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Nipah virus infection and glycoprotein targeting in endothelial cells.

Erbar S, Maisner A - Virol. J. (2010)

Bottom Line: Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all.We conclude that the NiV glycoprotein distribution is responsible for lateral virus spread in both, epithelial and endothelial cell monolayers.However, the prerequisites for correct protein targeting differ markedly in the two polarized cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, Philipps University of Marburg, Germany.

ABSTRACT

Background: The highly pathogenic Nipah virus (NiV) causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB) and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells.

Results: As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar), not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting.

Conclusion: We conclude that the NiV glycoprotein distribution is responsible for lateral virus spread in both, epithelial and endothelial cell monolayers. However, the prerequisites for correct protein targeting differ markedly in the two polarized cell types.

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Related in: MedlinePlus

NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.
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Figure 1: NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.

Mentions: Primary brain capillary endothelial cells have the closest resemblance to brain endothelia in vivo and exhibit excellent characteristics of the BBB at early passages. We therefore performed our initial studies in primary brain microvascular endothelial cells (PBMEC) freshly isolated from pig brains. Non-passaged PBMEC were cultivated on fibronectin-coated transwell filter supports with a pore size of 1 μm until full confluency and polarization were reached (6 days). Then, cells were infected with NiV at a multiplicity of infection (m.o.i.) of 0.5 under BSL-4 conditions. At 24 h p.i., the samples were inactivated with 4% PFA for 48 h. Virus-positive cells were immunostained with a NiV-specific polyclonal guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. To visualize cell junctions, cells were permeabilized and VE-cadherin was co-stained with a specific monoclonal antibody and an AlexaFluor 488-conjugated secondary antibody. In agreement with the in vivo studies in NiV-infected pigs [16,17], NiV infection caused a foci formation in the cultured primary porcine brain endothelia (Figure 1A). As observed previously in epithelial cells [18], cell junction staining was lost within the NiV-positive foci indicating a virus-induced cell-to-cell fusion (syncytia formation). Because brain microvascular endothelial cells as a major component of the BBB develop complete intercellular tight junction complexes, have no fenestrations, and are scarce of transcytotic vesicles [19,20], entry of most molecules from blood to brain parenchyma is impeded. To investigate the effect of NiV infection on the transendothelial permeability, we used a peroxidase (HRP) leak assay [21]. PBMEC were seeded on filter supports and were infected with NiV. At 6 h and 24 h p.i., the culture medium in the apical filter chamber was replaced by medium containing 5 μg HRP per ml. Apical-to-basolateral HRP passage through the endothelial monolayer was monitored over the time and is given as the relative HRP passage normalized to the HRP passage through mock-infected cells. As shown in Figure 1B, we did not observe a significant increase in HRP permeability in PBMEC infected for 6 h, a time point of infection at which virus replication is already ongoing but newly synthesized viral proteins and syncytia formation were not yet detectable (data not shown). In contrast, at 24 h p.i., when syncytia formation and the accompanying cytopathic effect were clearly detectable (Figure 1A), we found an about 2-fold increase in transendothelial permeability (Figure 1B; NiV 24 h p.i.). These findings indicate that NiV infection does not drastically influence endothelial permeability and barrier functions at early time points of infection. Only after productive replication and pronounced syncytia formation interfering with cell monolayer integrity, transendothelial permeability is increased.


Nipah virus infection and glycoprotein targeting in endothelial cells.

Erbar S, Maisner A - Virol. J. (2010)

NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991316&req=5

Figure 1: NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.
Mentions: Primary brain capillary endothelial cells have the closest resemblance to brain endothelia in vivo and exhibit excellent characteristics of the BBB at early passages. We therefore performed our initial studies in primary brain microvascular endothelial cells (PBMEC) freshly isolated from pig brains. Non-passaged PBMEC were cultivated on fibronectin-coated transwell filter supports with a pore size of 1 μm until full confluency and polarization were reached (6 days). Then, cells were infected with NiV at a multiplicity of infection (m.o.i.) of 0.5 under BSL-4 conditions. At 24 h p.i., the samples were inactivated with 4% PFA for 48 h. Virus-positive cells were immunostained with a NiV-specific polyclonal guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. To visualize cell junctions, cells were permeabilized and VE-cadherin was co-stained with a specific monoclonal antibody and an AlexaFluor 488-conjugated secondary antibody. In agreement with the in vivo studies in NiV-infected pigs [16,17], NiV infection caused a foci formation in the cultured primary porcine brain endothelia (Figure 1A). As observed previously in epithelial cells [18], cell junction staining was lost within the NiV-positive foci indicating a virus-induced cell-to-cell fusion (syncytia formation). Because brain microvascular endothelial cells as a major component of the BBB develop complete intercellular tight junction complexes, have no fenestrations, and are scarce of transcytotic vesicles [19,20], entry of most molecules from blood to brain parenchyma is impeded. To investigate the effect of NiV infection on the transendothelial permeability, we used a peroxidase (HRP) leak assay [21]. PBMEC were seeded on filter supports and were infected with NiV. At 6 h and 24 h p.i., the culture medium in the apical filter chamber was replaced by medium containing 5 μg HRP per ml. Apical-to-basolateral HRP passage through the endothelial monolayer was monitored over the time and is given as the relative HRP passage normalized to the HRP passage through mock-infected cells. As shown in Figure 1B, we did not observe a significant increase in HRP permeability in PBMEC infected for 6 h, a time point of infection at which virus replication is already ongoing but newly synthesized viral proteins and syncytia formation were not yet detectable (data not shown). In contrast, at 24 h p.i., when syncytia formation and the accompanying cytopathic effect were clearly detectable (Figure 1A), we found an about 2-fold increase in transendothelial permeability (Figure 1B; NiV 24 h p.i.). These findings indicate that NiV infection does not drastically influence endothelial permeability and barrier functions at early time points of infection. Only after productive replication and pronounced syncytia formation interfering with cell monolayer integrity, transendothelial permeability is increased.

Bottom Line: Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all.We conclude that the NiV glycoprotein distribution is responsible for lateral virus spread in both, epithelial and endothelial cell monolayers.However, the prerequisites for correct protein targeting differ markedly in the two polarized cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Virology, Philipps University of Marburg, Germany.

ABSTRACT

Background: The highly pathogenic Nipah virus (NiV) causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB) and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells.

Results: As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar), not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting.

Conclusion: We conclude that the NiV glycoprotein distribution is responsible for lateral virus spread in both, epithelial and endothelial cell monolayers. However, the prerequisites for correct protein targeting differ markedly in the two polarized cell types.

Show MeSH
Related in: MedlinePlus