the relevance of these findings, we studied whether PKCA25E and PKCA148E can induce HIV-1 LTR transcriptional activity in human primary CD4+ T cells. Purified human CD4+ T cells were transfected with either PKCA25E or PKCA148E, together with the luciferase reporter plasmids mentioned above. Similar to our results with Jurkat T cells, expression of either PKCA25E or PKCA148E in human primary CD4+ T cells induced HIV-LTR transcriptional activity via B sites (Fig. 1C and D).
Taken together these results demonstrate that both PKC and PKC can induce HIV LTR transcription preferentially through B sites in Jurkat and primary CD4+ T cells.
Suppression of either PKC or PKC inhibits HIV LTR activation by CD3/CD28 stimulation in Jurkat T cells. To identify which of the two PKC isoforms mediates CD3/CD28-induced HIV LTR activation, we utilized isoform-specific PKC suppression vectors (54), which direct synthesis of short hairpinned (sh) RNA molecules specific for targeting of either human PKC or human PKC. Jurkat T cells were transfected with either pFRT-PKC or pFRT-PKC together with luciferase reporter plasmids as mentioned above. The levels of PKC and PKC expression in transfected cells were analyzed 48 h later by immunoblotting with anti-PKC and anti-PKC antibodies. As shown in Fig. 2C, transfection of Jurkat T cells with either pFRT-PKC or pFRT-PKC resulted in selective and significant reduction of PKC and PKC expression. Neither of the shRNA constructs affected the levels of PKC?1. Cross-linking of transfected Jurkat T cells with CD3 and CD28 antibodies induced HIV LTR-dependent luciferase activity preferentially via B sites of LTR (Fig. 2A and B). Suppression of either PKC or PKC significantly impaired CD3/CD28-induced HIV LTR-dependent luciferase activity. In contrast, suppression of either PKC or PKC had no effect on a basal or prostratin-induced Sp1-dependent luciferase activity (Fig. 2D), suggesting that both PKCs are dispensable for Sp1 transcription. Altogether, these results indicate that both PKC isoforms are required for CD3/CD28-induced HIV LTR-dependent transcription via B sites in Jurkat T cells.
Constitutively active forms of both PKC and PKC reactivate HIV in latently infected J1.1 T cells. To further define the role of both PKCs in HIV reactivation, we studied HIV LTR-dependent transcription and HIV-1 production in the latently HIV-infected CD3+ J 1.1 T-cell line (42). J1.1 T cells were transfected with HA-PKCA25E or HA-PKCA148E or, as a positive control, with the constitutively active form of IKK?, HA-IKK?S177E,S181E, together with either HIV LTR (Fig. 3A) or HIV LTR (B)- luciferase reporters (Fig. 3B) and the tkRenilla reporter plasmid. Thirty-six hours later, supernatants were collected to measure p24 Gag levels and cells were harvested to assay luciferase activities. Expression of either PKCA148E or PKCA25E induced HIV LTR transcriptional activity in J1.1 T cells through both NF-B-dependent and non-NF-B-dependent pathways (Fig. 3A and B).
To test whether PKCA25E, HA-PKCA148E, or HA-IKK?S177E,S181E can reactivate HIV from latency, we measured p24 Gag levels in supernatants of transfected J1.1 cells. As shown in Fig. 3D, the expression of the constitutively active forms of PKC, PKC, or IKK? induce strong p24 Gag production in J1.1 T cells, which correlates with HIV LTR activation (Fig. 3A and B).
Taken together, these results demonstrate that activation of either PKC or PKC is sufficient for the activation of HIV LTR transcription and for subsequent HIV reactivation in latently infected T cells.
