Human cytomegalovirus infection is associated with increased expression of the lissencephaly gene PAFAH1B1 encoding LIS1 in neural stem cells and congenitally infected brains

peer reviewedCongenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these ndings, western blotting and immunouorescence analyses conrmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our ndings. Of note, PAFAH1B1 mutations (resulting in either haploinsufciency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufciency cause classic lissencephaly. Taken together, our ndings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infectio

Physiopathologie de l'infection par le cytomégalovirus sur les progéniteurs neuraux humains

Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1 % of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses. We investigated the outcome of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARg, a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARg levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARg agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARg activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARg was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV infected NSCs with the PPARg inhibitor T0070907 restored a normal rate of differentiation. The role of PPARg in the disease phenotype was strongly supported by the immunodetection of nuclear PPARg in brain germinative zones of congenitally infected fetuses (N=20), but not in control samples. We also identified LIS1 as one of the target genes for PPAR??in the infected brain. Levels of LIS1, the gene of classical lissencephaly, were strongly increased in infected NSC, presumably resulting from increased PPAR? activity. The relevance of this finding was further supported by our demonstration of a massive increase in the immunodetection in LIS1 fetal brains congenitally infected with HCMV (N = 6), relative to control cases (N = 3). Indeed, it is well known that overexpression of LIS1 is responsible for significant abnormalities of neural migration and development of a lissencephaly-like phenotype. Altogether, our findings reveal a key role for PPARg in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARg gene targets in the infected brain.L'infection congénitale par le cytomégalovirus humain (HCMV) est la première cause de séquelles acquises du système nerveux central (CNS). Elle est responsable de surdités neurosensorielles, de paralysies cérébrales ou d'anomalies neuro-développementales graves (0,1% des naissances) telles que des microcéphalies ou des anomalies de gyration. Pour étudier les effets de l'infection par le HCMV sur le développement cérébral, nous utilisons des cellules souches neurales (NSC) humaines dérivées de cellules souches embryonnaires (ES), ainsi que des coupes histologiques de cerveaux fœtaux infectés. Notre travail a porté sur l'analyse des conséquences de l'infection sur un facteur de transcription essentiel lors du développement cérébral, le Peroxisome Proliferator-Activated Receptor gamma (PPARg). Nous avons démontré que l'infection par le HCMV diminuait la neuronogénèse, en association avec une augmentation des niveaux d'expression et d'activité de PPARg. En accord avec ces résultats, nous avons montré que le niveau d'expression de l'acide 9-hydroxyoctadecadienoique (9-HODE), un agoniste connu de PPARg était augmenté dans les NSC infectées. En outre, l'ajout de 9-HODE dans les NSC reproduit l'effet de l'infection sur PPARg conduisant à une augmentation du nombre de cellules positives pour l'antigène viral IE parmi les NSC infectées. De plus, nous avons démontré que : (1) l'activation pharmacologique ou l'expression ectopique de PPARg suffisent pour perturber la neuronogénèse de NSC non infectées ; (2) le traitement de NSC non infectées par le 9-HODE diminue la différenciation des NSC ; (3) le traitement de NSC infectées par du T0070907, un inhibiteur de PPARg restaure un taux normal de différenciation. Le rôle crucial de PPARg dans les pathologies fœtales liées à l'infection a été souligné par la mise en évidence de sa translocation nucléaire au sein des zones germinatives de cerveaux fœtaux infectés congénitalement par le HCMV (N=20), mais pas dans les cas contrôles. Nous avons également identifié un des gènes cibles de PPARg dans le cerveau infecté: LIS1, le gène de la lissencéphalie classique, dont l'expression est également augmentée dans les NSC infectées, de façon dépendante de l'activité de PPARg. Nous avons mis en évidence que l'expression de LIS1 était augmentée de façon massive dans les cerveaux fœtaux infectés congénitalement par le HCMV (N=6) par rapport aux cas contrôles (N=3). Ceci pourrait jouer un rôle central dans la physiopathologie, car il est connu que toute perturbation de l'expression de LIS1 conduit à des anomalies importantes de la migration neurale et au développement d'un phénotype dit "lissencephaly-like". L'ensemble de nos données révèle le rôle clé de PPARg dans la neuronogénèse et la pathophysiologie de l'infection congénitale par le HCMV. Elles ouvrent la voie à une meilleure compréhension des mécanismes régissant les phénotypes pathologiques, notamment concernant le rôle de LIS1 dans les anomalies de la migration neurale

