References of "Lebrun, Marielle"
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See detailIdentification of VZV ORF9p potential cellular partners that could be important for the viral egress.
Lebrun, Marielle ULg; riva, laura; Rambout, Xavier ULg et al

Poster (2015, July 26)

ORF9p (homologous to HSV-1 VP22) is a VZV tegument protein essential for the viral replication. During the lytic cycle it is the mostly expressed gene. We have recently demonstrated that it is a substrate ... [more ▼]

ORF9p (homologous to HSV-1 VP22) is a VZV tegument protein essential for the viral replication. During the lytic cycle it is the mostly expressed gene. We have recently demonstrated that it is a substrate of the viral kinase ORF47p and that its ORF47p-dependent phosphorylation is important for the secondary envelopment process. We also have identified an acidic cluster (AC) within the protein that is important for its correct localization in the infected cells and for the interaction with ORF47p. The recombinant VZV expressing ORF9p-ΔAC presents an accumulation of capsids in the perinuclear space. ORF9p seems then to play an important role in several steps of the egress process. In this context, we sought to identify cellular partners of ORF9p that might be important for these functions. We performed a yeast two hybrid screen against the human ORFeome 5.1. and picked out 44 candidates among which 5 proteins playing roles in membrane organization and targeting. We currently are trying to confirm these interactions in infected cells and to assess the role of these interactions for the viral lytic cycle. [less ▲]

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See detailVaricella-Zoster Virus (VZV) assemblons interplay with PML bodies
Lebrun, Marielle ULg; Thiry, Marc ULg; Sadzot, Catherine ULg

Poster (2015, May 15)

Using a virus expressing the small capsid protein fused to an eGFP tag (eGFP-ORF23 VZV), we recently identified, in the nuclei of infected cells, the presence of dynamic capsid aggegrates. Because we ... [more ▼]

Using a virus expressing the small capsid protein fused to an eGFP tag (eGFP-ORF23 VZV), we recently identified, in the nuclei of infected cells, the presence of dynamic capsid aggegrates. Because we believe that these structures might represent sites of preferential caspid assembly and by analogy with HSV-1, we referred to them as “VZV assemblons”. Structures resembling these assemblons and identified as capsids entrapped in some “PML-cages” were recently described in the nuclei of wild-type VZV infected cells (Reichelt et al., 2011). We then wonder if there was a link between these independent observations. When we infected MeWo cells in which the expression of each PML subunit is downregulated by shRNA, VZV assemblons still formed. Immunostaining of MeWo cells infected by eGFP-ORF23 VZV with an antibody against the PML protein showed that VZV assemblons only partially colocalize with PML bodies. However, overexpression of PML-I-eGFP in HEK293 cells followed by infection with a tagRFP-T-ORF23 VZV, where the ORF23 protein is fused to a red tag, showed a complete colocalization is complete. The same result was obtained with all tested PML isoforms. This suggests that the partial colocalization in normal cells could be due to the expression level of PML proteins. Altogether, these results suggest that rather than a progressive accumulation of newly formed capsids within PML cages, it is likely that PML protein is recruited to the sites where VZV assemblons develop. It correlates with the fact that the number of PML bodies decreases with the infection. Obviously, even if this phenomenon might impede the egress of a substantial amount of capsids and, in this regard, limit the infection progression, all the tested cell lines are permissive to VZV. It would then be interesting to investigate the relationship between VZV assemblons and PML bodies in latent or non permissive VZV infection models. [less ▲]

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See detailRole of Kinases in VZV Nuclear Egress
Blondeau, Caroline ULg; Istaz, Bastienne; Lebrun, Marielle ULg et al

Poster (2015)

Like all Herpesviruses VZV expresses two conserved proteins forming the nuclear egress complex (NEC), namely ORF24p and ORF27p, respectively homologous to UL34 and UL31 in HSV-1. Many works described the ... [more ▼]

Like all Herpesviruses VZV expresses two conserved proteins forming the nuclear egress complex (NEC), namely ORF24p and ORF27p, respectively homologous to UL34 and UL31 in HSV-1. Many works described the role of this complex and viral kinases, the Alpha-herpesvirus specific kinase US3p, and hCMV UL97p (conserved in all Herpesviruses and homolog to UL13p in HSV-1), in the nuclear capsids translocation to the cytoplasm. Indeed, using a very complex mechanism, not yet fully characterized, nuclear capsids bud through the internal nuclear membrane, stay briefly in the perinuclear space and finally free the capsids into the cytoplasm through fusion of the primary envelope with the external nuclear membrane. In the cytoplasm, virions acquire the second and last envelopment before being released outside the cells. In order to better characterize the nuclear egress in the context of VZV, we focused our work on the NEC and on both viral kinases ORF47p and ORF66p respectively homologous to HSV-1 UL13p and US3p. By co-immunoprecipitation we identified a protein complex containing ORF24p, ORF27p and both viral kinases and are currently trying to determine the role of each kinase in the formation of this complex. We are also characterizing the phosphorylation status of these proteins depending on ORF66p and/or ORF47p. Immunofluorescence studies of cells infected with a deleted ORF66 or ORF47 virus showed mislocalisation of some viral proteins such as ORF24p as described for HSV-1. Finally electron microscopy analyses are in progress and will help to determine the role of both VZV kinases in the nuclear egress. [less ▲]

