Knockdown of SUMO-2/3 increases transactivation of KSHV lytic genes and reduces SUMO-2/3-specific modification located in the high SUMO-2/3 enrichment region during viral reactivation.

<p>(A) TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated with Dox (0.2 μg/ml) for 24 hours. Total cell lysates (TCLs) were analyzed by immunoblotting using anti-SUMO-2/3, anti-K-Rta, and anti-K-bZIP antibodies. Anti-GAPDH antibody was used for loading control. Ratio for each cell line is the SUMO-2/3/GAPDH signal observed for Dox treatment at 0 (for shSUMO-2/3) and 24 hour using TREx-F3H3-K-Rta BCBL-1 cells at 0 hour set as 1.0. (B) Total RNA isolated from cells treated as described in (A) was reverse transcribed using oligo-d(T)₁₈ primer. The expression level of four viral genes representing SUMO-2/3 enrichment region and four viral genes representing H3K9me3-rich region as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005051#ppat.1005051.g001" target="_blank">Fig 1</a> were quantified by real-time qPCR. All reactions were run in triplicate and normalized against GAPDH. The fold change was computed by comparing induced values to their non-induced controls. ctrl.; control, TREx-F3H3-K-Rta BCBL-1. **; P<0.005. NS; non-significant. (C) ChIP was performed using chromatin prepared from cells treated as described in (A) using anti-SUMO-2/3 antibody. Rabbit IgG was used as negative antibody control and enrichment is not visible in some plots. SUMO-2/3 binding to SUMO-2/3 enrichment and H3K9me3-rich regions were analyzed by real-time qPCR using primer pairs specific for the KSHV loci as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005051#ppat.1005051.g002" target="_blank">Fig 2</a>.