Kaposi's Sarcoma-Associated Herpesvirus LANA Modulates the Stability of the E3 Ubiquitin Ligase RLIM.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. In a previous study, we have identified LANA-interacting proteins using a protein array screen. Here, we explore the effect of LANA on the stability and activity of RLIM (RING finger LIM-domain-interacting protein, encoded by the RNF12 gene), a novel LANA-interacting protein identified in that protein screen. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Expression of LANA leads to downregulation of RLIM protein levels. This LANA-mediated RLIM degradation is blocked in the presence of the proteasome inhibitor, MG132. Therefore, the interaction between LANA and RLIM could be detected in coimmunoprecipitation assay only in the presence of MG132 to prevent RLIM degradation. A RING finger mutant RLIM is resistant to LANA-mediated degradation, suggesting that LANA promotes RLIM autoubiquitination. Interestingly, we found that LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. We also show that transcription regulation by RLIM substrates is modulated by LANA. RLIM substrates are assembled into multiprotein transcription regulator complexes that regulate the expression of many cellular genes. Therefore, our study identified another way KSHV can modulate cellular gene expression.IMPORTANCE E3 ubiquitin ligases mark their substrates for degradation and therefore control the cellular abundance of their substrates. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Here, we show that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA protein enhances the ubiquitin ligase activity of RLIM, leading to enhanced RLIM autoubiquitination and degradation. Interestingly, LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. In agreement with protein stability of RLIM substrates, we found that LANA modulates transcription by LHX3-LDB1 complex and suggest additional ways LANA can modulate cellular gene expression. Our study adds another way a viral protein can regulate cellular protein stability, by enhancing the autoubiquitination and degradation of an E3 ubiquitin ligase.

Pharmacologic Activation of Lytic Epstein-Barr Virus Gene Expression without Virion Production.

Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-positive Burkitt lymphoma cell line and confirmed the upregulation of all three branches of the UPR and activation of EBV lytic gene expression. While immediate early, early, and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. The use of clofoctol in combination with a protein kinase R-like endoplasmic reticulum kinase inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic protein upregulation were not limited to lymphoid tumor cell lines but also occurred in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but that does not lead to the production of infectious virions may have particular value for lytic induction strategies in the clinical setting.IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent; i.e., most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic, clofoctol, that is an activator of EBV lytic RNA and protein expression but that does not lead to virion production.

Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib.IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

UNLABELLED: The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE: KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

Phosphorylation of the chromatin binding domain of KSHV LANA.

The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

SUMO binding by the Epstein-Barr virus protein kinase BGLF4 is crucial for BGLF4 function.

An Epstein-Barr virus (EBV) protein microarray was used to screen for proteins binding noncovalently to the small ubiquitin-like modifier SUMO2. Among the 11 SUMO binding proteins identified was the conserved protein kinase BGLF4. The mutation of potential SUMO interaction motifs (SIMs) in BGLF4 identified N- and C-terminal SIMs. The mutation of both SIMs changed the intracellular localization of BGLF4 from nuclear to cytoplasmic, while BGLF4 mutated in the N-terminal SIM remained predominantly nuclear. The mutation of the C-terminal SIM yielded an intermediate phenotype with nuclear and cytoplasmic staining. The transfer of BGLF4 amino acids 342 to 359 to a nuclear green fluorescent protein (GFP)-tagged reporter protein led to the relocalization of the reporter to the cytoplasm. Thus, the C-terminal SIM lies adjacent to a nuclear export signal, and coordinated SUMO binding by the N- and C-terminal SIMs blocks export and allows the nuclear accumulation of BGLF4. The mutation of either SIM prevented SUMO binding in vitro. The ability of BGLF4 to abolish the SUMOylation of the EBV lytic cycle transactivator ZTA was dependent on both BGLF4 SUMO binding and BGLF4 kinase activity. The global profile of SUMOylated cell proteins was also suppressed by BGLF4 but not by the SIM or kinase-dead BGLF4 mutant. The effective BGLF4-mediated dispersion of promyelocytic leukemia (PML) bodies was dependent on SUMO binding. The SUMO binding function of BGLF4 was also required to induce the cellular DNA damage response and to enhance the production of extracellular virus during EBV lytic replication. Thus, SUMO binding by BGLF4 modulates BGLF4 function and affects the efficiency of lytic EBV replication.

A protein array screen for Kaposi's sarcoma-associated herpesvirus LANA interactors links LANA to TIP60, PP2A activity, and telomere shortening.