Molecular physiopathology of cytomegalovirus-infected human neural progenitors

L'infection congénitale par le cytomégalovirus humain (HCMV) est la première cause de séquelles acquises du système nerveux central (CNS). Elle est responsable de surdités neurosensorielles, de paralysies cérébrales ou d'anomalies neuro-développementales graves (0,1% des naissances) telles que des microcéphalies ou des anomalies de gyration. Pour étudier les effets de l'infection par le HCMV sur le développement cérébral, nous utilisons des cellules souches neurales (NSC) humaines dérivées de cellules souches embryonnaires (ES), ainsi que des coupes histologiques de cerveaux fœtaux infectés. Notre travail a porté sur l'analyse des conséquences de l'infection sur un facteur de transcription essentiel lors du développement cérébral, le Peroxisome Proliferator-Activated Receptor gamma (PPARg). Nous avons démontré que l'infection par le HCMV diminuait la neuronogénèse, en association avec une augmentation des niveaux d'expression et d'activité de PPARg. En accord avec ces résultats, nous avons montré que le niveau d'expression de l'acide 9-hydroxyoctadecadienoique (9-HODE), un agoniste connu de PPARg était augmenté dans les NSC infectées. En outre, l'ajout de 9-HODE dans les NSC reproduit l'effet de l'infection sur PPARg conduisant à une augmentation du nombre de cellules positives pour l'antigène viral IE parmi les NSC infectées. De plus, nous avons démontré que : (1) l'activation pharmacologique ou l'expression ectopique de PPARg suffisent pour perturber la neuronogénèse de NSC non infectées ; (2) le traitement de NSC non infectées par le 9-HODE diminue la différenciation des NSC ; (3) le traitement de NSC infectées par du T0070907, un inhibiteur de PPARg restaure un taux normal de différenciation. Le rôle crucial de PPARg dans les pathologies fœtales liées à l'infection a été souligné par la mise en évidence de sa translocation nucléaire au sein des zones germinatives de cerveaux fœtaux infectés congénitalement par le HCMV (N=20), mais pas dans les cas contrôles. Nous avons également identifié un des gènes cibles de PPARg dans le cerveau infecté: LIS1, le gène de la lissencéphalie classique, dont l'expression est également augmentée dans les NSC infectées, de façon dépendante de l'activité de PPARg. Nous avons mis en évidence que l'expression de LIS1 était augmentée de façon massive dans les cerveaux fœtaux infectés congénitalement par le HCMV (N=6) par rapport aux cas contrôles (N=3). Ceci pourrait jouer un rôle central dans la physiopathologie, car il est connu que toute perturbation de l'expression de LIS1 conduit à des anomalies importantes de la migration neurale et au développement d'un phénotype dit "lissencephaly-like". L'ensemble de nos données révèle le rôle clé de PPARg dans la neuronogénèse et la pathophysiologie de l'infection congénitale par le HCMV. Elles ouvrent la voie à une meilleure compréhension des mécanismes régissant les phénotypes pathologiques, notamment concernant le rôle de LIS1 dans les anomalies de la migration neurale.Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1 % of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses. We investigated the outcome of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARg, a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARg levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARg agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARg activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARg was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV infected NSCs with the PPARg inhibitor T0070907 restored a normal rate of differentiation. The role of PPARg in the disease phenotype was strongly supported by the immunodetection of nuclear PPARg in brain germinative zones of congenitally infected fetuses (N=20), but not in control samples. We also identified LIS1 as one of the target genes for PPAR??in the infected brain. Levels of LIS1, the gene of classical lissencephaly, were strongly increased in infected NSC, presumably resulting from increased PPAR? activity. The relevance of this finding was further supported by our demonstration of a massive increase in the immunodetection in LIS1 fetal brains congenitally infected with HCMV (N = 6), relative to control cases (N = 3). Indeed, it is well known that overexpression of LIS1 is responsible for significant abnormalities of neural migration and development of a lissencephaly-like phenotype. Altogether, our findings reveal a key role for PPARg in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARg gene targets in the infected brain

Cytomegalovirus Infection Triggers the Secretion of the PPARγ Agonists 15-Hydroxyeicosatetraenoic Acid (15-HETE) and 13-Hydroxyoctadecadienoic Acid (13-HODE) in Human Cytotrophoblasts and Placental Cultures

