FIG 3.
ORF20 interacts and colocalizes with ORF59 in KSHV replication compartments. (A) HEK293T cells were transiently transfected with HA-ORF20 (or mock) and streptavidin (strep)-ORF59 as indicated, and a co-IP was performed using anti-HA antibody. Reverse Co-IP was performed using anti-strep antibody. (B) Lysates of latently infected (Lat) or doxycycline (DOX)-reactivated (Lyt) KSHV-positive iSLK.WT cells were subjected to IP with an anti-ORF59 antibody, and then Western blotted (WB) for ORF20 or ORF59. (C) Reactivated KSHV-positive iSLK.WT or iSLK.ORF20STOP cells were transfected with Flag-ORF20 and, 24 h later, were subjected to immunofluorescence assay using an anti-Flag antibody (red) and DAPI staining to identify nuclei (blue). White dotted circles denote DAPI-deficient nuclear domain representing KSHV replication compartments. (D) DOX-, TPA-, and ionomycin (iono)-treated KSHV-positive TREX-BCBL1 cells were transfected with Flag-ORF20 and, 24 h, later subjected to immunofluorescence assay using an anti-Flag antibody (red), anti-ORF59 antibody (yellow), and DAPI staining to identify nuclei (blue).