Fig. 2

Functional BGLF2 proteins are transferred to the recipient cells via exosomes. A BGLF2 protein-containing exosomes were released by BGLF2-expressing cells. HEK293T cells were transfected with HA-tagged BGLF2-expression or empty plasmid. Exosomes were isolated from the cell supernatant. CD63 and CD9 served as exosome markers. B TEM image of the exosomes purified from BGLF2-expressing HEK293T cells. Scale bar represents 100 nm. C NanoSight profiles displaying size distribution for exosomes purified from BGLF2-expressing HEK293T cells. D Western blotting results showing the absence or underrepresentation of the negative exosome marker cytochrome-C (Cyt c) in isolated exosomes. E BGLF2 protein was incorporated into exosomes. Exosomes were purified from supernatant treated with or without 0.25% Triton-X 100 for 5 min at room temperature. F BGLF2 was delivered to the recipient cells via exosomes. HEK293T cells were treated with BGLF2-containing exosomes for the indicated number of hours, and then washed extensively, and harvested. Cell lysates were analyzed by immunoblotting. G BGLF2 transferred via exosomes activated the AP-1 promoter. Exosomes were purified from HEK293T cells transfected with BGLF2-expression plasmid or empty plasmid in the presence or absence of GW4869. HEK293T cells transfected with the pAP-1-Luc and promoterless-Rluc plasmids, and then treated with BGLF2-containing exosomes. Cell lysates were obtained at 24 h after treatment and analyzed using luciferase reporter assays. Values (mean ± SEs) were calculated from three independent experiments. Asterisks, p < 0.05; n.s., not significant. H The orthologs of BGLF2 are incorporated into exosomes. HEK293T cells were transfected with expression plasmids containing HA-tagged BGLF2 or its HSV-1 or HCMV orthologs. Exosomes were isolated from the cell supernatant