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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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Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.
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Figure 3: Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.

Mentions: Previous studies in polarized epithelial cells had shown that bipolar distribution of the NiV glycoproteins in infected epithelia is correlated with a predominant basolateral expression of the F and G proteins in the absence of virus infection ([18]; table 1). Upon single expression of the glycoproteins, basolateral sorting was shown to depend on cytoplasmic tyrosine-based targeting motifs: Y525 in the F protein and di-tyrosine Y28/29 in the G protein. Mutations in the two other potential basolateral sorting motifs, a di-tyrosine motif in the F protein (Y542/543) and a di-leucine motif in the G protein (L41/42) had no influence on basolateral sorting (table 1). Epithelial and endothelial cell types share common characteristics since they both form junctional complexes that seal off an apical surface area and both cell types support a vectorial exchange of substances between apical and basolateral compartments. However, sorting of membrane proteins not always follows the same rules. Several cellular proteins, such as the transferrin receptor, the polymeric immunoglobulin receptor and tissue factor, which are selectively expressed on the basolateral surface of epithelial cells are oppositely targeted to the apical membrane of endothelial cells [24-26]. It thus remains to be elucidated if the cytoplasmic tyrosine residues in the NiV glycoproteins, shown to act as basolateral sorting signals in epithelial cells, have the same function in endothelial cells. We therefore decided to analyze the sorting of F and G proteins with mutated potential tyrosine and leucine-dependent sorting signals in polarized endothelial cells. The cytoplasmic tail sequences of wildtype and mutant proteins are depicted in Figure 3A. Since transient expression in primary endothelial cells is extremely inefficient and often interferes with cell polarization, we generated PAEC clones stably expressing either wildtype or mutant NiV glycoproteins. To monitor the targeting of the expressed proteins, the cells were cultured on filter supports. At 5 days after seeding, the cells had formed confluent and polarized monolayers and were labeled without prior fixation with NiV-specific antibodies and AlexaFluor 568-conjugated secondary antibodies from both, the apical and basolateral side. Confocal vertical sections through the cell monolayers are shown in Figure 3B and 3C. As in the infection (Figure 2), wildtype F was expressed bipolar upon single expression (Figure 3B; Fwt). Interestingly, mutations in both Y-based signals in the F protein (Y525 and YY542/543) led to an apical F redistribution (Figure 3B; FY525A; FY542/543A). This contrasts with our recent findings in polarized epithelial cells which showed that polarized distribution of the NiV F protein only depends on Y525 but not on the di-tyrosine motif at position 542/543 ([18]; table 1). Also, the distribution of the G protein is differently affected by the cytoplasmic tail mutations. Mutant GY28/29A that was previously found to be sorted apically in polarized epithelial cells showed bipolar expression in PAEC as did the wildtype G protein (Figure 3C; Gwt; GY28/29A). Mutation in the di-leucine motif did also not affect the bipolar G distribution (Figure 3C; GL41/42A).


Nipah virus infection and glycoprotein targeting in endothelial cells.

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

Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.
Mentions: Previous studies in polarized epithelial cells had shown that bipolar distribution of the NiV glycoproteins in infected epithelia is correlated with a predominant basolateral expression of the F and G proteins in the absence of virus infection ([18]; table 1). Upon single expression of the glycoproteins, basolateral sorting was shown to depend on cytoplasmic tyrosine-based targeting motifs: Y525 in the F protein and di-tyrosine Y28/29 in the G protein. Mutations in the two other potential basolateral sorting motifs, a di-tyrosine motif in the F protein (Y542/543) and a di-leucine motif in the G protein (L41/42) had no influence on basolateral sorting (table 1). Epithelial and endothelial cell types share common characteristics since they both form junctional complexes that seal off an apical surface area and both cell types support a vectorial exchange of substances between apical and basolateral compartments. However, sorting of membrane proteins not always follows the same rules. Several cellular proteins, such as the transferrin receptor, the polymeric immunoglobulin receptor and tissue factor, which are selectively expressed on the basolateral surface of epithelial cells are oppositely targeted to the apical membrane of endothelial cells [24-26]. It thus remains to be elucidated if the cytoplasmic tyrosine residues in the NiV glycoproteins, shown to act as basolateral sorting signals in epithelial cells, have the same function in endothelial cells. We therefore decided to analyze the sorting of F and G proteins with mutated potential tyrosine and leucine-dependent sorting signals in polarized endothelial cells. The cytoplasmic tail sequences of wildtype and mutant proteins are depicted in Figure 3A. Since transient expression in primary endothelial cells is extremely inefficient and often interferes with cell polarization, we generated PAEC clones stably expressing either wildtype or mutant NiV glycoproteins. To monitor the targeting of the expressed proteins, the cells were cultured on filter supports. At 5 days after seeding, the cells had formed confluent and polarized monolayers and were labeled without prior fixation with NiV-specific antibodies and AlexaFluor 568-conjugated secondary antibodies from both, the apical and basolateral side. Confocal vertical sections through the cell monolayers are shown in Figure 3B and 3C. As in the infection (Figure 2), wildtype F was expressed bipolar upon single expression (Figure 3B; Fwt). Interestingly, mutations in both Y-based signals in the F protein (Y525 and YY542/543) led to an apical F redistribution (Figure 3B; FY525A; FY542/543A). This contrasts with our recent findings in polarized epithelial cells which showed that polarized distribution of the NiV F protein only depends on Y525 but not on the di-tyrosine motif at position 542/543 ([18]; table 1). Also, the distribution of the G protein is differently affected by the cytoplasmic tail mutations. Mutant GY28/29A that was previously found to be sorted apically in polarized epithelial cells showed bipolar expression in PAEC as did the wildtype G protein (Figure 3C; Gwt; GY28/29A). Mutation in the di-leucine motif did also not affect the bipolar G distribution (Figure 3C; GL41/42A).

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