Étude des mécanismes de l'intégration chromosomique des herpèsvirus humains 6A/B : caractérisation de la protéine précoce immédiate 1

Les herpèsvirus humains 6A/B (HHV-6A/B) sont deux virus ubiquitaires à l'échelle planétaire qui partagent 94% d'identité entre eux. Ces virus persistent tout au long de la vie de l'hôte en adoptant un état de latence. La latence d'HHV-6A/B est engendrée par l'intégration de leur génome entier aux télomères humains. Les télomères assurent la protection du génome cellulaire contre sa détérioration et dictent la durée de vie cellulaire. On estime que 1.1 % de la population mondiale possède la forme héritée du génome intégré d'HHV-6A/B (iciHHV-6A/B), ce qui signifie qu'environ 80 millions de personnes possèdent un génome d'HHV-6A/B dans un télomère de chaque cellule. Ceci signifie également que chez une personne iciHHV-6A/B, les virus peuvent se réactiver dans différents types de tissus cellulaires. Notamment, lors d'immunosuppression ou des traitements de chimiothérapie chez ces patients, la forme intégrée peut se réactiver et mène à de graves complications cliniques. Alors que les connaissances face à ces conséquences ont progressé, il demeure que les mécanismes fondamentaux utilisés par HHV-6A/B afin de s'intégrer aux télomères humains sont loin d'être élucidés. Les protéines précoces immédiates (IE) des herpèsvirus exercent plusieurs fonctions à des fins de réplications virales, dont celles d'être impliquées dans l'évasion du système immunitaire, dans la réponse aux dommages à l'ADN et dans le remodelage des protéines qui gouvernent les télomères. L'expression soutenue des protéines IE des herpèsvirus tout au long de leur infection afin d'établir la phase lytique et latente, font de ces protéines des protagonistes intéressantes dans la quête de la compréhension du déroulement de l'intégration d'HHV-6A/B. Les protéines IE s'associent à des corps nucléaires formés par la protéine PML (PML-NBs) et compromettent l'intégrité de ceux-ci afin de favoriser l'infection virale. Cependant, il a été documenté que lors de l'infection par HHV-6A/B, leur protéine IE, IE1 (IE1A et IE1B, respectivement), s'associe aux PML-NBs sans occasionner la dégradation ou la destruction de ceux-ci. D'un point de vue télomérique, les PML-NBs s'associent aux télomères endommagés. Notamment, les PML-NBs sont impliqués dans la recombinaison homologue des télomères. Dans le cadre de cette thèse, nous nous sommes intéressés à l'étude des mécanismes associés à l'intégration d'HHV-6A/B en portant une attention particulière à la caractérisation de la protéine IE1 d'HHV-6A/B (IE1A/B). Dans un premier temps, nous avons identifié les corps nucléaires de la protéine PML (PML-NBs) comme promoteurs de la SUMOylation d'IE1B. Ce résultat nous a conduits à identifier un motif d'interaction SUMO putatif d'IE1B, essentiel à la fois pour sa SUMOylation et pour son oligomérisation avec les PML-NB. Nous avons ensuite étudié le rôle des PML-NBs dans l'intégration d'HHV-6B et identifié que les cellules déficientes pour la protéine PML étaient moins susceptibles à l'intégration d'HHV-6B. Ces résultats corrèlent avec le résultat des PML-NBs qui influencent la localisation d'IE1B aux télomères. La deuxième étude est associée avec celle précédente dont l'objectif était de valider les résultats obtenus pour IE1B et HHV-6B avec IE1A et HHV-6A, dans un contexte de SUMOylation et d'absence de PML-NBs. Nous avons identifié des sites putatifs pour la SUMOylation d'IE1A, importants pour son oligomérisation. Par ailleurs, la localisation d'IE1A aux télomères et l'intégration d'HHV-6A furent influencées par la présence de PML-NBs. Ensemble, ces deux études ont mis en lumière l'importance de la SUMOylation pour l'oligomérisation d'IE1A/B, leur localisation aux PML-NBs et l'importance de ces derniers dans l'intégration d'HHV-6A/B. Lors de la troisième étude, nous nous sommes intéressés à étudier HHV-6A/B dans un contexte télomérique et ce, en nous penchant sur une protéine télomérique majeure, la protéine TRF2. Nous rapportons que la réplication de l'ADN d'HHV-6A/B augmente considérablement le nombre de répétitions télomériques dans les cellules infectées. En outre, nous démontrons que TRF2 se lient aux répétitions télomériques virales pendant l'infection et elle est importante pour l'intégration d'HHV-6A/B. La dernière étude de cette thèse avait l'objectif de caractériser la fonction d'IE1B dans un contexte de réparation de l'ADN. Nous avons mis en évidence qu'IE1B induit de l'instabilité génomique en inhibant la signalisation de la protéine détectrice de dommage de l'ADN, NBS1. Nous avons identifié deux régions protéiques d'IE1B responsables de l'interaction avec NBS1 et son inhibition. L'interaction/inhibition d'IE1B avec NBS1 mène à la diminution de la signalisation de dommage à l'ADN. Ces données corrèlent avec les évènements de différentes voies associées à la recombinaison homologue qui sont réduits en présence d'IE1B. Toutefois, NBS1 est importante pour l'intégration d'HHV-6B dans les cellules qui utilisent un mécanisme d'élongation des télomères basé sur la recombinaison homologue. Cette étude soulève la possibilité que d'autres protéines virales contrôlent l'action d'IE1B lors l'intégration afin de ne pas saturer l'instabilité génomique qui serait toxique à la survie cellulaire et virale. Dans l'ensemble, cette thèse identifie pour la première fois l'association de