Knockdown of KSHV PAN RNA reduces nuclear export of mRNA transcripts.

BCBL-1 TREx cells were electroporated with oligonucleotides antisense either to GFP mRNA (control KD) or to KSHV PAN RNA, followed by induction into the lytic phase with 1 μg/mL doxycycline. (A) 48 h after induction, cells were harvested for Western blot analysis of the early viral protein ORF6, and late viral proteins K8.1 and ORF65. (B) Seven days after induction, encapsulated viral DNA released into the media was harvested and quantified by qPCR; values were normalized to a control plasmid added at the onset of purification. (C) Seven days after induction, intracellular DNA was harvested from the cells and the level of viral DNA relative to host DNA was determined by qPCR. (D) 48 h after induction, a subset of the cells was harvested for RT-qPCR quantification of transcript levels in PAN RNA knockdown cells, relative to those in control knockdown cells. Additional transcript level quantifications are shown in S6 Fig. (E) 24 h after induction, the purity of cellular subfractions was assessed by Western blot for cytoplasmic (GAPDH) and nucleoplasmic (FUS) contents. (F) RT-qPCR analysis of fractionated BCBL-1 TREx cells. The fraction of each transcript present in the cytoplasm, relative to the nucleus, is plotted. (G) Representative Western blot analysis of BCBL-1 TREx cells treated with 100 μg/ml of the translation elongation inhibitor cycloheximide for the indicated times. (H) Quantification of data represented in panel (G). Degradation of the late viral protein ORF65 is slightly reduced following PAN RNA knockdown. Data are the average of three biological replicates and error bars represent standard deviations of the mean.