FIG 9.

Rescue of infectious KSHV production from iSLK/Bac16 cells containing an ORF57 KO KSHV genome. (A and B) Rescue of ORF59 expression from an ORF57 KO genome in the clone 2G1 cells by ectopic expression of ORF57. The 2G1 single-cell clone derived from iSLK/Bac16 cells containing an ORF57 KO KSHV-GFP genome was used for transfection of an empty (FLAG only) or ORF57-FLAG expression vector. After transfection, the virus was reactivated with Dox plus Bu treatment for 24 h. The expression of ORF59 or ectopic ORF57-FLAG was monitored by IF staining by using an anti-ORF59 specific antibody alone (A) or in combination with an anti-FLAG antibody for ORF57 expression (B). The WT and ORF57 KO 2G1 cells without transfection served as controls (A). Bar, 100 μm in panel A and 20 μm in panel B. (C and D) Ectopic expression of ORF57-FLAG in ORF57 KO 2G1 cells rescues the production of infectious virions. The 2G1 cells carrying an ORF57 KO genome were transfected with an empty (FLAG only) or ORF57-FLAG-expressing vector. The virus replication was induced by Dox plus Bu treatment, and the culture supernatant containing cell-free virions was collected 5 days after induction. The production of infectious virions was determined by inoculating HEK293T cells with the collected supernatant. The number of newly infected GFP⁺ cells was determined 48 h after infection using live fluorescence imaging (C) or by FACS analysis (D). The 2G1 cells with transfection of an empty FLAG vector served as negative controls. Bar, 100 μm.