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Viral Restriction Activity of Feline BST2 Is Independent of Its N-Glycosylation and Induction of NF-κB Activation.

Wang W, Wang J, Qu M, Li X, Zhang J, Zhang H, Wu J, Yu B, Wu H, Kong W, Yu X - PLoS ONE (2015)

Bottom Line: The N-terminal cytoplasmic tail of feline BST2 (fBST2) is characterized by a shorter N-terminal region compared to those of other known homologs.However, in contrast to human BST2, the wild-type fBST2 did not show the ability to activate NF-κB.The shorter N-terminal cytoplasmic region of fBST2 compared with human BST2 did not apparently affect its anti-viral activity, which is independent of its N-glycosylation and ability to activate NF-κB.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, Jilin Province, People's Republic of China.

ABSTRACT
BST2 (CD317, tetherin, HM1.24) is an interferon-inducible transmembrane protein which can directly inhibit the release of enveloped virus particles from infected cells, and its anti-viral activity is reported to be related to the specific topological arrangement of its four structural domains. The N-terminal cytoplasmic tail of feline BST2 (fBST2) is characterized by a shorter N-terminal region compared to those of other known homologs. In this study, we investigated the functional impact of modifying the cytoplasmic tail region of fBST2 and its molecular mechanism. The fBST2 protein with the addition of a peptide at the N-terminus retained anti-release activity against human immunodeficiency virus type-1 and pseudovirus based on feline immunodeficiency virus at a weaker level compared with the wild-type fBST2. However, the fBST2 protein with addition of a peptide internally in the ectodomain proximal to the GPI anchor still retained its anti-viral activity well. Notably, the N-glycosylation state and the cell surface level of the N-terminally modified variants were unlike those of the wild-type protein, while no difference was observed in their intracellular localizations. However, in contrast to human BST2, the wild-type fBST2 did not show the ability to activate NF-κB. Consistent with previous reports, our findings showed that adding a peptide in the cytoplasmic tail region of fBST2 may influence its anti-viral activity. The shorter N-terminal cytoplasmic region of fBST2 compared with human BST2 did not apparently affect its anti-viral activity, which is independent of its N-glycosylation and ability to activate NF-κB.

No MeSH data available.


Related in: MedlinePlus

Cell surface expression of BST2 variants.293T and CrFK cells were co-transfected with 500 ng of VR1012, fBST2, fBST2 N79A, fBST2 N119A, fBST2 N79/119A, fBST2-IHA, fBST2-NHA or fBST2* expression plasmid, along with 500 ng of pEGFP-N3 as a transfection marker. After 48 h, cells were harvested and stained with the anti-fBST2 pAb and Alexa 633 goat anti-rabbit IgG, followed by flow cytometric analysis. Cells only transfected with pEGFP-N3 were used as a negative control. The samples were gated on EGFP+ cells, and the surface BST2 levels are shown in the column diagram with mean fluorescent intensity values (A and B). This experiment was repeated three times, and the most representative data are shown.
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pone.0138190.g006: Cell surface expression of BST2 variants.293T and CrFK cells were co-transfected with 500 ng of VR1012, fBST2, fBST2 N79A, fBST2 N119A, fBST2 N79/119A, fBST2-IHA, fBST2-NHA or fBST2* expression plasmid, along with 500 ng of pEGFP-N3 as a transfection marker. After 48 h, cells were harvested and stained with the anti-fBST2 pAb and Alexa 633 goat anti-rabbit IgG, followed by flow cytometric analysis. Cells only transfected with pEGFP-N3 were used as a negative control. The samples were gated on EGFP+ cells, and the surface BST2 levels are shown in the column diagram with mean fluorescent intensity values (A and B). This experiment was repeated three times, and the most representative data are shown.

Mentions: Most researchers accept that BST2 must appear at the cell surface to exert its anti-viral function. In order to investigate the effect on cell surface localization by adding a peptide to different regions of fBST2 or mutating its glycosylation sites, the following FACS analysis was carried out. The EGFP+ encoding vector pEGFP-N3, used to verify the transfection efficiency for FACS analysis, was co-transfected with BST2 or VR1012 in 293T and CrFK cells. By flow cytometric analysis, cell surface protein levels were evaluated with the anti-fBST2 pAb and Alexa-633 conjugated anti-rabbit secondary antibody. Cells transfected with pEGFP-N3 and VR1012 were used as negative controls. Samples were gated on EGFP+ cells, and the surface BST2 levels were compared in histograms (Fig 6A and 6B). The cell surface distribution of the N-terminally modified mutants fBST2-NHA and fBST2* decreased remarkably compared with fBST2, while internally HA-tagged mutants fBST2-IHA was similar to fBST2. Surface levels of N-glycosylation site mutants were different, fBST2 N119A had a higher surface level than fBST2, while fBST2 N79A and fBST2 N79/119A only had a low level. Notably, fBST2, fBST2 N119A and fBST2-IHA showed better anti-viral activities than did the other three variants (Fig 4), suggesting that the cell surface appearance of fBST2 is a condition of its anti-viral function.


