Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.

peer reviewedDuring the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested

The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner

Varicella Zoster Virus Immediate Early 63 protein (IE63) has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-κB dependent genes such as IL-8, ICAM-1, and IκBα, it modulates transcription of these genes upon TNFα induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. Conclusion While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-κB dependent genes by the accelerated resynthesis of the inhibitor IκBα

La protéine Immediate Early 63 du Virus de la Varicelle et du Zona. Importance de sa phosphorylation dans le contrôle de son activité

Le Virus de Varicelle et du Zona (VZV) est un -herpesvirus responsable de deux maladies distinctes : la varicelle et le zona. Dans ce travail, nous nous sommes intéressés à la protéine Immediate Early 63 (IE63) du VZV. Cette protéine est essentielle pour la réplication virale et critique pour l’établissement de la latence du virus dans les ganglions sensoriels. Lors du cycle lytique, elle présente une localisation majoritairement nucléaire, alors que durant la latence, c’est dans le cytoplasme qu’elle se concentre pour finalement se répartir entre les deux compartiments une fois la réactivation du virus enclenchée. Ses propriétés régulatrices sont sujet à controverse depuis de nombreuses années. Les phénomènes de phosphorylation sont connus pour jouer un rôle clé dans la régulation de l’activité de nombreuses protéines. Dans la première partie de ce travail, nous nous sommes penchés sur la phosphorylation d’IE63 par deux types de kinases cellulaires importantes, la Protéine Kinase AMPc-dépendante (PKA) et les Cycline-dépendante Kinases (Cdks). Nous avons tenté de mettre en évidence l’impact de cette modification post-traductionnelle sur la localisation cellulaire et les propriétés régulatrices d’IE63. Nous avons pu montrer qu’IE63 est phosphorylée par la PKA. Il s’est avéré que cette phosphorylation n’était pas essentielle pour la localisation cellulaire correcte de la protéine, mais bien pour ses propriétés régulatrices. Ensuite, nous avons pu démontrer qu’IE63 est phosphorylée in vitro par la Cdk1 et la Cdk5, et in vivo par la Cdk1. Cette phosphorylation est apparue comme cruciale pour la localisation cellulaire et l’activité régulatrices d’IE63 en cellules Vero.A l’entame de la seconde partie de ce travail, une compréhension des fonctions globales d’IE63 sur l’expression de l’ensemble des gènes cellulaires manquait toujours. Dans ce contexte, nous avons décidé d’examiner les effets d’IE63 sur la transcription de l’ensemble du génome cellulaire par la technique de microarray. Nous avons pu montrer que : (i) en l’absence d’autres protéines virales, IE63 affecte l’expression d’un nombre limité de gènes, incluant des gènes impliqués dans la transduction de signaux, la transcription, la réponse immunitaire et la signalisation des protéines « Heat-Shock ». (ii) L’expression d’IE63 provoque suivant les cas une diminution ou une stimulation de la liaison de la RNA polymérase II sur les promoteurs réprimés et activés par la protéine, respectivement. (iii) En cellules HeLa, la phosphorylation correcte d’IE63 est cruciale pour ses propriétés régulatrices sur les promoteurs endogènes.Des travaux antérieurs émanant de notre laboratoire ont montré qu’IE63 était capable d’inhiber l’expression de certains gènes dépendants du NF-B comme l’IL-8 et l’IL-6. De plus, plusieurs études montrent que le VZV est capable d’échapper au système immunitaire via notamment une répression des gènes dépendant du NF-B. De manière surprenante, l’expression basale de ces gènes n’est pas affectée dans nos conditions, cela étant peut être du à un problème d’accessibilité de leur promoteur lié à l’ouverture de la chromatine. De manière à tester cette hypothèse, nous avons mesuré l’influence d’IE63 sur l’expression de certains de ces gènes (IL-8, IL-6, ICAM-1 et IB) après un traitement des cellules au TNF, une cytokine proinflammatoire connue pour provoquer l’ouverture de la chromatine au niveau du promoteur de nombreux gènes. Les résultats intéressants obtenus lors de ce travail sont : (i) dans des cellules stimulées au TNF, l’expression de certains gènes dépendant du NF-B est affectée par IE63 d’une manière dépendante du promoteur étudié, (ii) le niveau de phosphorylation de la protéine influence ces propriétés régulatrices, (iii) l’effet d’IE63 est corrélé à une modification de l’ouverture de la chromatine et enfin, (iv) IE63 est capable de modifier la liaison du NF-B sur les promoteurs testés

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance.

