Expression of ORF50 mRNA in HH-B2 cells. (A) Exon map of the region of KSHV DNA encompassing ORF50, K8, and K8.1. Arrow indicates a transcriptional start mapped in reference 40. The locations of the exons of ORF50 are defined in references 19, 40, and 45, of K8 in reference 16, and of K8.1 in reference 27. Numbers below the line are numbers in the KSHV sequence (32). Not shown is ORF49, located between exon 1 and exon 2 of ORF50 and transcribed in the opposite direction (32). polyA, polyadenylation site. Probes used to detect ORF50, K8, and K8.1 are shown above the map. (B) CHX resistance of the 3.6-kb ORF50 mRNA in HH-B2 cells. Cells were untreated or were treated for 8 h with 3 mM n-butyrate in the presence or absence of 33 μg of CHX/ml. At the indicated times, RNA was prepared and analyzed by probing a Northern blot with a single-stranded oligonucleotide complementary to the 3.6-kb ORF50 mRNA. (C) Autostimulation of expression of 3.6-kb ORF50 mRNA by transfection of KSHV Rta. HH-B2 cells were treated with chemical inducing agents (lanes 1 and 2), were untreated without (lane 3) or with (lane 4) electroporation, or were transfected with the plasmids indicated in lanes 5 to 12. KSHV gRta constructs derived from different KSHV strains beginning at nucleotide 71505 are designated with (L) for leader. KSHV gRta constructs beginning at nucleotide 71588 are designated with (A) for ATG. Twenty hours after transfection, total cellular RNA was harvested and analyzed by Northern blotting with a probe for K8 which detects both the K8 and ORF50 mRNAs.

Transfection of KSHV ORF50/Rta induces an increase in extracellular DNase-resistant viral DNA. Cells were untreated, treated with TPA, or transfected with KSHV gRta or KSHV gRta (rev) in reverse orientation or with a panel of ORF50 expression plasmids (see Fig. 1C). DNase-resistant viral DNA present in culture supernatant fluids harvested 96 h after transfection was analyzed by Southern blotting using a probe derived from KSHV ORF65.

Inhibition of KSHV Rta-induced late-gene expression by PAA. (A) HH-B2 cells were untreated (lanes 1 and 6), exposed to chemical inducing stimuli (lanes 2, 3, 7, and 8), or transfected with a KSHV ORF50 expression plasmid, gRta (lanes 4 and 9), or gRta mutant plasmids (lane 5 and 10). One-half of the cultures were exposed to 500 μM PAA at time zero immediately after electroporation (lanes 6 to 10). RNA harvested 30 h after transfection was analyzed by Northern blotting with probes for KSHV ORF65 (top) and K8 and K8.1 (bottom). RNaseP was used to control for RNA loading. (B) HH-B2 cells were transfected with vector pRTS or with vector containing ORF50/Rta from the BC-1 strain, in the presence or absence of PAA. Cell extracts prepared 48 h after transfection were analyzed by immunoblotting with antibodies to ORF65.