FIG 4.

Selection of BCBL-1 ORF57 KO single-cell clones expressing ORF50 (RTA, a viral transactivator), Cas9, and ORF56. (A) Western blot analysis of ORF57, ORF50, and Cas9 protein expression in selected BCBL-1 ORF57 KO clones after viral reactivation with sodium valproate (VA) for 24 h. Cellular β-tubulin served as a protein loading control. (B) Expression of ORF57 protein (red) as detected by immunofluorescence staining using anti-ORF57 antibody in BCBL-1 WT and ORF57 KO cells (clone 39) after KSHV reactivation with VA for 24 h. The cell nuclei were counterstained by DAPI. Bar, 10 μm. (C) RT-PCR analysis of ORF56 expression in ORF57 KO single-cell clone 39. Total RNA was prepared after viral reactivation with sodium valproate (VA) for 24 h, reverse transcribed in the presence (+) or absence (−) of reverse transcriptase (RT), and PCR amplified with ORF56-specific primer pair (see Table S1 in the supplemental material for details). Total RNA extracted from parental BCBL-1 cells (WT) or from a BCBL-1 cell stably transfected with a Cas9-only vector served as a control for ORF56 detection. GAPDH served as an internal RNA loading control.