Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and cerine residues in the Epstein-Barr Virus lytic switch protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon

First description of pestivirus disease in Rupicapra pyrenaica pyrenaica

Understanding the circulation of pestiviruses in wild ungulates is potentially important to explain variations in the number of animals in these species, and to implement pestivirus control programs in domestic animals. In 2002 in the French Pyrenees, symptoms of amyotrophy and weight loss, associated with bilateral alopecia with hairless and highly pigmented areas on the nose, around the eyes and the ear margins were found in 8 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) between 1 and 9 years old, 6 of which had been captured alive and 2 were found dead. These lesions were uncharacteristic. The intensity of abomasal or lung parasitism varied from one animal to the other. Pestiviruses were isolated in all 6 animals captured alive, but no anti-NS2/3 antibodies were found. Many questions remain on the transitory or persistent nature of the infection, and on the conditions of viral transmission within the Rupicapra genus.Comprendre la circulation de pestivirus chez les ongulés sauvages est potentiellement important pour expliquer les variations d'effectifs dans ces espèces et pour réaliser les programmes de contrôle des pestiviroses atteignant les animaux domestiques. En 2002 dans les Pyrénées ariégeoises, des symptômes d'amyotrophie et d'amaigrissement, associés à des signes d'alopécie bilatérale, avec des zones cutanées glabres et fortement pigmentées sur le chanfrein, le pourtour des yeux et les marges auriculaires ont été observés sur 8 isards (Rupicapra pyrenaica pyrenaica) âgés de 1 à 9 ans, dont 6 avaient été capturés vivants et 2 trouvés morts. Les lésions étaient peu caractéristiques. L'intensité du parasitisme abomasal ou pulmonaire variait d'un individu à l'autre. Un pestivirus a été mis en évidence sur les 6 animaux capturés vivants et un des deux récupérés morts (7 cas sur 8), mais aucun anticorps dirigé contre la protéine NS2/3 n'a été trouvé. De nombreuses questions persistent sur la nature transitoire ou persistante de l'infection et sur les modalités de transmission au sein du genre Rupicapra

X-box binding protein 1 induces the expression of the lytic cycle transactivator of Kaposi's sarcoma-associated herpesvirus but not Epstein–Barr virus in co-infected primary effusion lymphoma

Cells of primary effusion lymphoma (PEL), a B-cell non-Hodgkin's lymphoma, are latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV), with about 80 % of PEL also co-infected with Epstein–Barr virus (EBV). Both viruses can be reactivated into their lytic replication cycle in PEL by chemical inducers. However, simultaneous activation of both lytic cascades leads to mutual lytic cycle co-repression. The plasma cell-differentiation factor X-box binding protein 1 (XBP-1) transactivates the KSHV immediate–early promoter leading to the production of the replication and transcription activator protein (RTA), and reactivation of KSHV from latency. XBP-1 has been reported to act similarly on the EBV immediate–early promoter Zp, leading to the production of the lytic-cycle transactivator protein BZLF1. Here we show that activated B-cell terminal-differentiation transcription factor X-box binding protein 1 (XBP-1s) does not induce EBV BZLF1 and BRLF1 expression in PEL and BL cell lines, despite inducing lytic reactivation of KSHV in PEL. We show that XBP-1s transactivates the KSHV RTA promoter but does not transactivate the EBV BZLF1 promoter in non-B-cells by using a luciferase assay. Co-expression of activated protein kinase D, which can phosphorylate and inactivate class II histone deacetylases (HDACs), does not rescue XBP-1 activity on Zp nor does it induce BZLF1 and BRLF1 expression in PEL. Finally, chemical inducers of KSHV and EBV lytic replication in PEL, including HDAC inhibitors, do not lead to XBP-1 activation. We conclude that XBP-1 specifically reactivates the KSHV lytic cycle in dually infected PELs

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

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed

Carbonyl reductase 1 catalyzes 20β-reduction of glucocorticoids, modulating receptor activation and metabolic complications of obesity

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH-dependent production of 20 beta-dihydrocortisol (20 beta-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20 beta-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20 beta-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity

Optimization based on the study of trajectories in a random environment : application to the control of production systems

Dans le contexte international actuel, les entreprises doivent être capables de développer des stratégies leur permettant d’augmenter leurs performances et d'être plus compétitives. Cet environnement très évolutif introduit de nombreuses incertitudes et contraintes qui rendent beaucoup plus difficile la détermination de la meilleure stratégie à adopter selon les objectifs fixés. Le travail développé dans cette thèse, s’inscrit dans ce cadre et nous nous intéressons plus précisément à l'optimisation du pilotage de systèmes de production soumis à aléas (comme les pannes des machines) de façon à minimiser les coûts globaux. Pour la modélisation, le modèle à flux continus est choisi afin de représenter le flux de matières transitant dans le système. Ce modèle, nous permet également d'intégrer les délais de transfert et de transport entre les différents éléments qui composent le système.La méthode de résolution analytique utilisée est issue des méthodes d’analyse de sensibilité et correspond à la méthode d’analyse des perturbations infinitésimales (IPA). Cette méthode nous permet de déduire à partir d'une étude de trajectoires, un gradient du coût global pour chacune des études menées. Nous prouvons alors que ces gradients ne sont pas biaisés. Cela nous permet de les utiliser dans des simulations numériques. Ces simulations nous permettent de déterminer les variables de décision des stratégies de pilotage du système considéré. Le pilotage intègre la maintenance à la production. Pour le pilotage des systèmes considérés, nous considérons également des contraintes liées aux trois piliers du développement durable. Ces contraintes sont intégrées à nos modèles sous forme de coûts. Ainsi, les coûts globaux peuvent comporter en plus de coûts purement économiques, des coûts environnementaux et sociaux. Nous montrons donc que l'approche de résolution proposée peut être utilisée pour optimiser d'autres objectifs dans un cadre de durabilitéIn the current international context, companies need to be able to develop strategies to increase their performance and become more competitive. This rapidly changing environment introduces many uncertainties and constraints, making much more difficult to determine the best strategy according to the objectives set. The work developed in this thesis falls within this context and, more precisely, we are interested in the optimization of the control of production systems subject to uncertainties (such as machine failures) in order to minimize the overall costs. For modeling, the continuous-flow model is chosen to represent the material flow moving through the system. This model allows us to integrate transfer and transportation delays between the different components of the system. The analytical resolution method used is based on the sensitivity analysis methods and corresponds to the infinitesimal perturbation analysis method (IPA). This method allows us to deduce, based on learning from sample-paths, a gradient of the overall cost for each of the studies conducted. We prove that these gradients are unbiased, which allows us to use them in numerical simulations. The simulations allow us to determine the decision variables of control strategies of the studied systems. The control integrates the maintenance to the production. For the control of the considered systems, we also take into account constraints linked to the three pillars of sustainable development. These constraints are integrated into our models in terms of costs. Thus, the overall costs may not only include purely economic costs, but also environmental and social costs. We show that the proposed resolution approach may be used to optimize other objectives within a sustainability framewor