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. To identify novel LANA protein-cell protein interactions that could contribute to these activities, we performed a proteomic screen in which purified, adenovirus-expressed Flag-LANA protein was incubated with an array displaying 4,192 nonredundant human proteins. Sixty-one interacting cell proteins were consistently detected. LANA interactions with high-mobility group AT-hook 1 (HMGA1), HMGB1, telomeric repeat binding factor 1 (TRF1), xeroderma pigmentosum complementation group A (XPA), pygopus homolog 2 (PYGO2), protein phosphatase 2A (PP2A)B subunit, Tat-interactive protein 60 (TIP60), replication protein A1 (RPA1), and RPA2 proteins were confirmed in coimmunoprecipitation assays. LANA-associated TIP60 retained acetyltransferase activity and, unlike human papillomavirus E6 and HIV-1 TAT proteins, LANA did not reduce TIP60 stability. The LANA-bound PP2A B subunit was associated with the PP2A A subunit but not the catalytic C subunit, suggesting a disruption of PP2A phosphatase activity. This is reminiscent of the role of simian virus 40 (SV40) small t antigen. Chromatin immunoprecipitation (ChIP) assays showed binding of RPA1 and RPA2 to the KSHV terminal repeats. Interestingly, LANA expression ablated RPA1 and RPA2 binding to the cell telomeric repeats. In U2OS cells that rely on the alternative mechanism for telomere maintenance, LANA expression had minimal effect on telomere length. However, LANA expression in telomerase immortalized endothelial cells resulted in telomere shortening. In KSHV-infected cells, telomere shortening may be one more mechanism by which LANA contributes to the development of malignancy.

Bortezomib induction of C/EBPß mediates Epstein-Barr virus lytic activation in Burkitt lymphoma.

Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinß (C/EBPß) in a variety of tumor cell lines. C/EBPß activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPß inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPß knock-down. Therefore, C/EBPß mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.

Comparison of humoral immune responses to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus using a viral proteome microarray.

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). METHODS: To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi's sarcoma or lymphoma. RESULTS: In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1-positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. CONCLUSIONS: The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.

A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency.

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.

Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4.

A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an Epstein-Barr virus (EBV) protein array to evaluate the targets of the EBV protein kinase BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrecognized substrates for BGLF4, illustrating the broad role played by this protein kinase. Approximately half of the BGLF4 targets were also in vitro substrates for the cellular kinase CDK1/cyclin B. Unexpectedly, EBNA1 was identified as a substrate and binding partner of BGLF4. EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency. BGLF4 did not prevent EBNA1 binding to sites in the EBV latency origin of replication, oriP. Rather, we found that BGLF4 was recruited by EBNA1 to oriP in cells transfected with an oriP vector and BGLF4 and in lytically induced EBV-positive Akata cells. In cells transfected with an oriP vector, the presence of BGLF4 led to more rapid loss of the episomal DNA, and this was dependent on BGLF4 kinase activity. Similarly, expression of doxycycline-inducible BGLF4 in Akata cells led to a reduction in episomal EBV genomes. We propose that BGLF4 contributes to effective EBV lytic cycle progression, not only through phosphorylation of EBV lytic DNA replication and virion proteins, but also by interfering with the EBNA1 replication function.

p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease.

We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington's disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

Notch and Wnt signaling: mimicry and manipulation by gamma herpesviruses.

A small number of fundamental cell signaling pathways are key to the regulation of proliferation and differentiation responses during normal development. Two of these pathways, the Notch and Wnt pathways, have proven to be attractive targets for virus interaction and manipulation. In general, viral gene expression and replication are intimately linked to the differentiation state of the infected cell and, in the case of the gamma herpesviruses, establishment of a lifelong persistent infection in the host is also dependent on the proliferative expansion of an infected B cell population. This review examines the ways in which the gamma herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) have exploited the Notch and Wnt pathways to advance their own life cycles. The virus-pathway interactions are compared with the mechanisms and outcome of cellular Notch and Wnt signaling.

MAGE-A1 interacts with adaptor SKIP and the deacetylase HDAC1 to repress transcription.

