Figure 6

Depletion of O -GlcNAc transferase (OGT) led to enhancement of KSHV reactivation. Lentivirus-based short hairpin RNAs (shRNA) targeting luciferase (Luc), or OGT were expressed in ERKV cells for 48 h, followed by doxycycline (50 ng/ml)-induction of KSHV reactivation for additional 48 h. (A) Western blot analysis of protein extracts from shRNA-treated cells by using α-O- GlcNAc antibody (RL2). Compared to control shLuc, shOGT decreases the global O-GlcNAcylation state in ERKV cells. β-actin served as a loading control. (B) K-RTA interacts with less PARP1 in shOGT-treated ERKV cells. Protein extracts from shRNA-treated ERKV cells were subjected to co-immunoprecipitation (Co-IP) assay using α-K-RTA antibody. Equal aliquots of each Co-IP were resolved on SDS-PAGE followed by western blot analysis using indicated antibodies. (C) (upper) Western blot analysis of K-bZIP and OGT expressions in shRNA-treated ERKV cells. An enhanced expression of K-bZIP is present in the OGT knockdown cells relative to that in the control shLuc group. The expression pattern of K-bZIP is inversely correlated with the cellular OGT level. α-tubulin served as a loading control. (lower) KSHV particles released from shLuc and shOGT-treated ERKV cells. Copy numbers of DNase I-resistant, encapsidated viral DNAs in each filtrated culture supernatants were determined by comparative quantitative PCR of KSHV DNA polymerase gene (ORF9). Data are presented as means ± SD from three PCR assays in an experiment. Similar results were obtained from two independent experiments; one set of data is shown.