protéines cellulaires, PML, TRF2 et NBS1, dans l'intégration d'HHV-6A/B. Elle permet de mieux comprendre l'implication d'IE1B lors de l'infection d'HHV-6B et elle permet de percevoir une conséquence possible de l'iciHHV-6B. L'implication et l'importance d'IE1B dans l'instabilité génomique suggèrent qu'IE1B serait une cible thérapeutique pertinente dans le traitement des réactions d'HHV-6A/B.Human herpesviruses 6A/B (HHV-6A/B) are two ubiquitous viruses distributed worldwide that share 94% identity between them. These viruses persist throughout the host's life through a state of latency. The latency of HHV-6A/B is generated by integrating their entire genome into human telomeres. Telomeres protect the host's genome from its deterioration and dictate the cell lifespan. It is estimated that 1.1% of the world's population has the inherited form of the integrated HHV-6A/B genome (iciHHV-6A/B), meaning that about 80 million people have an HHV-6A/B genome in one telomere of each cell. In an iciHHV-6A/B+ individual, viruses can reactivate in different types of cell tissue. Particularly, during immunosuppression or chemotherapy treatments in these patients, the integrated form can reactivate and lead to serious clinical complications. While knowledge about these consequences has grown, the fact remains that the fundamental mechanisms used by HHV-6A/B to integrate at human telomeres are far from clear. The immediate early proteins (IE) of herpesviruses perform several functions for viral replication purposes, including those involved in evading the immune system, responding to DNA damage, and remodeling the proteins that govern telomeres. The sustained expression of the IE proteins of herpesviruses throughout their infection in order to establish the lytic and latent phase make these proteins interesting protagonists in the quest to a better understanding of the process leading to the integration of HHV-6A/B. The IE proteins associate with nuclear bodies formed by the PML protein (PML-NBs) to compromise their integrity and promote viral infection. However, it has been documented that upon infection of HHV-6A/B, their IE protein, IE1 (IE1A/B), associates with PML-NBs without causing their degradation or destruction. From a telomeric perspective, PML-NBs associate at damaged telomeres. In particular, the PML-NBs are involved in the homologous recombination of telomeres. As part of this thesis, we were interested in studying the mechanisms associated with the integration of HHV-6A/B with particular interest in the characterization of the IE1 protein of HHV-6A/B. We first identified the PML NBs as promoters of IE1B SUMOylation. This result led us to identify a putative SUMO interaction motif of IE1B that is essential for both its SUMOylation and for the oligomerization of IE1B with PML-NBs. We then investigated the role of PML-NBs in the integration of HHV-6B and identified that cells deficient for PML were less susceptible to HHV-6B integration. These results correlate with the result that PML-NBs influence the localization of IE1B to telomeres. The second study is associated with the previous one whose objective was to validate the results obtained for IE1B and HHV-6B with IE1A and HHV-6A, in a context of SUMOylation and absence of PML-NBs. We have identified putative sites for IE1A SUMOylation, important for its oligomerization. Furthermore, the localization of IE1A to telomeres and the integration of HHV-6A was influenced by the presence of PML-NBs. Together, these two studies highlighted the importance of SUMOylation for the oligomerization of IE1A/B, their localization to PML-NBs and the importance of these in the integration of HHV-6A/B. In the third study, we were interested in studying HHV-6A/B in a telomeric context by studying a major telomeric protein, the TRF2 protein. We report that the replication of HHV-6A/B DNA dramatically increases the number of telomeric repeats in infected cells. In addition, we demonstrate that TRF2 binds to viral telomeric repeats during infection and is important for the integration of HHV-6A/B. The last study of this thesis aimed to characterize the function of IE1B in the context of DNA repair. We have shown that IE1B induces genomic instability by inhibiting the function of the DNA damage sensor protein, NBS1. We have identified two protein regions of IE1B responsible for the interaction with NBS1 and its inhibition. Interaction/inhibition of IE1B with NBS1 leads to decreased DNA damage signaling. These data correlate with the events of different pathways associated with homologous recombination which are reduced in the presence of IE1B. However, NBS1 is important for the integration of HHV-6B into cells that use a mechanism of telomere elongation based on homologous recombination. This study raises the possibility that other viral proteins control the action of IE1B during integration to not saturate genomic instability that would be toxic to cell and viral survival. Taken together, this thesis identifies for the first time the association of cellular proteins, PML, TRF2 and NBS1, in the integration of HHV-6A/B. It provides a better understanding of the implication of IE1B in the infection of HHV-6B and in identifying a possible consequence of iciHHV-6B. The implication and importance of IE1B in genomic instability suggests that IE1B may be an interesting therapeutic target in the treatment of HHV-6B reactivations

