Table 1.
qPCR primers and probes used to evaluate BACmid gene expression.
Table 2.
Oligonucleotides used for ChIRP.
Figure 1.
Deletion of the duplicated region within BAC36.
(A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.
Figure 2.
Production of infectious virus from BAC36CR induced cell lines.
BAC36 and BAC36CR cell lines were induced with TPA/n-butyrate for 5 days and supernatant virus was analyzed using qPCR. The amount of virus DNA accumulation was compared to DNA accumulation from uninduced BAC36. The error bars are the standard deviation of the mean from 3 separate experiments.
Figure 3.
Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN.
(A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.
Figure 4.
BAC36CRΔPAN fails to produce supernatant virus.
Supernatant virus was harvested from cell lines harboring BAC36CR, BAC36CRΔPAN or BAC36CRΔPANrevt DNA 5 days post induction. Relative amounts of viral DNA was analyzed using qPCR and is reported as fold increase in viral DNA level compared to uninduced BAC36CR.
Figure 5.
Decreased gene expression in the absence of PAN RNA expression.
qPCR analysis of mRNA accumulation from genes encoding K-Rta, K7, K-bZIP, ORF25 and ORF57 from BAC36CR, BAC36CRΔPAN or BAC36CRΔPANrevt cells treated with TPA/n-butyrate for 4 days. Values are compared to mRNA accumulation from uninduced BAC36CR containing cells. Each experiment was performed 3 times and error bars are the standard deviation from the mean. Boxed Panel. Western blot analysis of protein extracts reacted with anti-K-Rta, anti-K-bZIP, anti-LANA or anti-actin specific antibodies from induced and uninduced cell lines containing BAC36CR or BAC36CRΔPAN.
Figure 6.
K-Rta mRNA accumulation is decreased at early times post induction in the absence of PAN RNA expression.
(A) qPCR analysis of K-Rta mRNA accumulation from BAC36CR or BAC36CRΔPAN containing cells treated with TPA/n-butyrate (TPA/BT) for 15 hr. The experiment was performed 3 times and error bars are the standard deviation from the mean. (B) Expression of PAN RNA in trans activates K-Rta transcription BACmid harboring cell lines. qPCR analysis of K-Rta mRNA accumulation from BAC36CR or BAC36CRΔPAN containing cells transfected with a PAN RNA expression plasmid with or without treatment with TPA/n-butyrate (TPA/BT). Total RNA was harvested 15 h post induction. The experiment was performed 3 times and error bars are the standard deviation from the mean.
Figure 7.
Overexpression of K-Rta cannot complement BAC36CRΔPAN.
(A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.
Figure 8.
PAN RNA physically interacts with the ORF50 promoter.
(A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.
Figure 9.
JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression.
Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.
Figure 10.
PAN RNA interacts with demethylases and the histone methyltransferase MLL2.
(A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.