Fig. 3

Exosomal BGLF2 enhances the infectivity of EBV. A and B Akata(-) cells were infected with wildtype (A) and BGLF2-KO EBV (B) in the presence of BGLF2-containing exosomes. After 2 days, GFP positivity was determined by FACS. Results are presented as the mean ± SE of at least three independent experiments and as the relative infectivity to control treatment with PBS (infectivity value of 1). Double asterisks, p < 0.01; n.s., not significant. C The workflow of sample collection for RNA-seq/qRT-PCR analyses. Akata(-) cells were pretreated with exosomes for 16 h, and then infected with BGLF2-KO EBV. The infected cells were collected via FACS at 24 hpi. Total RNA extracted from the sorted cells was subjected to RNA-seq and qRT-PCR. D Read counts of EBV genes in BGLF2-KO EBV-infected cells that were treated with BGLF2-containing or control exosomes by RNA-seq. E Gene expression levels were normalized as counts per million followed by log₂-transformation with a pseudo-count of 1. Each bar indicates the log₂ fold change of a viral gene expression between treatment with exosomes with or without BGLF2. Viral gene expression kinetics are categorized into five groups: latent, immediate early, early, leaky late, and late [53]. F Validation of enhanced EBV gene expression by qPCR. Data are presented as the mean ± SE. Samples were tested in duplicate. Asterisk, p < 0.05; double asterisks, p < 0.01; n.s., not significant. G DAVID analysis of RNA-seq data from BGLF2-KO EBV-positive cells treated with BGLF2-containing or control exosomes. FDR, false discovery rate. H HEK293 cells were treated with BGLF2-containing exosomes for 16 h. Cells were challenged with transfection of poly(I:C) (2 μg/ml) for 2.5 h. RT-qPCR was conducted. Data are presented as the mean ± SE of three independent experiments. Asterisk, p < 0.05; n.s., not significant. H Exosomal BGLF2 inhibited type I IFN signaling. AGS/EBV-EGFP cells were pretreated with exosomes for 16 h, and then infected with or without IFN alpha (1000 Unit/mL) for 1 h. Lysates were analyzed by immunoblotting using the indicated antibodies. BGLF2-HA was detected using the anti-HA antibody. J Type I IFN inhibited EBV infection. Akata(-) cells were infected with EBV-EGFP in the presence of IFN alpha and beta (100 or 500 Unit/mL). After 2 days, GFP positivity was determined by FACS. Results are presented as the mean ± SE of three independent experiments. Double asterisks, p < 0.01; n.s., not significant