Fig 2: PNUTS-mediated suppression of ORF59-GFP correlates with its PP1-dependent transcriptional functions.a Representative northern blot and quantification of ORF59-GFP from the reporter line treated with the indicated siRNAs. Values were normalized to GAPDH and quantified relative to siControl. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-tests compared to single siPNUTS knockdown: (siControl ***p = 0.0007, siZFC3H1 **p = 0.0025, siMTR4 *p = 0.0164, siARS2 n.s. p = 0.1733, siPNUTS/siZFC3H1 *p = 0.0278, siPNUTS/siMTR4 *p = 0.0428, siPNUTS/siARS2 n.s. p = 0.2810), (n = 3). b Representative northern blot and quantification of ORF59-GFP RNA from HEK293A-TOA cells treated with siRNAs and then transiently transfected with pAAVS1-ORF59-GFP-Hyg and empty vector, Fl-PNUTS, or Fl-W401A (both with siRNA-refractory silent mutations). RNA was harvested at 72 h post-transfection with siRNAs and 24 h post-transfection of DNA plasmids. ORF59-GFP RNA was normalized to GAPDH and is graphed relative to siPNUTS with empty vector. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (siControl vs siPNUTS 1 ****p = 1.4 × 10-6, siControl vs Fl-PNUTS n.s. p = 0.5284, siControl vs Fl-W401A **p = 0.0092, siPNUTS 1 vs FL-PNUTS ***p = 0.0008, Fl-PNUTS vs Fl-W401A **p = 0.0092), (n = 5). c Representative northern blot and quantification of ORF59-GFP RNA, as in (a). Values were normalized to GAPDH and relative to siPNUTS. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test compared to single siControl sample: (siPNUTS *p = 0.0116, siSETD1A **p = 0.0032, siCDK12 *p = 0.0132, siCycK *p = 0.0370, siPNUTS/siSETD1A *p = 0.0116, siPNUTS/siCDK12 *p = 0.0158, siPNUTS/siCycK *p = 0.0160), (n = 3). Source data are provided as a Source Data file.

Fig 3: Depletion of PNUTS enhances KSHV viral replication.a vDNA content relative to host genome was quantified by qPCR at various timepoints from 0 to 48hpi in siControl, siPNUTS 1 or siPNUTS 2-treated cells. Error bars are mean with standard deviation, (n = 3). b Schematic of viral replication assay from iSLK cells after PNUTS depletion. c Quantification of the percent of recipient cells infected with virus produced from non-targeting control versus PNUTS-depleted cells after 48 h of lytic induction. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (siControl vs siPNUTS 1 ***p = 0.0001, siControl vs siPNUTS 2 ****p = 2.4 × 10-5), (n = 3). d as in c, except from virus harvested at 24hpi from iSLK cells. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (siControl vs siPNUTS 1 *p = 0.0289, siControl vs siPNUTS 2 **p = 0.0087), (n = 3). e Representative flow cytometry results of recipient HCT116 cells after infection assay. f Schematic of TREx-shRNA-BCBL1 virus production assay. Expression of the shRNAs was first induced by treatment with doxycycline, and lytic reactivation was induced by sodium butyrate. g LANA expression as determined by RT-qPCR in recipient cells 24 h after infection with supernatant from shControl or shPNUTS TREx-shRNA-BCBL1 cells. Signal was first normalized to beta-actin and is displayed relative to shControl. Error bars are mean with standard deviation, asterisk denotes Student’s two-tailed unpaired t-test: *p = 0.0172, (n = 3). Source data are provided as a Source Data file.