Viral Restriction Activity of Feline BST2 Is Independent of Its N-Glycosylation and Induction of NF-κB Activation.

Wang W, Wang J, Qu M, Li X, Zhang J, Zhang H, Wu J, Yu B, Wu H, Kong W, Yu X - PLoS ONE (2015)

Cell surface expression of BST2 variants.293T and CrFK cells were co-transfected with 500 ng of VR1012, fBST2, fBST2 N79A, fBST2 N119A, fBST2 N79/119A, fBST2-IHA, fBST2-NHA or fBST2* expression plasmid, along with 500 ng of pEGFP-N3 as a transfection marker. After 48 h, cells were harvested and stained with the anti-fBST2 pAb and Alexa 633 goat anti-rabbit IgG, followed by flow cytometric analysis. Cells only transfected with pEGFP-N3 were used as a negative control. The samples were gated on EGFP+ cells, and the surface BST2 levels are shown in the column diagram with mean fluorescent intensity values (A and B). This experiment was repeated three times, and the most representative data are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4574558&req=5

pone.0138190.g006: Cell surface expression of BST2 variants.293T and CrFK cells were co-transfected with 500 ng of VR1012, fBST2, fBST2 N79A, fBST2 N119A, fBST2 N79/119A, fBST2-IHA, fBST2-NHA or fBST2* expression plasmid, along with 500 ng of pEGFP-N3 as a transfection marker. After 48 h, cells were harvested and stained with the anti-fBST2 pAb and Alexa 633 goat anti-rabbit IgG, followed by flow cytometric analysis. Cells only transfected with pEGFP-N3 were used as a negative control. The samples were gated on EGFP+ cells, and the surface BST2 levels are shown in the column diagram with mean fluorescent intensity values (A and B). This experiment was repeated three times, and the most representative data are shown.
Mentions: Most researchers accept that BST2 must appear at the cell surface to exert its anti-viral function. In order to investigate the effect on cell surface localization by adding a peptide to different regions of fBST2 or mutating its glycosylation sites, the following FACS analysis was carried out. The EGFP+ encoding vector pEGFP-N3, used to verify the transfection efficiency for FACS analysis, was co-transfected with BST2 or VR1012 in 293T and CrFK cells. By flow cytometric analysis, cell surface protein levels were evaluated with the anti-fBST2 pAb and Alexa-633 conjugated anti-rabbit secondary antibody. Cells transfected with pEGFP-N3 and VR1012 were used as negative controls. Samples were gated on EGFP+ cells, and the surface BST2 levels were compared in histograms (Fig 6A and 6B). The cell surface distribution of the N-terminally modified mutants fBST2-NHA and fBST2* decreased remarkably compared with fBST2, while internally HA-tagged mutants fBST2-IHA was similar to fBST2. Surface levels of N-glycosylation site mutants were different, fBST2 N119A had a higher surface level than fBST2, while fBST2 N79A and fBST2 N79/119A only had a low level. Notably, fBST2, fBST2 N119A and fBST2-IHA showed better anti-viral activities than did the other three variants (Fig 4), suggesting that the cell surface appearance of fBST2 is a condition of its anti-viral function.

Bottom Line: The N-terminal cytoplasmic tail of feline BST2 (fBST2) is characterized by a shorter N-terminal region compared to those of other known homologs.However, in contrast to human BST2, the wild-type fBST2 did not show the ability to activate NF-κB.The shorter N-terminal cytoplasmic region of fBST2 compared with human BST2 did not apparently affect its anti-viral activity, which is independent of its N-glycosylation and ability to activate NF-κB.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, Jilin Province, People's Republic of China.

ABSTRACT
BST2 (CD317, tetherin, HM1.24) is an interferon-inducible transmembrane protein which can directly inhibit the release of enveloped virus particles from infected cells, and its anti-viral activity is reported to be related to the specific topological arrangement of its four structural domains. The N-terminal cytoplasmic tail of feline BST2 (fBST2) is characterized by a shorter N-terminal region compared to those of other known homologs. In this study, we investigated the functional impact of modifying the cytoplasmic tail region of fBST2 and its molecular mechanism. The fBST2 protein with the addition of a peptide at the N-terminus retained anti-release activity against human immunodeficiency virus type-1 and pseudovirus based on feline immunodeficiency virus at a weaker level compared with the wild-type fBST2. However, the fBST2 protein with addition of a peptide internally in the ectodomain proximal to the GPI anchor still retained its anti-viral activity well. Notably, the N-glycosylation state and the cell surface level of the N-terminally modified variants were unlike those of the wild-type protein, while no difference was observed in their intracellular localizations. However, in contrast to human BST2, the wild-type fBST2 did not show the ability to activate NF-κB. Consistent with previous reports, our findings showed that adding a peptide in the cytoplasmic tail region of fBST2 may influence its anti-viral activity. The shorter N-terminal cytoplasmic region of fBST2 compared with human BST2 did not apparently affect its anti-viral activity, which is independent of its N-glycosylation and ability to activate NF-κB.

No MeSH data available.


Related in: MedlinePlus