MAGE-A1 belongs to a family of 12 genes that are active in various types of tumors and silent in normal tissues except in male germ-line cells. The MAGE-encoded antigens recognized by T cells are highly tumor-specific targets for T cell-oriented cancer immunotherapy. The function of MAGE-A1 is currently unknown. To analyze it, we attempted to identify protein partners of MAGE-A1. Using yeast two-hybrid screening, we detected an interaction between MAGE-A1 and Ski Interacting Protein (SKIP). SKIP is a transcriptional regulator that connects DNA-binding proteins to proteins that either activate or repress transcription. We show that MAGE-A1 inhibits the activity of a SKIP-interacting transactivator, namely the intracellular part of Notch1. Deletion analysis indicated that this inhibition requires the binding of MAGE-A1 to SKIP. Moreover, MAGE-A1 was found to actively repress transcription by binding and recruiting histone deacetylase 1 (HDAC1). Our results indicate that by binding to SKIP and by recruiting HDACs, MAGE-A1 can act as a potent transcriptional repressor. MAGE-A1 could therefore participate in the setting of specific gene expression patterns for tumor cell growth or spermatogenesis.

Manipulation of glycogen-synthase kinase-3 activity in KSHV-associated cancers.

The Kaposi's sarcoma-associated herpesvirus, KSHV, is associated with cancers that have increased incidence in patients who are also HIV positive or who have undergone organ transplantation. It has recently been observed that beta-catenin is overexpressed in two KSHV-associated cancers, Kaposi's sarcoma and primary effusion lymphoma. Investigation of the underlying defect in beta-catenin regulation revealed that the KSHV-encoded LANA protein stabilizes beta-catenin by binding to the negative regulator GSK-3, causing a cell-cycle-dependent nuclear accumulation of GSK-3. Thus, redistribution of GSK-3 has been identified as yet another mechanism through which beta-catenin can be dysregulated and contribute to human cancer.

Targeted therapy for Epstein-Barr virus-associated gastric carcinoma using low-dose gemcitabine-induced lytic activation.

The constant presence of the viral genome in Epstein-Barr virus (EBV)-associated gastric cancers (EBVaGCs) suggests the applicability of novel EBV-targeted therapies. The antiviral nucleoside drug, ganciclovir (GCV), is effective only in the context of the viral lytic cycle in the presence of EBV-encoded thymidine kinase (TK)/protein kinase (PK) expression. In this study, screening of the Johns Hopkins Drug Library identified gemcitabine as a candidate for combination treatment with GCV. Pharmacological induction of EBV-TK or PK in EBVaGC-originated tumor cells were used to study combination treatment with GCV in vitro and in vivo. Gemcitabine was found to be a lytic inducer via activation of the ataxia telangiectasia-mutated (ATM)/p53 genotoxic stress pathway in EBVaGC. Using an EBVaGC mouse model and a [125I] fialuridine (FIAU)-based lytic activation imaging system, we evaluated gemcitabine-induced lytic activation in an in vivo system and confirmed the efficacy of gemcitabine-GCV combination treatment. This viral enzyme-targeted anti-tumor strategy may provide a new therapeutic approach for EBVaGCs.

Hypermethylation of the interferon regulatory factor 5 promoter in Epstein-Barr virus-associated gastric carcinoma.

Interferon regulatory factor-5 (IRF-5), a member of the mammalian IRF transcription factor family, is regulated by p53, type I interferon and virus infection. IRF-5 participates in virus-induced TLR-mediated innate immune responses and may play a role as a tumor suppressor. It was suppressed in various EBV-infected transformed cells, thus it is valuable to identify the suppression mechanism. We focused on a promoter CpG islands methylation, a kind of epigenetic regulation in EBV-associated Burkitt's lymphomas (BLs) and gastric carcinomas. IRF-5 is not detected in most of EBV-infected BL cell lines due to hypermethylation of IRF-5 distal promoter (promoter-A), which was restored by a demethylating agent, 5-aza-2'-deoxycytidine. Hypomethylation of CpG islands in promoter-A was observed only in EBV type III latent infected BL cell lines (LCL and Mutu III). Similarly, during EBV infection to Akata-4E3 cells, IRF-5 was observed at early time periods (2 days to 8 weeks), concomitant unmethylation of promoter-A, but suppressed in later infection periods as observed in latency I BL cell lines. Moreover, hypermethylation in IRF-5 promoter-A region was also observed in EBV-associated gastric carcinoma (EBVaGC) cell lines or primary gastric carcinoma tissues, which show type I latent infection. In summary, IRF-5 is suppressed by hypermethylation of its promoter-A in most of EBV-infected transformed cells, especially BLs and EBVaGC. EBV-induced carcinogenesis takes an advantage of proliferative effects of TLR signaling, while limiting IRF-5 mediated negative effects in the establishment of EBVaGCs.