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Interleukin-9 stimulates the production of interleukin-5 in CD4+ T cells.

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Mapping the global depth to bedrock for land surface modeling

Depth to bedrock serves as the lower boundary of land surface models, which controls hydrologic and biogeochemical processes. This paper presents a framework for global estimation of depth to bedrock (DTB). Observations were extracted from a global compilation of soil profile data (ca. 1,30,000 locations) and borehole data (ca. 1.6 million locations). Additional pseudo-observations generated by expert knowledge were added to fill in large sampling gaps. The model training points were then overlaid on a stack of 155 covariates including DEM-based hydrological and morphological derivatives, lithologic units, MODIS surface reflectance bands and vegetation indices derived from the MODIS land products. Global spatial prediction models were developed using random forest and Gradient Boosting Tree algorithms. The final predictions were generated at the spatial resolution of 250 m as an ensemble prediction of the two independently fitted models. The 10–fold cross-validation shows that the models explain 59% for absolute DTB and 34% for censored DTB (depths deep than 200 cm are predicted as 200 cm). The model for occurrence of R horizon (bedrock) within 200 cm does a good job. Visual comparisons of predictions in the study areas where more detailed maps of depth to bedrock exist show that there is a general match with spatial patterns from similar local studies. Limitation of the data set and extrapolation in data spare areas should not be ignored in applications. To improve accuracy of spatial prediction, more borehole drilling logs will need to be added to supplement the existing training points in under-represented areas.</p

The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner-5

<p><b>Copyright information:</b></p><p>Taken from "The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner"</p><p>http://www.biomedcentral.com/1471-2199/8/99</p><p>BMC Molecular Biology 2007;8():99-99.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2176069.</p><p></p>asing times (from 0 to 2 h) with TNFα at a final concentration of 200 U/mL. (A) Total RNA extracts were isolated and analyzed by Real-time RT-PCR using primers for the IL-8 mRNA, IL-6 mRNA, ICAM-1 mRNA, and IκBα mRNA. (B) The IκBα degradation. HeLa cells expressing IE63wt or control cells were treated for increasing times (from 0 to 2 h) with TNFα (200 U/mL). IκBα degradation was followed by Western Blotting on total cellular extracts. β-actin Western Blotting detection was used as loading control (lower panel). ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (Inv ; p-value < 0.05) ; **, not significantly different from control (Inv ; p-value ≥ 0.05)

The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner-4

<p><b>Copyright information:</b></p><p>Taken from "The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner"</p><p>http://www.biomedcentral.com/1471-2199/8/99</p><p>BMC Molecular Biology 2007;8():99-99.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2176069.</p><p></p>t IE63 and normalized using the β2-microglobuline transcripts. Experiments were done at least in triplicate. Differences (n-fold) between samples were calculated using the standard-curve method and the 2method. ρ-values were calculated using the graphpad quickcalcs software [59] : **, not significantly different from control (Inv ; p-value ≥ 0.05)

The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner-7

<p><b>Copyright information:</b></p><p>Taken from "The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner"</p><p>http://www.biomedcentral.com/1471-2199/8/99</p><p>BMC Molecular Biology 2007;8():99-99.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2176069.</p><p></p> lines that stably express the protein IE63 wild-type (HeLa-IE63), in inverted orientation (HeLa-Inv) or mutated (HeLa-S224/T222A, HeLa-Full). One week after infection, cells were harvested. (A) Cells were lysed in radioimmunoprecipitation assay buffer and used for immunoblotting with mouse monoclonal antibody to the ORF63 protein or with rabbit polyclonal antibody to the EGFP. (B) Forty-eight hours post-seeding, immunostaining analysis was carried out using a monoclonal antibody (9A12) directed against IE63. Secondary antibody used is conjugated with Texas Red. (C) HeLa cells (HeLa-IE63, and HeLa-Inv) were transfected with 1 μg of pPol-Luc. 24 hours post-transfection, cells were harvested and the reporter gene activity was measured. Results are presented as a percentage of stimulation with respect to the basal expression of the promoter (= 100%). Data from luciferase assays were collected from six independent transfection experiments. ρ-values were calculated using the graphpad quickcalcs software [59]: *, significantly different from control (p-value < 0.05)