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See detailDeletion of the ORF9p acidic cluster impairs the nuclear egress of Varicella-zoster virus capsids.
Riva, Laura ULg; Thiry, Marc ULg; Lebrun, Marielle ULg et al

in Journal of virology (2014)

The protein encoded by the ORF9 is essential for Varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment ... [more ▼]

The protein encoded by the ORF9 is essential for Varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment. In this work, we deleted the ORF9p acidic cluster, destroying its interaction with ORF47p and resulting in a nuclear accumulation of both proteins. This phenotype results to an accumulation of primary enveloped capsids in the perinuclear space, reflecting a capsid de-envelopment defect. [less ▲]

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See detailVaricella-zoster virus induces the formation of dynamic nuclear capsid aggregates.
Lebrun, Marielle ULg; Thelen, Nicolas ULg; Thiry, Marc ULg et al

in Virology (2014), 454-455

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains ... [more ▼]

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. [less ▲]

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See detailInteractomic map of the Ets factors family : Identification of unexpected functions in mRNA processing
Rambout, Xavier ULg; Simonis, Nicolas; Brohée, Sylvain et al

Poster (2013, January 28)

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets ... [more ▼]

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets factors in order to better define their roles and regulations in normal and oncogenic processes. The Ets interactome was built on a high-throughput yeast-two hybrid (Y2H) approach, and a literature and database curation. We identified 431 PPIs and 276 different protein partners. Clustering of the Ets interactome divided it into 24 functional subnetworks classified on their novelty index and their size. Cluster#1 was exclusively composed of newly identified interaction partners and was highly connected to the Erg subfamily of Ets factors. Gene ontology enrichment analysis revealed that it was associated to mRNA processing. In support of this result, we observed in HeLa cells that ERG and the components of cluster#1 localized in p-bodies and stress granules, physically linked cytoplasmic sites of mRNA degradation and silencing. Hence, we hypothesized that Erg proteins might have a role in post-transcriptional gene regulation and be involved in cellular mRNAs degradation. To test this hypothesis, we performed a MS2-based tethering assay and showed that the recruitment of ERG on a mRNA reporter promoted inhibition of its expression via a two-fold decrease of its half-life. ERG controls degradation of target mRNAs via different mechanisms including polysome stability, mRNA deadenylation, and p-bodies aggregation. A microarray-based appraoch identified 321 endogeneous genes whose mRNA decay rate was lowered in ERG silenced cells. Results point out the Nter domain of ERG as the predominant domain required for mRNA degradation. Importantly, oncogenic TET-Erg fusions described in AML and Ewing’s sarcoma exhibited diminished ability to degrade target mRNAs, concomitantly with the loss of the ERG Nter domain. This reinforces the important role of Erg proteins in mRNA degradation in cancer. [less ▲]

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See detailORF9p phosphorylation by ORF47p is crucial for the formation and egress of the Varicella-zoster virus (VZV) viral particles.
Riva, Laura ULg; Thiry, Marc ULg; BONTEMS, Sébastien ULg et al

in Journal of Virology (2013), 87(5), 2868-2881

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully ... [more ▼]

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The Varicella-zoster virus (VZV) protein coded by ORF9 (ORF9p) is an essential tegument protein and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a BAC containing the complete VZV genome creating viruses expressing mutant versions of ORF9p.We showed that ORF9p is hyper-phosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultra-structural analysis revealed that the mutation of this consensus site (Glutamate 85 to Arginine) strongly affects viral assembly and release, reproducing ORF47 kinase dead VZV phenotype. It also slightly diminishes the infectivity towards immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress. [less ▲]

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See detailPP2A regulatory subunit Balpha controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.
Martin, Maud ULg; Geudens, Ilse; Bruyr, Jonathan et al

in EMBO Journal (2013)

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens ... [more ▼]

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Balpha regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Balpha in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Balpha in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Balpha controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Balpha, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Balpha/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion. [less ▲]

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See detailInteractomic map of the Ets factors family : Identification of unexpected functions in mRNA processing
Rambout, Xavier ULg; Simonis, Nicolas; Demoitié, Pauline et al

in Keystone symposium - Protein-RNA Interactions in Biology and Disease (C1) (2012, March 05)

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain, the ETS domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the ... [more ▼]

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain, the ETS domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets factors in order to better define their roles and regulations in normal and oncogenic processes. The Ets interactome was built on a high-throughput yeast-two hybrid (Y2H) approach, and a literature and database curation of confident interactions which led us to the identification of 602 PPIs and 369 different protein partners. Clusterization using the Network Analysis Tool box (NeAT) divided the ETS interactome into 39 functional sub-networks. Among these, we identified Cluster16 as highly connected to the Erg ETS subfamily. A gene ontology (GO) enrichment analysis revealed that Cluster16 was associated to various aspects of mRNA processing. We therefore hypothesized that Erg factors might have a role in post-transcriptional gene regulation. This would constitute a entirely new and undisclosed role for ETS factors, which are so far firmly established as transcription factors. In support of our hypothesis, we observed that ERG localized in p-bodies, cytoplasmic sites of mRNA decay. Interestingly, under various cellular stresses, a portion of ERG and its partners from Cluster16 localized in stress granules, cytoplasmic sites of mRNA silencing physically linked to p-bodies. Hence, we hypothesized that Erg proteins might be involved in cellular mRNAs degradation. To test this, we performed a MS2-based tethering assay and showed that the recruit-ment of Erg factors promoted degradation of a reporter mRNA, mainly via its N-ter domain. Very importantly, oncogenic TET-Erg fusions described in AML and Ewing’s sarcoma exhibited diminished ability to degrade target mRNAs, concomitantly with the loss of the N-ter domain of the corresponding Erg protein. This re-inforces the important role of Erg proteins in mRNA degradation in cancer. Our efforts are now concentrated on identifying the molecular determinants behind this new function of Erg proteins. [less ▲]

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See detailThe inositol phosphatase SHIP-1 inhibits NOD2-induced NF-κB activation by disturbing the interaction of XIAP with RIP2
Condé, Claude ULg; Rambout, Xavier ULg; Lebrun, Marielle ULg et al

in PLoS ONE (2012)

SHIP-1 is an inositol phosphatase predominantly expressed in hematopoietic cells. Over the ten past years, SHIP-1 has been described as an important regulator of immune functions. Here, we characterize a ... [more ▼]

SHIP-1 is an inositol phosphatase predominantly expressed in hematopoietic cells. Over the ten past years, SHIP-1 has been described as an important regulator of immune functions. Here, we characterize a new inhibitory function for SHIP-1 in NOD2 signaling. NOD2 is a crucial cytoplasmic bacterial sensor that activates proinflammatory and antimicrobial responses upon bacterial invasion. We observed that SHIP-1 decreases NOD2-induced NF-κB activation in macrophages. This negative regulation relies on its interaction with XIAP. Indeed, we observed that XIAP is an essential mediator of the NOD2 signaling pathway that enables proper NF-κB activation in macrophages. Upon NOD2 activation, SHIP-1 C-terminal proline rich domain (PRD) interacts with XIAP, thereby disturbing the interaction between XIAP and RIP2 in order to decrease NF-κB signaling. [less ▲]

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See detailThe varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3.
Vandevenne, Patricia ULg; Lebrun, Marielle ULg; El Mjiyad, Nadia et al

in PLoS ONE (2011), 9(2),

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by ... [more ▼]

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-kappaB, is a key regulator of IFN-beta expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-beta and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-beta and ISG15. [less ▲]

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See detailVaricella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway.
Ote, Isabelle ULg; Vandevenne, Patricia ULg; Bontems, Sébastien ULg et al

in PLoS ONE (2009), 4(11), 7882

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level ... [more ▼]

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export. [less ▲]

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See detailTransgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules.
Liao, Shan; Bentley, Kevin; Lebrun, Marielle ULg et al

in Proceedings of the National Academy of Sciences of the United States of America (2007), 104(11), 4577-82

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid ... [more ▼]

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the beta-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTbetaR-Ig (lymphotoxin-beta receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity. [less ▲]

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See detailMultiple roles of Hoxc8 in skeletal development
Juan, A. H.; Lei, H. Y.; Bhargava, P. et al

in Annals of the New York Academy of Sciences (2006), 1068

We are interested in investigating the function of Hoxc8 in skeletogenesis during mouse development. Previous studies have shown that deregulation of Hoxc8 expression in the mouse leads to several ... [more ▼]

We are interested in investigating the function of Hoxc8 in skeletogenesis during mouse development. Previous studies have shown that deregulation of Hoxc8 expression in the mouse leads to several skeletal defects, such as homeotic transformation in the thoracic vertebrae, abnormal development of the rib cage, and overproliferation of chondrocytes in the hypertrophic area. By deleting a crucial enhancer of Hoxc8 in vivo, we found that precise temporal expression of Hoxc8 is important for determining the correct identity of the vertebral column in early embryos. We also identified downstream targets of Hoxc8 relevant to osteoblast differentiation at later developmental stages. [less ▲]

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