Epstein-Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), but the function of these miRNAs has been obscure. Nasopharyngeal carcinoma (NPC) is associated with EBV infection, and the EBV-encoded LMP1 is believed to be a key factor in NPC development. However, detection of LMP1 protein in NPC is variable. Here, we report that EBV-encoded BART miRNAs target the 3 UTR of the LMP1 gene and negatively regulate LMP1 protein expression. These miRNAs also modulate LMP1-induced NF-B signaling and alleviate the cisplatin sensitivity of LMP1-expressing NPC cells. Consistent with a previous study on the NPC C666-1 cell line and C15 xenograft, we found abundant expression of BART miRNAs in NPC tissues. Furthermore, DNA sequencing revealed that the 3 UTR of LMP1 is highly conserved in NPC-derived EBV isolates. The data provide insight into the discrepancy between LMP1 transcript and protein detection in NPC and highlight the role of the EBV miRNAs in regulating LMP1 downstream signaling to promote cancer development. Epstein-Barr virus ͉ nasopharyngeal carcinoma

Summary Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α-, β-, and γ-herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase critical for virus replication and spread. The extent of substrate conservation and the key common cell signalling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective anti-viral strategies.

No abstract

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.Herpesviruses encode two families of serine/threonine protein kinases, one of which, the BGLF4 (Epstein-Barr virus [EBV])/UL97 (human cytomegalovirus)/UL13 (herpes simplex virus)/ORF36 (Kaposi's sarcoma-associated herpesvirus)/ORF47 (varicella-zoster virus) family, is the sole protein kinase encoded by beta and gamma herpesviruses. The protein kinases phosphorylate both viral and host proteins (16,21,42) and are necessary for efficient virus lytic replication. Consequently, these kinases have been of interest as potential targets for antiviral drug development (37), and the compound 1263W94 (maribavir), which inhibits the cytomegalovirus UL97 protein (3), has been used in phase I clinical trials (27,31,47).EBV infection is prevalent worldwide, and primary infection in adolescence or early adulthood is associated in 30 to 40% of cases with infectious mononucleosis. EBV efficiently infects B cells in the lymphoid tissues of the Waldeyer ring (43). EBV infection of B cells is biased toward establishment of latency with limited viral-gene expression (49). During latent infection, EBV genomes are maintained as extrachromosomal episomes. Replication of episomal genomes utilizes the latency origin of replication, oriP. The only EBV-encoded protein required is the origin binding protein EBNA1. All other essential replication factors are provided by the cell. Expression of the EBV replicative cycle and production of progeny virus take pla...

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) protein is functionally pleiotropic. LANA contributes to KSHV-associated pathogenesis, in part, by increasing entry of cells into S phase through a process that is driven by LANA interaction with the serine-threonine kinase glycogen synthase kinase 3 (GSK-3) and stabilization of ␤-catenin. Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered in lesions of Kaposi's sarcoma using differential display (12) and was subsequently recognized to also be associated with primary effusion lymphoma and multicentric Castleman's disease (10, 18, 52, 59). The KSHV latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in latently infected cells and in KSHV-associated malignancies (47). LANA is encoded by KSHV ORF73 and has unique N-terminal and C-terminal domains separated by three sets of repeated sequences that represent approximately half of the total protein sequence. These repeats function similarly to the central repeat region of the Epstein-Barr virus EBNA-1 protein by inhibiting antigen presentation and allowing tumor cells expressing LANA to escape immune surveillance (2,16,70).LANA is a multifunctional protein that is essential for the replication (5, 20, 29, 34) and maintenance (4) of KSHV episomal DNA during latent infection. LANA binds to the terminal repeats of the KSHV genome (14, 25); links the genomes to the cell chromosomes through interactions with chromatinassociated proteins such as the core histones H2A and H2B, DEK, HP1, Brd4, and MeCP2 (6,28,37,69); and recruits cellular DNA replication machinery to the terminal repeats (45,60,62,64). Expression of LANA in a transgenic mouse generated activated, hyperproliferative B cells, and mice developed lymphoma with a long latency (19). LANA has multiple properties that could contribute to tumorigenesis. These include inhibition of p53-mediated apoptosis (9, 21), stimulation of S-phase entry through stabilization of ␤-catenin and upregulation of cyclin D1 (24) and through induction of Rb/ E2F-regulated genes (1, 49), and overcoming G 1 cell cycle arrest mediated by p16 (1) and BRD4 and BRD2 (46). LANA is also responsible for promoting KSHV latency gene expression at the expense of lytic induction and for some of the reprogramming of cell gene expression that occurs in KSHVinfected cells (1,57,65,66). Targeting of LANA to DNA either through the use of Gal4-LANA fusion proteins (38, 53) through binding of LANA to the KSHV terminal repeats (25) or through LANA recruitment to cell (57) or viral promoters (39, 42) leads to transcriptional repression. LANA binds to histone deacetylase-associated corepressors (38) and is also capable of recruiting de novo DNA methyltransferases and the histone methyl transferase SUV39H1 to downregulate targeted cell promoters through CpG methylation (50, 57).LANA has also been reported to increase expression of genes regulated by a variety of transcription factors (40,44,61,63). A source...

