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 which 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 (RanGEF) 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 FRET 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

Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection

Un vaccin contre le virus d'Epstein-Barr : une réalité pour demain, mais pour qui ?

International audiencePlus de 95% de la population mondiale adulte est infectée par le virus d'Epstein-Barr (EBV), (virus appartenant à la famille des herpesviridae qui infecte principalement les lymphocytes B). Le virus, transmis par la salive, infecte généralement son hôte au moment de la petite enfance et cette infection est largement asymptomatique. Si l'infection intervient plus tardivement, à l'adolescence ou chez le jeune adulte, elle conduit dans environ 40% des cas au développement d'une pathologie aiguë appelée mononucléose infectieuse (MNI). Rien qu'aux États-Unis, 125 000 cas de MNI sont déclarés chaque année. L'agent causal de la MNI est aussi associé au développement de nombreux cancers dérivés soit de lymphocytes soit de cellules épithéliales (Tableau 1). On estime, d'une part, qu'environ 10% des cancers associés à une infection virale le sont avec EBV ; et, d'autre part, que, chaque année en moyenne, environ 200 000 nouveaux cas de cancers associés à EBV sont diagnostiqués dans le monde. Par ailleurs, EBV est aussi associé au développement de maladies auto-immunes comme la sclérose en plaques (1). Le cycle biologique du virus est assez complexe et passe par une phase d'infection latente durant laquelle le virus induit l'activation, la prolifération et la différenciation des cellules B primaires en cellules B mémoires. Durant cette phase, l'infection entraîne une réponse immunitaire humorale et cellulaire dirigée contre les protéines de la phase de latence. Lors de la différentiation terminale en plasmocytes des cellules infectées, le cycle viral productif est activé est des virions sont produits qui vont pouvoir infecter des cellules épithéliales capables de produire une grande quantité de virions. Les nombreuses protéines virales exprimées au cours du cycle productif sont également des cibles importantes de la réponse immunitaire cellulaire. Les cellules Natural Killer et les lymphocytes T cytotoxiques (CTL) anti-EBV sont les principaux acteurs de la réponse immunitaire qui contrôle efficacement l'infection (2). Le rôle primordial des CTL anti-EBV serait de contrôler la prolifération des cellules B infectées de façon latente

Le virus d’Epstein-Barr: Un acteur clé dans le développement de la sclérose en plaques

International audienceNo abstract availabl

Etude de la fonction de facteur d'export EB2 au cours du cycle productif du virus d'Epstein-Barr (EBV)

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Etude des mécanismes permettant l'accumulation cytoplasmique de certains ARNm viraux par la protéine EB2 du virus d'Epstein-Barr (rôle des facteurs cellulaires TAP/NFX1 et SRp20)

La protéine EB2 du virus d'Epstein-Barr (EBV) est une protéine du cycle réplicatif du virus indispensable à la production de particules virales. Elle permet l accumulation dans le cytoplasme de certains ARNm viraux issus de gènes dépourvus d intron. Pour mettre en évidence les mécanismes qui permettent à EB2 d exporter ses ARNm cibles dans le cytoplasme, nous avons identifié différents partenaires cellulaires d EB2 et nous avons étudié certaines de ces interactions d un point de vue fonctionnel. Nous avons pu montrer qu EB2 recrute directement le facteur général d'export des ARNm, TAP/NXF1, ce qui lui permet d être exportée du noyau vers le cytoplasme. Puis nous avons montré qu EB2 interagit avec SRp20, une protéine impliquée notamment dans la régulation de l'épissage et l'export des ARNm cellulaires. Cette interaction entre EB2 et SRp20 est indispensable pour l accumulation dans le cytoplasme de certains ARNm cibles d EB2, notamment parce que SRp20 semble permettre le recrutement d'EB2 sur ces ARNm. Enfin, nous avons montré qu EB2 forme un dimère et nous avons caractérisé le domaine de la protéine responsable de cette interaction. La dimérisation d'EB2 semble essentielle pour que la protéine interagisse avec certains de ses partenaires comme SRp20 ou encore REF.The Epstein-Barr virus (EBV) protein EB2 is an early protein essential for the production of infectous virions. EB2 allows the cytoplasmic accumulation of a subset of viral mRNAs derived from intronless genes. To highlight the mecanisms by which EB2 exports his targets mRNA, we identified cellular partners and studied the functional role of some of these interactions. We showed that EB2 recruits directly the cellular mRNA export factor TAP/NXF1 and this interaction allows EB2 s shuttling between the nucleus and the cytoplasm. The we showed that EB2 interacts with SRp20, a cellular protein implicated in splicing regulation and mRNA export. This interaction is essential for the efficient cytoplasmic accumulation of some EB2 target mRNAs, partly because SRp20 appears to be able to recruit EB2 on these mRNAs. Then we showed that EB2 dimerises and we characterized the domain necessary for this interaction. This dimerisation appears to be essential for EB2 s interaction with several partners, including SRp20 and REF.LYON-ENS Sciences (693872304) / SudocSudocFranceF

EBV/KSHV co-infection: an effective partnership

International audienceKaposi's sarcoma human herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL) in which Epstein-Barr virus (EBV) is also very often present. By using a humanized mouse model, Pr. Münz's team has been able to demonstrate that EBV/KSHV co-infection increases KSHV persistence and cell transformation through the stimulation of EBV replication. This is the first model of PEL in small animals, opening up exciting prospects for future studies on this unique lymphoma

The Epstein-Barr virus (EBV) protein EB2 is an mRNA export factor essential for virus production

International audienceThe EBV early protein EB2 (aka Mta, SM and BMLF1) shares properties with mRNA export factors. It shuttles between the cytoplasm and the nucleus, and interacts with RNA both in vitro and in vivo but with no apparent sequence specificity. EB2 induces the cytoplasmic accumulation of mRNAs generated from intronless and intron-containing genes, likely through interactions with cellular export factors of the TAP/p15 pathway. Using a cell line carrying a viral genome with the EB2 gene deleted, it has been shown that EB2 is essential for the production of infectious virions by facilitating the nuclear export of a subset of early and late viral mRNAs, a function regulated by CK2 phosphorylation of EB2. There aredocking sites for both CK2 subunits and for the heterotetrameric enzyme in the EB2 N- and C-terminal domains. Accordingly, EB2 and CK2 copurify as a complex in which CK2 phosphorylates EB2. CK2 phosphorylation of EB2 at one of the Ser-55, Ser-56 and ser-57 is critical for its mRNA export function and as a consequence, for infectious virus production

Herpesvirus Late Gene Expression: A Viral-Specific Pre-initiation Complex Is Key

International audienceDuring their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E), and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the transcription start site (TSS). Moreover, in the case of the β-and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies (β and γ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors-especially ICP4-play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein