Fig 1.
The ORF24-RNA pol II interaction is essential for late gene expression and contributes to viral DNA replication.
iSLK cells harboring WT BAC16, the ORF24RAAAG mutant or the ORF24RAAAG mutant rescue (MR) were reactivated with doxycycline and sodium butyrate for 72 hours. (A) Western blot showing expression of an early protein (ORF59) and a late protein (K8.1) in WT, ORF24RAAAG and ORF24RAAAG-MR. Vinculin was used as a loading control. (B) Progeny virion production was measured by supernatant transfer on to 293T cells and quantified by flow cytometry. (C) DNA replication of different mutants and MRs was measured by qPCR and normalized to the respective uninduced sample. The fold induction of all mutants was normalized to the WT to get the relative change in DNA replication compared to WT.
Fig 2.
Differential expression analysis of ORF24-Pol II interaction mutant and mutant rescue.
iSLK cells harboring ORF24RAAAG and ORF24RAAAG -MR were reactivated with doxycycline and sodium butyrate for 24 and 48 h, whereupon total RNA was extracted and subjected to library preparation and RNA sequencing. (A) Bar plot showing log2 fold change of the viral genes in the ORF24RAAAG -MR at 48 h relative to 24 h. Genes were color coded by kinetic class as defined in (30). (B) Bar plot showing log2 fold change of the viral genes in the mutant relative to the MR. Genes were color coded by kinetic class as defined in (30). (C) Heatmap of the scaled log2 fold change of the genes induced in the MR between 24 to 48 h and genes repressed in the mutant compared to MR at 48 h. Gene clusters with different kinetics and dependence on ORF24-Pol II interaction are indicated. (D) Consensus motif identified by MEME analysis of promoters from 14 genes that showed greater than one standard deviation repression in the mutant relative to the MR.
Table 1.
Occurrence of TATTWAA motif in KSHV.
ORFs shown in red do not have a known Transcription Start Site (TSS).
Fig 3.
ChIP-Seq with HA-ORF34 shows multiple binding sites across the viral genome.
(A) ChIP-Seq was performed using an anti-HA antibody to immunoprecipitate DNA prepared from iSLK HA-ORF34 cells reactivated for 48 h with doxycycline and sodium butyrate. iSLK-WT cells lacking the HA tag and input DNA were used as negative controls. The alignment files were converted to the tiled data file (tdf) format and the ChIP background from the untagged cells was subtracted and visualized on the Integrated Genome Viewer (IGV) (B) ChIP-qPCR was performed using an anti-HA antibody to immunoprecipitate DNA prepared from iSLK HA-ORF24 cells reactivated for 48 h with doxycycline and sodium butyrate. The DNA was quantified by qPCR with primers specific to the promoter regions of the indicated genes. Data were normalized to the level of input DNA and are presented as percentages of the input. Data shown are from three biological replicates. (C) Consensus motif identified by MEME analysis of promoters from 19 genes whose promoters had a ChIP peak.
Table 2.
Features of ChIP-Seq targets.
Fig 4.
Identifying direct targets of the vPIC.
(A) Heatmap showing genes repressed in the ORF24RAAAG mutant compared to the MR adjacent to the list of genes that show ORF34 binding at their promoter by ChIP-Seq analysis. Also indicated are viral genes that were identified to have the TATTWAA motif by FIMO analysis. The direct targets were identified by their combined dependence on the ORF24-Pol II interaction and the presence of a ChIP signal at the promoter. (B) Consensus motif identified by MEME analysis of promoters from 13 direct targets.
Fig 5.
The motif identified from direct targets is important for transcription from the promoter.
(A) Schematic of plasmid-based promoter activation assay. iSLK-WT or ORF24RAAAG mutant cells reactivated with doxycycline and sodium butyrate were transfected with reporter plasmid carrying the indicated promoter sequence upstream of the firefly luciferase gene and a control renilla luciferase expressing plasmid. 48 h post transfection and reactivation, the cells were assessed for luciferase activity. (B) Reactivated iSLK-WT cells were transfected with the K8.1 promoter reporter plasmid with or without the KSHV left lytic origin of replication cloned in cis and assessed for luciferase activity. (C) Reactivated iSLK-WT cells were transfected with the K8.1 and ORF57 promoter reporter plasmids in the presence or absence of DNA replication inhibitor (PAA) and assessed for luciferase activity. (D) Reactivated iSLK-WT and iSLK- ORF24RAAAG mutant cells were transfected with K8.1 or ORF57 promoter reporter plasmids and assessed for luciferase activity. (E-F) iSLK-WT cells were transfected with plasmids containing the WT K8.1 promoter or the indicated single or double point mutant promoters and assessed for luciferase activity. The data for each mutant was normalized to the WT K8.1 promoter. All data shown are average of 3–5 biological replicates.
Fig 6.
The expanded motif is important for DNA binding by vPIC.
(A) Schematic of the transient ChIP assay. iSLK-WT or iSLK-HAORF34 cells reactivated with doxycycline and sodium butyrate were transfected with plasmid carrying the indicated promoter sequence. 48 h post transfection and reactivation, cells were subjected to ChIP with an anti-HA antibody, and the associated DNA was quantified using primers specific to the promoter on the plasmid and the genomic K8.1 promoter locus. (B) iSLK-HAORF34 cells were transfected with K8.1 promoter containing plasmid, mutant plasmids or ORF57 promoter plasmid (as a control). iSLK-WT cells without the HA tag transfected with K8.1 plasmid served as a control for the IP. The ChIP DNA was quantified using plasmid specific primers (left panel) and K8.1 promoter specific primers (right panel), normalized to the level of input DNA and presented as percent input. Data shown are an average of 3–6 biological replicates.
Fig 7.
The ORF24-34 binding element at the origin is essential for late gene activation.
(A) (Top panel) Schematic of the left lytic origin of replication of the KSHV genome. (Bottom panel) IGV image of the ChIP peak for HA-ORF34 at the origin of replication. (B) iSLK-WT cells were transfected with the K8.1 promoter plasmid containing either the full length Ori (WT), the Ori lacking the ORF24-34 binding element (Delta ORF24-34 BE), or the Ori lacking the minimal Ori-lyt and retaining only the ORF24-34 binding element (ORF24-34 BE). 48 h post transfection and reactivation, the cells were assessed for luciferase activity. (C) iSLK-HAORF34 cells were transfected with a K8.1 promoter containing plasmid containing either the full-length Ori (WT), the Ori lacking the ORF24-34 binding element (Delta ORF24-34 BE), or no Ori. Untagged iSLK-WT cells transfected with K8.1 plasmid served as a control for the IP. The ChIP DNA was quantified using plasmid specific primers (left panel) and genomic K8.1 promoter specific primers (right panel), normalized to the level of input DNA and presented as percent input. Data shown are an average of 3–4 biological replicates.