Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF

Etude du contrôle transcriptionnel du gène de la thyroglobuline et clonage d'un facteur de transcription

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Cloning of a putative transcription factor recognizing the thyroglobulin gene promoter

info:eu-repo/semantics/publishe

Création d’un espace collaboratif dans Moodle : retour d’expérience des bibliothèques universitaires de Paris Descartes

Les environnements numériques d’apprentissage peuvent servir d’appui aux formations documentaires offertes par les bibliothèques universitaires tant à leur personnel qu’à leurs usagers. Ainsi, à l’Université Paris Descartes, la mise en place de la plateforme Moodle a permis aux formateurs des bibliothèques de disposer d’un espace commun contenant l’ensemble des outils nécessaires à la réalisation des cours de recherche documentaire et favorisant les retours d’expériences entre formateurs. Résultant de ces échanges, une base de questions accessible dans tous les cours de la plateforme a été créée pour accompagner les formations aux étudiants et personnels de l’Université et favoriser l’appropriation des contenus

Cloning and sequence analysis of TFE, a helix-loop-helix transcription factor able to recognize the thyroglobulin gene promoter in vitro

A cDNA that encodes a transcription factor able to recognize the thyroglobulin gene promoter in vitro was isolated from a dog thyroid cDNA expression library in lambda gt11. The library was screened with a multimerized 20 bp-oligonucleotide probe corresponding to the -126 to -107 bp region of the bovine thyroglobulin gene promoter. The specificity of DNA sequence recognition was demonstrated by DNA binding experiments realized with beta-galactosidase-fusion protein immobilized on nitrocellulose filters and various unlabelled multimerized competing DNA fragments. The encoded protein, TFE, appears to be the canine counterpart of a recently cloned human transcription factor, ITF-2, that binds to the mu E5 kappa E2 motif found in both immunoglobulin heavy and light chains genes enhancers and belongs to the basic-Helix-Loop-Helix family of transcription factors. When TFE protein was produced in a rabbit reticulocyte lysate, it displayed the same specificity of DNA sequence recognition as the beta-galactosidase fusion protein and immobilization of the translation product on nitrocellulose still appeared to be essential for detecting in vitro DNA binding activity. Functional data failed to assign a role for TFE in the control of thyroglobulin gene transcription in vitro, suggesting that the selection of TFE clone resulted from the fortuitous presence of a high affinity binding site in the probe used for screening the expression library.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction

A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cell-type Specificity of Inhibition of Glycolysis By 5-amino-4-imidazotecarboxamide Riboside - Lack of Effect in Rabbit Cardiomyocytes and Human Erythrocytes, and Inhibition in Fto-2b Rat Hepatoma-cells

The nucleoside AICAriboside (5-amino-4-imidazolecarboxamide riboside) has been shown to inhibit glycolysis in isolated rat hepatocytes [Vincent, Bontemps and Van den Berghe (1992) Biochem. J. 281, 267-272]. The effect is mediated by AICAribotide (ZMP), the product of the phosphorylation of AICAriboside by adenosine kinase. To assess the cell-type specificity of the effect, studies were conducted in rabbit cardiomyocytes, human erythrocytes and rat hepatoma FTO-2B cells. AICAriboside had no effect on glycolysis in cardiomyocytes, and a slight stimulatory effect in erythrocytes, but inhibited glycolysis by 65% at 250 mu M concentration in FTO-2B cells, although only when tissue-culture medium was replaced by Krebs-Ringer bicarbonate buffer. At 500 mu M AICAriboside, ZMP remained undetectable in cardiomyocytes, but reached 0.65 mM in erythrocytes and 5 mM in FTO-2B cells. In the latter, AICAriboside provoked up to 2-fold elevations of glucose 6-phosphate and fructose 6-phosphate, accompanied by a decrease in fructose 1,6-bisphosphate. This indicated inhibition of 6-phosphofructo-1-kinase (PFK-1). Accordingly, in FTO-2B cell-free extracts, the activity of PFK-1, measured under physiological conditions, was inhibited by approx. 70% by 5 mM ZMP. ZMP had a less pronounced effect on the activity of PFK-1 in normal rat liver; it did not influence the activity of PFK-1 in rat muscle, rabbit heart and human erythrocytes. It is concluded that the inhibitory effect of AICAriboside on glycolysis is dependent on both (1) the capacity of the cells to accumulate ZMP and (2) the presence of target enzymes which are sensitive to ZMP

cAMP-dependent binding of a trans-acting factor to the thyroglobulin promoter

We have investigated the interaction of a nuclear factor(s) with the promoter region of the thyroglobulin (Tg) gene, which is only expressed in differentiated thyroid cells under the positive control of the pituitary hormone thyrotropin (TSH) via a cAMP-dependent pathway. Using the mobility shift assay, we first demonstrated that a thyroid nuclear factor interacts with a short segment of 60 bp (-136 - -77) which is conserved among species in the regulatory region of the Tg gene. A specific binding site was then localized in a subfragment of 20 bp located between -126 bp and -107 bp relative to the transcription initiation site. The corresponding nuclear factor is absent in a tissue which does not express the Tg gene. This factor differs from previously identified factors shown to mediate a direct cAMP response since the observed binding is neither competed out by the cAMP responsive element (CRE) nor by the activator protein 2 (AP2) binding site. This trans-acting factor represents a new candidate intermediate in the regulation of transcription by a cAMP dependent mechanism.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Towards the development of an effective vaccine against malignant catarrhal fever

peer reviewedAlcelaphine herpesvirus 1 (AlHV-1) persists in wildebeest asymptomatically but induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease upon transmission to several ruminants, including cattle. The significant socio-economic impact of MCF in East-Africa urges for the development of new vaccination strategies. We have previously shown using the experimental rabbit model that the latency-associated gene ORF73 was essential for MCF induction while a ORF73-deficient (ORF73−) strain C500 could induce sterile immunity against a wild-type (WT) challenge. In the present study, we first infected 4-month-old calves with WT or ORF73− virus. Intranasal infection with the WT virus induced typical MCF clinical signs and lesions. However, ORF73− virus did not cause any clinical sign but induced a complete protection against an intranasal challenge with the WT virus. These results were encouraging for future prospects of vaccination against MCF. However, AlHV-1 is highly cell-associated and viral titres remain low, potentially hampering effective vaccine production. Interestingly, attenuated strain WC11 of AlHV-1 displays increased viral growth and cell-free infectious particles. Whole-genome sequencing of strain WC11 revealed few genomic changes including full deletion of the gene A7. A7 is a positional homolog of Epstein-Barr virus (EBV) BZLF2 encoding the C-type lectin-like glycoprotein gp42. Gp42 is expressed in the envelope of EBV virions and mediates entry into B cells. Hence, we used the C500 BAC clone to generate an A7STOP recombinant strain. We observed that a lack of A7 expression resulted in significant increased viral growth in fibroblasts in vitro. Also, the plaque size over time and the morphology of the plaques were modified in absence of A7. Finally, infection of rabbits demonstrated that A7 is essential for the development of MCF. In conclusion, the lack of A7 significantly alters the replication of the virus in vitro and the development of MCF in rabbits. Thus, joined impairments of A7 and ORF73 could lead to an optimized vaccine