Figure 4
R-loop formation in Kaposi sarcoma-associated herpesvirus (KSHV) genome. Total nucleic acid from iSLK cells latently infected with KSHV, undergoing lytic viral replication, and uninfected cells (mock) were subjected DNA-RNA immunoprecipitation with S9.6 antibody. RNase H digestion was used as a negative control to ensure S9.6 was specific for DNA-RNA hybrids. A mouse IgG served as an isotype control to ensure DNA-RNA binding to S9.6 was specific. (A) Schematic representation of KSHV genome. Selected loci of RLFS and non-RLFS for experimental validation are shown in pink and green mark. The blue triangles represent the positions of RLFS. The boxes represent the repeat regions in the genome. (B) qPCR was performed to measure specific immunoprecipitation of KSHV ORF16 promoter by S9.6, cMYC was used as a positive control, and a region of KSHV ORF45 was used as a negative control. RNase H digestion was used as a treatment control to ensure S9.6 was specific for DNA:RNA hybrids. (C) R-loops were detected in the terminal repeat region of KSHV genomes by PCR. *indicates lytic BAC16. Reactivated (lytic) samples were treated with sodium butyrate and doxycycline to induce KSHV lytic replication 48 h prior to cell lysis. Data are presented as mean of technical duplicates. Error bars represent SD. Experiments were performed in biological duplicates, only one replicate shown.