<div><p>Introduction</p><p>Congenital infection by human cytomegalovirus (HCMV) is a leading cause of congenital abnormalities of the central nervous system. Placenta infection by HCMV allows for viral spread to fetus and may result in intrauterine growth restriction, preeclampsia-like symptoms, or miscarriages. We previously reported that HCMV activates peroxisome proliferator-activated receptor gamma (PPARγ) for its own replication in cytotrophoblasts. Here, we investigated the molecular bases of PPARγ activation in infected cytotrophoblasts.</p><p>Results</p><p>We show that onboarded cPLA₂ carried by HCMV particles is required for effective PPARγ activation in infected HIPEC cytotrophoblasts, and for the resulting inhibition of cell migration. Natural PPARγ agonists are generated by PLA₂ driven oxidization of linoleic and arachidonic acids. Therefore, using HPLC coupled with mass spectrometry, we disclosed that cellular and secreted levels of 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE) were significantly increased in and from HIPEC cytotrophoblasts at soon as 6 hours post infection. 13-HODE treatment of uninfected HIPEC recapitulated the effect of infection (PPARγ activation, migration impairment). We found that infection of histocultures of normal, first-term, human placental explants resulted in significantly increased levels of secreted 15-HETE and 13-HODE.</p><p>Conclusion</p><p>Our findings reveal that 15-HETE and 13-HODE could be new pathogenic effectors of HCMV congenital infection They provide a new insight about the pathogenesis of congenital infection by HCMV.</p></div

Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles.

International audienceDespite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness

Name and abbreviation of each metabolite quantified in our study.

<p>Name and abbreviation of each metabolite quantified in our study.</p

Onboarded cPLA₂ is required for PPARγ activation and inhibition of cytotrophoblast migration.

<p>(A) TLC analysis of bodipy-phosphatidylcholine (B-PC) incubated in the presence of buffer (mock), purified PLA₂ (PLA2), native HCMV particles (HCMV), HCMV particles pre-treated by MAFP (HCMV+MAFP) or UV-irradiated (HCMV-UV). (B) PPARγ activity luciferase assay performed with HIPEC in various conditions, using a reporter plasmid responsive to PPARγ (pGL4-PPRE) or a control plasmid (PGL4), 48 h pi or post treatment onset. HCMV: HIPEC infected by live HCMV particles; vi: the viral inoculum was treated beforehand by MAFP; cm: control with 50 nM MAFP in the culture medium; GW: PPARγ inhibitor GW9662; Rosi: PPARγ agonist rosiglitazone. RLU: relative luciferase units; **: p < 0.01 (Kruskal-Wallis test). The assay was repeated twice.(C) Wound-healing assays. HCMV+MAFP: the viral inoculum was treated beforehand by MAFP; HCMV+GW: the HIPEC were infected in the presence of GW9662; HCMV-UV: the viral inoculum was UV-irradiated. Results are expressed as the percent variation relative to the control. The figure shows results of a representative experiment, out of two independent experiments, each comprising triplicate measures. NI: non-infected.</p

Retention time (RT), line equation, coefficient of determination (r2), maximum percent residual, limit of detection (LOD), limit of quantification (LOQ) and attributed internal standard (IS) for each metabolite.

<p>Retention time (RT), line equation, coefficient of determination (r2), maximum percent residual, limit of detection (LOD), limit of quantification (LOQ) and attributed internal standard (IS) for each metabolite.</p

Proposed model of PPARγ activation in HCMV infection of placental cells.

<p>HCMV particles (HCMV) carry onboarded cPLA2 (oPLA2) which catalyses linoleic acid (LA) and arachidonic acid (AA) release from host membrane phospholipids (PL). AA and LA undergo oxidization driven by 15-lipoxygenase (15-LOX), which generates 15HETE and 9-HODE, respectively. 15HETE and 9-HODE are activating ligands of PPARγ, which dimerizes with RXR to regulate the expression of the host and virus genomes, resulting in impaired migration abilities in vitro, and enhanced IE transcription and viral replication. M: cell membrane, C: cytoplasm, N: nucleus.</p

Increased amounts of 15-HETE and 13-HODE secreted by from early placenta explants infected by HCMV.

<p>(A) LC-MS/MS analysis of the amounts of PUFA-derived lipids secreted from histocultures from 12 first trimester placentas either infected by HCMV (CMV) or uninfected (NI). *: p < 0.05; **: p < 0.01 (Wilcoxon test). (B) Immunostaining analysis of HCMV antigen IE in infected (HCMV) or uninfected (NI) placental explants cultured ex vivo. Representative views are shown. Note the positive, nuclear IE staining in the cytotrophoblast layer (ST) surrounding the villus stroma (S).</p