<i>N</i>-linked high-mannose glycosylation of EBV gB is required for FBXO2 interaction.

<p><b>(A)</b> Analysis of glycan modifications of gB. gB-SFB purified from CNE2 cells stably expressing the protein was treated with endoglycosidase H (Endo H) or PNGase H or a Deglycosylation Mix before electrophoresis on 8% polyacrylamide gels. The sizes of molecular mass markers (M) are shown in kDa. <b>(B)</b> CNE2 cells stably expressing gB-SFB were treated with tunicamycin at different concentrations as indicated overnight before harvest. Con A agarose was used to enrich <i>N</i>-linked glycoproteins. TCL: total cell lysates. <b>(C-D)</b> Inhibition of N-linked glycosylation of gB by tunicamycin abrogates the gB-FBXO2 association. HEK293T cells transfected with plasmids encoding Myc-FBXO2 and gB-SFB were treated with 0.25 μg/mL tunicamycin or DMSO for 24 h, and the cells were harvested and subjected to immunoprecipitation by S-protein agarose <b>(C)</b> or anti-Myc agarose <b>(D)</b>. <b>(E)</b> Sequential pull-down of glycosylated gB by GST-FBXO2 followed by Con A agarose pull-down. Eight percent of the TCL of cells stably expressing gB was loaded as input; F-T: 8% of the flow-through. <b>(F)</b> Schematic diagram of glycosylation sites on gB and its N-to-Q mutants. The signal peptide (SP), furin cleavage site and transmembrane (TM) domain of gB are indicated. <b>(G)</b> Ribbon diagrams of the monomeric EBV gB structure, illustrated with the PyMol program using PDB 3FVC as the template. The glycosylation sites as shown in <b>(F)</b> are colored in magenta. <b>(H-I)</b> Con A agarose pull-down of gB single <b>(H)</b> or multiple <b>(I)</b> N-to-Q mutants. <b>(J-K)</b> Co-IP of FBXO2 with glycosylation-defective gB mutants. HEK293T cells were co-transfected with Myc-FBXO2 and gB N-to-Q mutants as indicated, and the cell lysates were subjected to immunoprecipitation by S-protein agarose <b>(J)</b> or anti-Myc agarose <b>(K)</b> and immunoblotted with anti-Myc and anti-FLAG antibodies.