Implications des particules défectives et de la protéine HBSP du virus de l'Hépatite B dans la pathogenèse virale

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Représentation de la convection dans les modèles globaux et régionaux (concepts, équations, études de cas)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade

International audienceSyncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. Importance: Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the features of a Syncytin: it is specifically expressed in the placenta of the woodchuck Marmota monax, at the level of cells fusing into a syncytium; it can trigger cell-cell and virus-cell fusion ex vivo; and it has been conserved for >25 million years of evolution, suggesting an essential role in its host physiology. Remarkably, syncytin-Mar1 is unrelated to all other Syncytin genes identified thus far in mammals (primates, muroids, carnivores, and ruminants). These results extend the range of retroviral envelope gene "domestication" in mammals and show that these events occurred independently, on multiple occasions during evolution to improve placental development in a process of convergent evolution

Strong association between a new marker of hemolysis and glomerulopathy in sickle cell anemia.

International audienceTo perform a precise evaluation of the hemolytic status of patients with sickle cell anemia (SCA), advanced red blood cell parameters provided by the last generation analyzers were investigated in a series of SCA patients. The search for precise markers of hemolysis was performed to identify if patients so exposed develop organic complications related to a postulated hemolysis-linked endothelial dysfunction. Red blood cell survival was evaluated by the ratio between mature red blood cell (RBC) and reticulocyte (RET) hemoglobin (RBC-Hb/RET-Hb). In comparison with serum lactate dehydrogenase (LDH) and total bilirubin, the log (RBC-Hb/RET-Hb) was identified as the most discriminant hematological parameter to evaluate hemolysis. Furthermore, by combining this parameter with LDH, we defined a composite variable, which we called CVar, that is highly correlated with albuminuria and might constitute a powerful new marker of risk for this complication

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice

International audienceSyncytins are envelope genes from endogenous retroviruses, " captured " for a role in pla-centation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncy-tiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucle-ated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20–40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals

Phenotype of 12 week old SynB^-/- and WT mice.

<p>(<i>A</i>) Body weight. (<i>B</i>) Representative WT and SynB^-/- male (m) and female (f) mice. (<i>C</i>) Muscle mass of three skeletal muscles located in the hindlimb, TA (<i>Tibialis Anterior</i>), SOL (<i>Soleus</i>), EDL (<i>Extensor Digitorum Longus</i>). (<i>D</i>) Mass of heart and kidney, length of tibia and femur. Data are the mean ± SEM (number of mice per sex and per genotype: 13–17 in <i>A</i>, 5–11 in <i>C</i>, 4–7 in <i>D</i>; * p<0.05, ** p<0.01, Mann and Whitney test).</p

Impact of murine syncytins and myomaker overexpression on myoblast cell-cell fusion <i>ex vivo</i>.

<p>C2C12 myoblasts were co-transfected with an empty (cont), syncytin-A (synA),–B (synB) or myomaker expression vector, supplemented with a GFP expression vector. Differentiation was induced 2 days later (D0). Cells were fixed at D0 or after four days of differentiation (D4) in 4% PFA, stained with an anti-desmin antibody (muscle cell marker), and nuclei were counterstained with DAPI. (<i>A</i>) The fusion index at D0 was calculated as the percentage of nuclei in GFP-positive cells with at least 2 nuclei. (<i>B</i>) The fusion index at D4 of differentiation was calculated as the percentage of nuclei in desmin-positive cells with at least 2 nuclei. (<i>C</i>) Representative images of desmin- and DAPI-labelled cells transfected with the control, syncytin-A, syncytin-B or myomaker vectors at D4. Data are the mean ± SEM (three independent experiments; * p<0.05, ** p<0.01, Student’s t-test).</p

Murine syncytin expression and impact of their knockdown on myoblast cell-cell fusion <i>ex vivo</i>.

<p>Primary myoblast cells were transfected with control siRNA (si cont) or siRNAs specifically designed against <i>syncytin-B</i> (si synB),–<i>A</i> (si synA) or myomaker (si myomaker), and differentiation was induced 2 days later. Following two days of differentiation, cells were fixed in 4% PFA, stained with an anti-desmin antibody (muscle cell marker), and nuclei were counterstained with DAPI. (<i>A</i>) Level of expression of <i>syncytin-B</i> and <i>-A</i> (arbitrary units, A. U.) during myoblast fusion in murine primary myoblasts, either actively proliferating (P: proliferating non confluent cells, D0: proliferative confluent cells), or in differentiation and fusion (D2 to D4), as analyzed by quantitative RT-PCR. All quantifications were normalized by RPL0, and the fold-change in gene expression expressed relative to the values of proliferating myoblasts, arbitrarily settled to one. (<i>B</i>) Efficiency of siRNA knockdown measured by quantitative RT-PCR. (<i>C</i>) Representative images of desmin- and DAPI-labelled myoblast cells transfected with control, <i>syncytin-B</i> or <i>syncytin-A</i> siRNAs. Representative myotubes are indicated by white arrows whereas white triangles correspond to mononucleated myoblast cells. (<i>D</i>) Fusion index (number of nuclei in cells with ≥2 nuclei per total number of nuclei). (<i>E</i>) Nuclei distribution per myotube. Data are the mean ± SEM (four independent experiments; * p<0.05, ** p<0.01, Mann and Whitney test).</p