Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.

Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein.

Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.

Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection.

The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in the differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with that in EBV-negative sporadic Burkitt lymphoma-derived cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B-cell transformation. We show that infection of B cells with EBV leads to downregulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.IMPORTANCE In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with those leading to sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, and among them was the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study, we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.

Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G.

Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.

Epstein-Barr virus nuclear antigen 1 interacts with regulator of chromosome condensation 1 dynamically throughout the cell cycle.

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.

Epstein-Barr virus down-regulates tumor suppressor DOK1 expression.

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2'-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.

TLR9 transcriptional regulation in response to double-stranded DNA viruses.

The stimulation of TLRs by pathogen-derived molecules leads to the production of proinflammatory cytokines. Because uncontrolled inflammation can be life threatening, TLR regulation is important; however, few studies have identified the signaling pathways that contribute to the modulation of TLR expression. In this study, we examined the relationship between activation and the transcriptional regulation of TLR9. We demonstrate that infection of primary human epithelial cells, B cells, and plasmacytoid dendritic cells with dsDNA viruses induces a regulatory temporary negative-feedback loop that blocks TLR9 transcription and function. TLR9 transcriptional downregulation was dependent on TLR9 signaling and was not induced by TLR5 or other NF-κB activators, such as TNF-α. Engagement of the TLR9 receptor induced the recruitment of a suppressive complex, consisting of NF-κBp65 and HDAC3, to an NF-κB cis element on the TLR9 promoter. Knockdown of HDAC3 blocked the transient suppression in which TLR9 function was restored. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR9, immune induction, and inflammation against viruses.

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

The mycotoxin aflatoxin B1 stimulates Epstein-Barr virus-induced B-cell transformation in in vitro and in vivo experimental models.

Although Epstein-Barr virus (EBV) infection is widely distributed, certain EBV-driven malignancies are geographically restricted. EBV-associated Burkitt's lymphoma (eBL) is endemic in children living in sub-Saharan Africa. This population is heavily exposed to food contaminated with the mycotoxin aflatoxin B1 (AFB1). Here, we show that exposure to AFB1 in in vitro and in vivo models induces activation of the EBV lytic cycle and increases EBV load, two events that are associated with an increased risk of eBL in vivo. AFB1 treatment leads to the alteration of cellular gene expression, with consequent activations of signaling pathways, e.g. PI3K, that in turn mediate reactivation of the EBV life cycle. Finally, we show that AFB1 triggers EBV-driven cellular transformation both in primary human B cells and in a humanized animal model. In summary, our data provide evidence for a role of AFB1 as a cofactor in EBV-mediated carcinogenesis.

Epstein-Barr virus late gene transcription depends on the assembly of a virus-specific preinitiation complex.

UNLABELLED: During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. IMPORTANCE: Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC).

Epstein - Barr virus transforming protein LMP-1 alters B cells gene expression by promoting accumulation of the oncoprotein ΔNp73α.

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.

Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs.

The Epstein-Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human ß-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the ß-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.

The Epstein-Barr virus BcRF1 gene product is a TBP-like protein with an essential role in late gene expression.

That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly, a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover, our results suggest that BcRF1 acts via interaction with other viral proteins.

Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1).

We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties.

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

Post-transcriptional regulation of Herpesvirus productive cycle genes expression: importance of EB2 factor from Epstein-Barr virus (EBV) and its similar proteins.

During viral infection, the amount of viral mRNAs expressed is not only a reflection of the viral gene transcription level: mRNA stability and nucleo-cytoplasmic export are also important for optimal viral gene expression during the productive cycle. It is now well established that herpesviruses express a protein absolutely required for the cytoplasmic accumulation of some viral mRNAs transcribed from intronless genes. This family of proteins comprises the EB2 factor from Epstein-Barr virus (EBV), and its similar proteins: ICP27 from HSV-1, UL69 from cytomegalovirus (CMV), ORF57 from KSHV and IE4 from VZV. These proteins are able to stabilize their target mRNAs in the nucleus and, by interacting with various cellular factors (TAP/NXF1, SR proteins, RBM15 etc), promote mRNA export to the cytoplasm, where they are also involved in the translation efficiency of these viral mRNAs. On the basis of their essential role in the viral productive cycle, these multifunctional viral factors should be considered as important targets for therapeutic approaches.

The nuclear and adherent junction complex component protein ubinuclein negatively regulates the productive cycle of Epstein-Barr virus in epithelial cells.

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.

Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2.

The Epstein-Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.

Epstein-Barr virus protein EB2 contains an N-terminal transferable nuclear export signal that promotes nucleocytoplasmic export by directly binding TAP/NXF1.

The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), which allows the nuclear export of a subset of early and late viral mRNAs derived from intronless genes, is essential for the production of infectious virions. An important feature of mRNA export factors is their capacity to shuttle continuously between the nucleus and the cytoplasm. In a previous study, we identified a novel CRM1-independent transferable nuclear export signal (NES) at the N terminus of EB2, between amino acids 61 and 146. Here we show that this NES contains several small arginine-rich domains that cooperate to allow efficient interaction with TAP/NXF1. Recruitment of TAP/NXF1 correlates with this NES-mediated efficient nuclear export when it is fused to a heterologous protein. Moreover, the NES can export mRNAs bearing MS2 RNA-binding sites from the nucleus when tethered to the RNA via the MS2 phage coat protein RNA-binding domain.