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Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

Scordel C, Huttin A, Cochet-Bernoin M, Szelechowski M, Poulet A, Richardson J, Benchoua A, Gonzalez-Dunia D, Eloit M, Coulpier M - PLoS Pathog. (2015)

Bottom Line: Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis.We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis.Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1161, Maisons-Alfort, France; ANSES, UMR Virologie, Maisons-Alfort, France; Université Paris-Est, Ecole Nationale Vétérinaire d'Alfort, UMR Virologie, Maisons-Alfort, France.

ABSTRACT
It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.

No MeSH data available.


Related in: MedlinePlus

Lentiviral transduction and establishment of transgenic hNPCs.(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ns, non-significant (p > 0.5). Scale bar, 20 μm.
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ppat.1004859.g001: Lentiviral transduction and establishment of transgenic hNPCs.(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ns, non-significant (p > 0.5). Scale bar, 20 μm.

Mentions: To identify the viral proteins responsible for BDV-induced alteration of neurogenesis, we chose to study the phosphoprotein and the X protein (henceforth referred to P and X), as they have been previously described to interact with many cellular pathways in neural cells. We thus established transgenic populations of hNPCs expressing either bdv-p or bdv-x gene, or as a control, the gfp gene. At 10 weeks of expansion, adherent hNPCs were transduced with highly purified lentiviral vectors encoding the different genes of interest and amplified for a further 2 to 4 week period before epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) withdrawal and analysis of the effect of the transgene on neural differentiation (Fig 1A). The level of expression of gfp, bdv-p or bdv-x genes in transduced hNPCs was first verified. In undifferentiated cells (day 0) more than 90% of hNPCs were GFP- (91.28 +/- 2.9%) or P-positive (96 +/- 2%) and approximately 80% were X-positive (79 +/- 4.3%), as determined by enumeration of cells labeled with antibodies directed against the P or X viral proteins (Fig 1B and 1C). A similar percentage of transgene-expressing hNPCs was observed after 14 days of differentiation (87.6 +/- 1.1%, 96.2 +/- 1.5%, and 79.9 +/- 3.3% of cells expressing the gfp, bdv-p or bdv-x gene, respectively) (Fig 1B and 1C). Thus, high efficiencies of transduction were obtained with lentiviral vectors and differentiation did not affect gfp, bdv-p and bdv-x gene expression. In keeping with the presence of a nuclear localization signal [33], P was strictly nuclear, whereas X, which contains both a nuclear localization signal and a short helix responsible for mitochondrial targeting [34,35], was observed in both nuclear and cytoplasmic structures, in undifferentiated and differentiated hNPCs (Fig 1B).


Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

Scordel C, Huttin A, Cochet-Bernoin M, Szelechowski M, Poulet A, Richardson J, Benchoua A, Gonzalez-Dunia D, Eloit M, Coulpier M - PLoS Pathog. (2015)

Lentiviral transduction and establishment of transgenic hNPCs.(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ns, non-significant (p > 0.5). Scale bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414417&req=5

ppat.1004859.g001: Lentiviral transduction and establishment of transgenic hNPCs.(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ns, non-significant (p > 0.5). Scale bar, 20 μm.
Mentions: To identify the viral proteins responsible for BDV-induced alteration of neurogenesis, we chose to study the phosphoprotein and the X protein (henceforth referred to P and X), as they have been previously described to interact with many cellular pathways in neural cells. We thus established transgenic populations of hNPCs expressing either bdv-p or bdv-x gene, or as a control, the gfp gene. At 10 weeks of expansion, adherent hNPCs were transduced with highly purified lentiviral vectors encoding the different genes of interest and amplified for a further 2 to 4 week period before epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) withdrawal and analysis of the effect of the transgene on neural differentiation (Fig 1A). The level of expression of gfp, bdv-p or bdv-x genes in transduced hNPCs was first verified. In undifferentiated cells (day 0) more than 90% of hNPCs were GFP- (91.28 +/- 2.9%) or P-positive (96 +/- 2%) and approximately 80% were X-positive (79 +/- 4.3%), as determined by enumeration of cells labeled with antibodies directed against the P or X viral proteins (Fig 1B and 1C). A similar percentage of transgene-expressing hNPCs was observed after 14 days of differentiation (87.6 +/- 1.1%, 96.2 +/- 1.5%, and 79.9 +/- 3.3% of cells expressing the gfp, bdv-p or bdv-x gene, respectively) (Fig 1B and 1C). Thus, high efficiencies of transduction were obtained with lentiviral vectors and differentiation did not affect gfp, bdv-p and bdv-x gene expression. In keeping with the presence of a nuclear localization signal [33], P was strictly nuclear, whereas X, which contains both a nuclear localization signal and a short helix responsible for mitochondrial targeting [34,35], was observed in both nuclear and cytoplasmic structures, in undifferentiated and differentiated hNPCs (Fig 1B).

Bottom Line: Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis.We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis.Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1161, Maisons-Alfort, France; ANSES, UMR Virologie, Maisons-Alfort, France; Université Paris-Est, Ecole Nationale Vétérinaire d'Alfort, UMR Virologie, Maisons-Alfort, France.

ABSTRACT
It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.

No MeSH data available.


Related in: MedlinePlus