This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBP␣ binding sites. Finally, in C/EBP␣-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G 1 arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBP␣ is essential for at least one pathway of ZTA-induced G 1 arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBP␣ leading to greatly enhanced C/EBP␣ and p21 levels through both transcriptional and posttranslational mechanisms.In cytomegalovirus, herpes simplex virus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV) infections, viral DNA replication appears to take place only in G 1 -arrested host cells (21,53). The cell cycle arrest may be imposed by herpesviruses during viral lytic-cycle DNA replication to prevent competition with host cell DNA synthesis for limited free nucleotides and to provide nuclear space for progeny viral DNA storage. Herpesviruses in general encode many of their own viral DNA replication proteins and nucleotide synthesis enzymes, thus partially obviating the need for cellular S-phase-associated replication machinery (54, 59). These virusencoded proteins always include six core DNA replication proteins (DNA polymerase, polymerase processivity factor, helicase, primase, primase-associated factor, and single-stranded DNA binding protein), together with an origin binding initiator protein, and may also include enzymes such as thymidylate synthase, thymidine kinase, ribonucleotide reductase, uracil DNA glycosylase, and dihydrofolate reductase.

E pstein-Barr virus (EBV), first discovered in association withBurkitt's lymphoma (27), is linked to a variety of human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease (105). EBV infection results in either lytic replication or the establishment of viral latency. Both latent and lytic EBV gene products have been implicated in the development of cancer (28,51,72,105). EBV can be reactivated from latency by various reagents, such as 5-bromodeoxyuridine (39, 46), phorbol esters (110), anti-Ig antibodies (23,92,97), sodium butyrate (71), methotrexate (28), bortezomib (33, 34), thapsigargin (88, 95), and arsenic trioxide (89). The transition from latency to lytic replication is mediated by two EBV immediate-early genes, BZLF1 and BRLF1. The encoded proteins, ZTA and RTA, function as transcriptional activators that regulate the expression of EBV lytic cycle genes and lytic viral DNA replication (16,21,22,31,36,69,70,83,86,96). The lytic induction of EBV has been postulated as a therapeutic strategy for the treatment of virus-associated tumors (29,30,33,77).The small ubiquitin-related modifier (SUMO) was first identified as a posttranslational modifier of RanGAP1 (73,75). Similarly to the ubiquitination pathway, SUMOylation involves a series of sequential enzymatic reactions. The SUMO precursor protein is first cleaved by sentrin-specific proteases (SENPs) to generate a C-terminal diglycine motif. This then forms an E1ϳSUMO thioester, which is transferred to the E2-conjugating enzyme UBC9. E2ϳSUMO directly transfers SUMO to the substrate at lysine residues to form an isopeptide bond. E3 SUMO protein ligases facilitate this process by recruiting E2ϳSUMO to specific substrates and by enhancing the transfer process. SUMOylated targets can be de-SUMOylated by the SENP removal of SUMO (37). SUMOylation has been implicated in a variety of cellular processes, including transcriptional regulation, cell cycle regulation, signal transduction, the DNA damage response (DDR), and the regulation of protein-protein interactions (38). Both latent and lytic EBV proteins interact with the SUMO system. EBNA3C is SUMOylated (84), while LMP1 modulates the SUMOylation processes by interaction with UBC9 (6). SUMOylation regulates the transcriptional activity of ZTA and RTA (10,13,14,45,47,80). Noncovalent SUMO-protein interactions can also occur through a SUMO interaction motif (SIM) in the target proteins (3,57,90,91,93). EBNA3C contains a SIM motif and upregulates EBNA2-mediated gene activation by binding to a SUMOylated protein (84).In this study, we used an EBV protein microarray to identify additional EBV proteins that bind to SUMO. One of the identified proteins was the conserved protein kinase BGLF4. BGLF4 is present in the virion and expressed at an early stage of the lytic cycle (40,41,99). BGLF4 phosphorylates multiple EBV proteins, including BMRF1 (18, 42), EBNA2 (106), EBNA-LP (55), ZTA (4), EBNA1, and virion proteins (108). BGLF4 also phosp...

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5 termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EBPs was characterized by electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter assays. More-abundant BART expression in epithelial cells correlated with the relative expression of positive and negative regulators in these cells.