Caspase inhibition activates HIV in latently infected cells. Role of tumor necrosis factor receptor 1 and CD95

Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.     

Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type I TNF receptor [corrected]

Agonist antibodies (Ab) to the two TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), have been shown to signal many of the distinct functions induced by TNF-alpha. We have found that anti-TNF-R1, but not anti-TNF-R2, Ab trigger antiviral activity in human hepatoma Hep-G2 cells and enhance the antiviral activity of IFN-gamma in human lung fibroblast A549 cells. Likewise, anti-human-TNF-R1 Ab had antiviral enhancing activity on murine L929 cells engineered to express human TNF-R1. However, L929 cells that express human TNF-R1 lacking most of the intracellular domain fail to respond to anti-human-TNF-R1 Ab. This demonstrates that the intracellular domain of TNF-R1 is necessary to generate antiviral activity. TNF-R1 but not TNF-R2 also signals killing of virus-infected cells by TNF-alpha. Thus, all the known antiviral activities of TNF-alpha are mediated through TNF-R1.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amniotic fluid levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 before and after the onset of labor in normal pregnancies.

Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells and promotes the regulation of trophoblast growth and invasion. Tumor necrosis factor receptor 1 (TNF-R1) is a receptor for TNF-alpha, and soluble TNF-R1 (sTNF-R1) is present in amniotic fluid after receptor shedding. We evaluated whether amniotic fluid TNF-alpha and sTNF-R1 levels during labor differed from those before the onset of labor in normal pregnancies. This study enrolled 34 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these subjects, 17 went into labor and had subsequent term deliveries (the labor group), and the other 17 underwent cesarean section without labor (the nonlabor group). The average gestational age at entry was 38-39 weeks of gestation. Maternal ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the TNF-alpha and sTNF-R1 levels were determined by the ELISA method. Each of these levels was compared between the two groups. There was a significant increase in amniotic fluid TNF-alpha levels in the labor group. However, amniotic fluid sTNF-R1 levels did not differ significantly between the two groups. Amniotic fluid TNF-alpha may promote the onset of labor at term and/or term labor contributing to subsequent delivery may induce the production and secretion of TNF-alpha into the amniotic cavity. There was no pregnancy-associated increase in receptor shedding or cell apoptosis at the onset of labor.     

TRUSS, a novel tumor necrosis factor receptor 1 scaffolding protein that mediates activation of the transcription factor NF-kappaB

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.     

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.     

Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.     

Epstein-Barr virus BHRF1 protein protects intestine 407 epithelial cells from apoptosis induced by tumor necrosis factor alpha and anti-Fas antibody.

Tumor necrosis factor (TNF) and cytotoxic T lymphocytes, which utilize Fas to induce apoptosis in target cells, are known to play a critical role in the host defense against viral infection. In this study, the Epstein-Barr virus BHRF1 protein was stably expressed in intestine 407 cells which were susceptible to cell death mediated through both the TNF receptor and Fas. WST-1 conversion assays and acridine orange staining showed that vector-transfected control cells were killed by TNF-alpha or anti-Fas antibody in a dose-dependent manner, whereas BHRF1-expressing cells were resistant to apoptosis induced by these mediators. DNA fragmentation, a characteristic of apoptosis induced by TNF-alpha and the anti-Fas antibody, was suppressed in BHRF1-expressing cells. These results indicate that the BHRF1 protein protects cells from apoptosis mediated by the TNF receptor and Fas. The role of BHRF1 as an inhibitor of cytokine-induced apoptosis during the Epstein-Barr virus lytic cycle in vivo is discussed.     

Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death

Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.     

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.     

Expression and regulation of tumor necrosis factor (TNF) and TNF-receptor family members in the macaque corpus luteum during the menstrual cycle

Members of the tumor necrosis factor (TNF)-receptor (R) family may be involved in the tissue remodeling that occurs in the primate corpus luteum (CL) during development and regression. As a first step towards addressing this issue, studies assessed TNF ligand-R expression and regulation in CL collected from monkeys during the early (ECL, Days 3-5), mid (MCL, Days 7-8), mid-late (MLCL, Days 10-11), late (LCL, Days 14-16), and very late (VLCL, menses) luteal phase of the menstrual cycle. CL were also collected after gonadotropin and/or steroid ablation and replacement (with hLH and the progestin R5020) for 3 days at mid-late luteal phase. TNF-alpha, -beta, FAS ligand (FASL), and TNF-R1 mRNA levels were two- to sixfold greater (P < 0.05) at the MLCL or LCL phase as compared to earlier (ECL, MCL). In contrast, TNF-R2 and FAS mRNA levels did not change during the luteal phase. Immunohistochemical staining for TNF-beta, TNF-R1, TNF-R2, FAS, and FASL was observed in luteal cells, whereas only TNF-beta staining was observed in endothelial cells. Several TNF-R components were influenced by LH and/or steroid ablation; notably, steroid ablation reduced (P < 0.05) luteal TNF-alpha, but not TNF-beta, mRNA levels, which was prevented by progestin treatment. In contrast, steroid ablation increased (P < 0.05) luteal cell immunostaining for FAS and FASL, which was reduced by progestin treatment. Thus, several members of the TNF R-ligand family are expressed in the primate CL in an LH- and/or progestin-dependent manner. Peak expression in the late luteal phase may signify a role for the TNF-R system in death receptor-mediated apoptosis during luteolysis.     

Mass spectrometric analysis of tumor necrosis factor receptor-associated factor 1 ubiquitination mediated by cellular inhibitor of apoptosis 2

Signaling complexes formed on tumor necrosis factor receptor 2 (TNF-R2) contain adaptor proteins TNF-R-associated factors (TRAFs) 1 and 2, and cellular inhibitors of apoptosis (cIAPs) 1 and 2 which function as regulators of programmed cell death. TRAF2, cIAP1 and cIAP2 all have RING finger domains known to possess E3 ubiquitin ligase activity, implying that ubiquitination may play an important role in the TNF signaling pathway. In this report, we have shown that cIAP2 specifically mediated ubiquitination and proteasome-dependent degradation of TRAF1. To identify the sites for cIAP2-mediated ubiquitination of TRAF1, we used high pressure liquid chromatography coupled with tandem mass spectrometry. Lys185 and Lys193 of TRAF1 were found to be modified with ubiquitin chains. Mutation of Lys185 and Lys193 to Arg almost completely blocked cIAP2-mediated ubiquitination of TRAF1, indicating that they are the major, if not the only, sites of TRAF1 ubiquitination. Our data suggest that cIAP2 may regulate the turnover of TRAF1 by adding polyubiquitin chains on Lys185 or Lys193 following its recruitment to TNF-R signaling complexes.     

EBV encoded miR-BHRF1-1 potentiates viral lytic replication by downregulating host p53 in nasopharyngeal carcinoma

miRNAs (microRNAs) are a class of non-coding small RNAs. The Epstein-Barr-virus (EBV) encoded miR-BHRF1-1 is barely expressed in most nasopharyngeal carcinoma (NPC) cells with EBV latent infection. Here, we used a strategy of overexpression and inhibition of miR-BHRF1-1 and showed that miR-BHRF1-1 is involved in TPA-induced accumulation of EBV lytic proteins and viral copies in late lytic cycle. The data further suggested that the miR-BHRF1-1-potentiated induction of EBV lytic replication was accompanied by inhibiting p53 expression. Our results demonstrated that the EBV original pathogen miR-BHRF1-1 is involved in the control of EBV late lytic replication by directly targeting the host p53 gene.