Suppression of either PKC or PKC inhibits HIV reactivation by CD3/CD28 stimulation in J1.1 T cells. To confirm which of the two PKC isoforms is required for the CD3/CD28-induced HIV reactivation, J1.1 T cells were transfected with control vector, pFRT-PKC alone, pFRT-PKC alone, or pFRT-PKC together with pFRT-PKC. HIV LTR or HIV LTR (B) luciferase plasmids and Renilla were cotransfected. Forty-eight hours later cells were cross-linked with anti-CD3 and anti-CD28 antibodies and then cells and supernatants were harvested to assay luciferase activity (Fig. 4A), PKC expression (Fig. 4B), and p24 Gag concentrations (Fig. 4C). As shown in Fig. 4A, stimulation of J1.1 T cells with anti-CD3 anti-CD28 antibodies induced a threefold increase in HIV LTR transcription. Deletion of two B sites in the LTR resulted in a 50% decrease of CD3/CD28-induced HIV LTR transcription. Suppression of PKC inhibited B-specific CD3/CD28-induced HIV LTR transcription and resulted in a twofold decrease of p24 levels in CD3/CD28-treated J1.1 T cells. Conversely, PKC suppression inhibited both B-dependent and to a lesser degree B-independent CD3/CD28-induced HIV LTR transcription, potentially altering AP-1 (4, 56) and NF-AT (2, 43, 56) activation. Simultaneous suppression of both PKCs did not have a greater effect on CD3/CD28-induced HIV LTR transcription and p24 production than suppression of PKC or PKC alone (Fig. 4A and C), confirming that PKC and PKC act in sequence following CD3/CD28 stimulation (54).
Both PKC and PKC are required for HIV reactivation by phorbol esters in J 1.1 T cells. Having shown that both PKC isoforms are required for CD3/CD28-mediated HIV reactivation, we addressed the role of both PKCs in phorbol ester-mediated HIV reactivation from latency. Isoform-specific PKC suppression vectors were expressed in J1.1. T cells that were then stimulated with two different phorbol esters, PMA or prostratin. Stimulation of J1.1 T cells with PMA or prostratin induced 12-fold increases in HIV LTR-dependent luciferase activity that was reduced by deletion of the B sites of HIV LTR [Fig. 5A, HIV-LTR (B)] or suppression of PKC. PKC suppression resulted in a twofold decrease of p24 Gag production induced by PMA or prostratin (Fig. 5B). It also blocked 50% of HIV LTR-dependent transcription induced by either PMA or prostratin. As with CD3/CD28 stimulation, suppression of PKC had no effect on PMA- or prostratin-induced HIV LTR (B) transcription (Fig. 5A). Suppression of PKC blocked almost 50% of prostratin PMA-mediated HIV LTR activation via B sites and resulted in a twofold decrease of prostratin- or PMA-induced HIV LTR (B) transcription. PKC suppression also significantly decreased p24 production induced by prostratin (P < 0.05) (Fig. 5B). Similar to CD3/CD28 stimulation, suppression of both PKC and PKC together was no more effective in blocking PMA- or prostratin-induced HIV LTR transcription and p24 production than suppression of PKC alone.
DISCUSSION
In this study we have demonstrated a specific requirement for both PKC and PKC in HIV reactivation following TCR ligation or stimulation with the non-tumor-promoting ester prostratin in T lymphocytes. We conclude this based on observations that selective suppression of PKC, PKC, or both PKC isoforms inhibits HIV LTR transcription and HIV reactivation following CD3/CD28 cross-linking or prostratin treatment in Jurkat and J1.1 T cells. Several interesting features of HIV reactivation by these stimuli are underscored by our results. First, HIV reactivation by either CD3/CD28 engagement or prostratin stimulation requires both PKC and PKC, demonstrating that both PKC isoforms are necessary. Second, suppression of PKC alone or PKC alone has the same inhibitory effect on HIV LTR transcription as suppression of both PKC isoforms together, suggesting that PKC and PKC act in sequence to activate HIV LTR transcription. These findings are consistent with our previous observation that PKC acts upstream of PKC to activate NF-B following TCR ligation (54). Exactly how PKC upregulates PKC is undefined, yet potential direct phosphorylation of PKC by PKC at the plasma membrane may be required for either full PKC activation or to prolong PKC signaling following either CD3/CD28 engagement or prostratin stimulation. The murine PKC undergoes inducible phosphorylation at the C terminus on two adjacent (Thr-692 and Thr-703) residues following TCR or phorbol ester stimulation, which is required for NF-B activation (52). We observed that the C-terminal phosphorylation of PKC occurs in a PKC-dependent manner (data not shown), suggesting that PKC can directly phosphorylate the two adjacent serine residues, S685 and S703, in human PKC (unpublished data). Therefore, PKC-mediated phosphorylation prolongs PKC activation which, as we have previously demonstrated (53), is required for efficient NF-B transcription. Consequently, persistent PKC activation may be necessary for efficient HIV LTR transcription and subsequent HIV reactivation from latency. This