Fig 4: PNUTS depletion increases transcriptional output of a viral gene reporter by decreasing promoter-proximal pausing.a Schematic of the modified reporter used for ChIP-seq and 4SU experiments. A and B mark the 4SU-qPCR amplicon locations. b RT-qPCR of 4SU-labeled RNA following a 10 min 4SU pulse before sample harvesting and 4SU selection. Amplicon levels were normalized to 4SU-labeled beta-actin and quantified relative to siControl. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (ORF59 siControl vs siPNUTS 1 **p = 0.0060, siControl vs siPNUTS 2 **p = 0.0066; Readthrough siControl vs siPNUTS 1 ***p = 0.0009, siControl vs siPNUTS 2 n.s. p = 0.0684), (n = 3). c Representative IGV images of human genes GAPDH and MYC ChIP-sequencing profiles in non-targeting control (dark gray), siPNUTS 1 (dark blue), and siPNUTS 2 (teal). Bottom tracks are relative (Log2 ratio) pol II occupancy comparing PNUTS-depleted samples to non-targeting control; black and red denote higher and lower signal in PNUTS-depleted samples, respectively. d Metagene centered on TES for uniquely annotated (n = 6383) protein-coding genes at least 1 kb long. Due to extensive signal from pol II readthrough of adjacent genes, we filtered our dataset of protein coding genes to include only those that have =10 kb flanking the neighboring genes. e Same as d, except metagenes centered on the transcription start site (TSS). f IGV browser image of ChIP-seq reads from the integrated reporter construct. Labeling as in panel (c). g Box plot showing the PI of the integrated reporter under the indicated siRNA treatments. Black line inside the boxplot indicates the mean values. The black dots show data points from two replicates. h Violin plot showing the PI (Log2 scale) of human protein-coding genes (n = 1358) under the indicated siRNA conditions. Thick dashed line indicates median, thin dashed lines below and above show 1st and 3rd quartile respectively. Genes (as in d) having low read coverage in the promoter-proximal region were excluded from the analysis. Two-sided Wilcoxon rank sum tests were used for pairwise comparisons, (siControl vs siPNUTS 1 n.s. p = 0.0975, siControl vs siPNUTS 2 ****p < 0.0001). Source data are provided as a Source Data file.

Fig 5: Depletion of PNUTS broadly enhances KSHV gene expression.a Northern blot of ORF59 with total RNA harvested from iSLK cells at 24 hpi. For quantification, signal was first normalized to GAPDH then quantified relative to siControl. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (siControl vs siPNUTS 1 **p = 0.0057, siControl vs siPNUTS 2 **p = 0.0035), (n = 3). b RT-qPCR of PAN RNA levels in iSLK cells at 8, 24, and 48 hpi, normalized to beta-actin. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (siControl vs siPNUTS 1 8hpi *p = 0.0348, 24hpi **p = 0.0032, 48hpi ***p = 0.0009), (n = 3). c Heatmap of Log2-fold changes (Log2FC) in KSHV gene expression in siPNUTS 1 relative to siControl. RNA-seq from three biological replicates in iSLK cells at four timepoints after lytic reactivation. d Heatmap of average Log2 median viral gene counts of RNA-seq data from indicated samples. e Western blot for PNUTS protein and CPSF73 loading control in TREx-shRNA-BCBL1 lines after 7 days of shRNA induction and 0, 24, or 72 h lytic induction with sodium butyrate (NaB). f Quantification of relative PNUTS protein levels at multiple timepoints of lytic induction for shControl and shPNUTS cell lines; values were first normalized to CPSF73 and quantified relative to shControl. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test, n.s. not significant: (shControl vs shPNUTS 0hpi **p = 0.0036, 24hpi p = 0.1952, 72hpi **p = 0.0019), (n = 4). g Northern blot of ORF59 expression from TREx-shRNA-BCBL1 shControl and shPNUTS cells at 0, 24, and 72hpi. h Quantification of ORF59 northern blot data from TREx-shRNA-BCBL1 cells as in (g). Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (shControl vs shPNUTS 0hpi *p = 0.0128, 24hpi ***p = 0.0009, 72hpi ****p = 6.3 × 10-6), (n = 3). i RT-qPCR of lytic genes at 0, 24, and 72hpi from TREx-shRNA-BCBL1 lines, normalized to beta-actin mRNA. Note that expression of several KSHV lytic genes peaks at ~24–48hpi, while relative expression is highest at 72hpi, likely due to host shut-off. Error bars are mean with standard deviation, asterisks denote Student’s two-tailed unpaired t-test: (shControl vs shPNUTS ORF50 0hpi **p = 0.0049, 24hpi ****p = 9.3 × 10-5, 72hpi n.s. p = 0.0905; K5 0hpi **p = 0.0078, 24hpi ***p = 0.0004, 72hpi n.s. p = 0.0614; T1.4 0hpi **p = 0.0048, 24hpi ***p = 0.0007, 72hpi n.s. p = 0.1073; PAN 0hpi *p = 0.0118, 24hpi *p = 0.0202, 72hpi n.s. p = 0.1011), (n = 3). Source data are provided as a Source Data file.