Expression of vIRF2 in KSHV-infected cells.

(A) The vIRF genes are located between ORF57 and ORF58 in the KSHV genome. The vIRF2 protein is encoded by the two exons K11.1 and K11. (B) Schematic diagram of the vIRF2 open reading frame, shown in the opposite orientation to its position in the viral genome. ATG start codons are indicated by red amino acid numbers at the positions aa 1, 47, 103, 108 110, 131, 134 in K11.1 and aa 213, 311, 350, 382, 408, 440, 484, 584, 593 in K11. Dotted lines indicate binding sites for siRNAs used in this study: aa7-13 (1), aa193-199 (2) and aa609-615 (3), DBD: DNA binding domain (aa7-114), NLS: nuclear localization signal (aa146-159), R1/R2: repeat region 1/2, E1/2: antibody epitopes. (C) Expression of vIRF2 in different KSHV positive cell lines. The lytic cycle in the PEL-derived cell lines BC1, BC3 and BCBL1 was induced by 100 ng/ml TPA, HuARLT.rKSHV.219 cells were induced using 1.67 mM SB and 10% tissue culture supernatant containing RTA-expressing baculovirus, the BJAB.rKSHV.219 using 2.5 μg/ml goat anti human-IgM. At the indicated time points after induction the cells were lysed and protein expression was analyzed by WB. (D) Subcellular localization of vIRF2 was analyzed by nuclear and cytoplasmic extraction of BJAB or BJAB.rKSHV.219 cells with or without induction of the lytic cycle using 2.5 μg/ml goat anti human-IgM. WB analysis was performed for vIRF2, Calnexin (cytoplasmic marker; C) and Lamin A/C (nuclear marker; N).