contention is supported by the fact that the constitutively active form of PKC provides prolonged signaling and, therefore, it induces HIV LTR transcription in primary CD4+ T cells (Fig. 1C) and efficiently reactivates HIV from latency (Fig. 3D). Finally, we demonstrated that suppression of either PKC or PKC inhibits prostratin-mediated or CD3/CD28-mediated HIV LTR transcription via B sites. However, suppression of PKC also has a significant effect on prostratin-induced HIV LTR (B) transcription and a smaller effect on CD3/CD28-induced HIV LTR (B) transcription, potentially due to inhibition of other transcription factors, such as AP-1. Previously it was shown that prostratin induces both NF-B- and AP-1-dependent transcription (57). Our data are in agreement, since constitutively active forms of both PKC and PKC can induce transcription of HIV LTR lacking B sites (Fig. 3B) and potently induce AP-1-dependent transcription in J1.1 T cells (data not shown). However, it is noteworthy that the magnitude of AP-1 activation by PKC is much greater than that by PKC. Therefore, selective inhibition of CD3/CD28- and prostratin-induced HIV LTR (B) transcription by suppression of PKC but not PKC indicates that PKC and PKC pathways are divergent. This is in agreement with the previous observation that PKC activates NF-B, but not AP-1, in a PKC-dependent manner (54). It is possible that the divergence in the PKC and PKC signaling pathway occurs on the level of Ste20-related proline- and alanine-rich kinase, which recently has been shown to mediate PKC, but not PKC, signaling to AP-1 (34). Therefore, while both PKC isoforms induce HIV LTR transcription via B sites, PKC also contributes in HIV LTR transcription via AP-1 sites. The conclusion that both PKC and PKC are required for HIV reactivation is in agreement with recently published data demonstrating that CD3/CD28 costimulation and prostratin treatment reactivate HIV from latency in the SCID-HU Thy/Liv model (9) in both a PKC- and NF-B-dependent manner. Similarly, a role for PKC in mediating prostratin-induced HIV reactivation is further supported by observations that prostratin induces translocation of several conventional, novel, and atypical PKC isoforms to the cell membrane, suggesting that all these PKC isoforms can contribute to HIV reactivation (57). That suggestion is supported by observations that simultaneous inhibition of conventional and novel PKC isoforms together completely blocks prostratin-induced HIV reactivation (9, 57). However, inhibition of conventional PKC isoforms or PKC with inhibitor TER14687 which blocks PKC translocation, demonstrated that these drugs failed to prevent prostratin-induced HIV reactivation from latency (47, 57). These data are in sharp contrast to our results obtained with specific suppression constructs (Fig. 5), where we showed that PKC suppression abrogates both prostratin- and CD3/CD28-induced HIV LTR reactivation. Whether the previous report succeeded in achieving complete inhibition of conventional PKC isoforms is untested. Similarly, although TER14687has been shown to prevent PKC translocation to the membrane following OKT3 stimulation by interfering with PKC-Fyn interactions (47), it is not clear whether the PKC-Fyn interaction is required for prostratin-induced PKC translocation and activation. Therefore, direct suppression of PKC with shRNA seems to be a more reliable approach to address the requirement for PKC function in prostratin-induced HIV reactivation. In doing so, we have demonstrated the absolute requirement for PKC in both TCR- and prostratin-induced HIV reactivation.
The sequential nature of PKC and PKC activation is probably not limited to TCR or prostratin signaling pathways. Studies with another non-tumor-promoting phorbol ester, 12-deoxyphorbol 13-phenylacetate (DPP), suggest that DPP induces the same set of genes as prostratin and PMA (8), implying a similar PKC- and PKC-dependent signal transduction pathway. In contrast to phorbol esters, synthetic diacylglycerol (DAG) lactones can reactivate HIV with the same efficiency as DPP without causing T-cell proliferation and upregulation of tumor necrosis factor alpha (24). Of note, these DAG lactones have 20- to 100-fold lower PKC binding affinity than DPP, raising the possibility that DAG lactones can preferentially target PKC, thereby avoiding PKC-mediated T-cell activation.
Altogether our data provide novel insights on the role of PKC and PKC in HIV reactivation in T cells. The inability to efficiently reactivate HIV from latency represents a major barrier to HIV eradication. Identification of PKC and PKC as necessary for HIV reactivation following TCR and phorbol ester stimulation indicates that these cellular targets may provide a key for the development of new therapies. Moreover, since PKC activation has been implicated in tumorigenesis (reviewed in reference 39), selection of PKC as a therapeutic target may yield clinically relevant new agents.
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