FIG 5.

SIRT1 mediates KSHV latency. (A) SIRT1 binds to both RTA and LANA promoters. ChIP-qPCR was performed with BCBL-1 cells using an antibody to SIRT1 and a control antibody. (B) Short hairpin RNA knockdown of SIRT1 induces the expression of KSHV lytic protein ORF65. BCBL-1 cells infected with lentiviruses expressing shRNAs to SIRT1 or a scrambled control shRNA for 3 days were examined for the expression of SIRT1 and ORF65 proteins by Western blotting. (C) Expression of SIRT1, RTA, ORF65, and LANA transcripts in BCBL-1 cells following knockdown of SIRT1, as described in the legend to panel B. Viral transcripts were detected by RT-qPCR, and their levels were normalized to the level of cellular GAPDH. (D and E) Quantification of the H3K4me3 and H3K27me3 marks in RTA (D) and LANA (E) promoters by ChIP-qPCR assays following SIRT1 knockdown. Cells infected with lentiviruses expressing shRNAs to SIRT1 or a scrambled control shRNA as described in the legend to panel B were subjected to ChIP using an antibody to H3K4me3 or H3K27me3 or a control antibody. The ChIP DNAs were quantified by qPCR.