Characterization of human herpesvirus 6A/B U94 as ATPase, helicase, exonuclease and DNA-binding proteins

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integratio

Influence of Pre-Training Predator Stress on the Expression of c-fos mRNA in the Hippocampus, Amygdala, and Striatum Following Long-Term Spatial Memory Retrieval

We have studied the influence of pre-training psychological stress on the expression of c-fos mRNA following long-term spatial memory retrieval. Rats were trained to learn the location of a hidden escape platform in the radial-arm water maze, and then their memory for the platform location was assessed 24 h later. Rat brains were extracted 30 min after the 24-h memory test trial for analysis of c-fos mRNA. Four groups were tested: (1) Rats given standard training (Standard); (2) Rats given cat exposure (Predator Stress) 30 min prior to training (Pre-Training Stress); (3) Rats given water exposure only (Water Yoked); and (4) Rats given no water exposure (Home Cage). The Standard trained group exhibited excellent 24 h memory which was accompanied by increased c-fos mRNA in the dorsal hippocampus and basolateral amygdala (BLA). The Water Yoked group exhibited no increase in c-fos mRNA in any brain region. Rats in the Pre-Training Stress group were classified into two subgroups: good and bad memory performers. Neither of the two Pre-Training Stress subgroups exhibited a significant change in c-fos mRNA expression in the dorsal hippocampus or BLA. Instead, stressed rats with good memory exhibited significantly greater c-fos mRNA expression in the dorsolateral striatum (DLS) compared to stressed rats with bad memory. This finding suggests that stressed rats with good memory used their DLS to generate a non-spatial (cue-based) strategy to learn and subsequently retrieve the memory of the platform location. Collectively, these findings provide evidence at a molecular level for the involvement of the hippocampus and BLA in the retrieval of spatial memory and contribute novel observations on the influence of pre-training stress in activating the DLS in response to long-term memory retrieval

Welfare issues and potential solutions for laying hens in free range and organic production systems: A review based on literature and interviews

In free-range and organic production systems, hens can make choices according to their needs and desires, which is in accordance with welfare definitions. Nonetheless, health and behavioral problems are also encountered in these systems. The aim of this article was to identify welfare challenges observed in these production systems in the EU and the most promising solutions to overcome these challenges. It is based on a review of published literature and research projects complemented by interviews with experts. We selected EU specific information for welfare problems, however, the selected literature regarding solutions is global. Free range use may increase the risk of infection by some bacteria, viruses and parasites. Preventive methods include avoiding contamination thanks to biosecurity measures and strengthening animals' natural defenses against these diseases which can be based on nutritional means with new diet components such as insect-derived products, probiotics and prebiotics. Phytotherapy and aromatherapy can be used as preventive and curative medicine and vaccines as alternatives to antibiotics and pesticides. Bone quality in pullets and hens prevents keel deviations and is favored by exercise in the outdoor range. Free range use also lead to higher exposure to variable weather conditions and predators, therefore shadow, fences and guard animals can be used to prevent heat stress and predation respectively. Granting a free range provides opportunities for the expression of many behaviors and yet many hens usually stay close to the house. Providing the birds with trees, shelters or attractive plants can increase range use. Small flock sizes, early experiences of enrichment and personality traits have also been found to enhance range use. Severe feather pecking can occur in free range production systems, although flocks using the outdoor area have better plumage than indoors. While many prevention strategies are facilitated in free range systems, the influence of genetics, prenatal and nutritional factors in free range hens still need to be investigated. This review provides information about practices that have been tested or still need to be explored and this information can be used by stakeholders and researchers to help them evaluate the applicability of these solutions for welfare improvement

The Pristine Survey – VI. The first three years of medium-resolution follow-up spectroscopy of Pristine EMP star candidates★

We present the results of a 3-year long, medium-resolution spectroscopic campaign aimed at identifying very metal-poor stars from candidates selected with the CaHK, metallicity-sensitive Pristine survey. The catalogue consists of a total of 1007 stars, and includes 146 rediscoveries of metal-poor stars already presented in previous surveys, 707 new very metal-poor stars with [Fe/H]<−2.0⁠, and 95 new extremely metal-poor stars with [Fe/H]<−3.0⁠. We provide a spectroscopic [Fe/H] for every star in the catalogue, and [C/Fe] measurements for a subset of the stars (10% with [Fe/H]<−3 and 24% with −3<[Fe/H]<−2⁠) for which a carbon determination is possible, contingent mainly on the carbon abundance, effective temperature and S/N of the stellar spectra. We find an average carbon enhancement fraction ([C/Fe] ≥ +0.7) of 41 ± 4% for stars with −3<[Fe/H]<−2 and 58 ± 14% for stars with [Fe/H]<−3⁠, and report updated success rates for the Pristine survey of 56 % and 23 % to recover stars with [Fe/H]<−2.5 and [Fe/H]<−3⁠, respectively. Finally, we discuss the current status of the survey and its preparation for providing targets to upcoming multi-object spectroscopic surveys such as WEAVE

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data