The predicted KSHV circRNAs in individual RNA-seq samples described in Fig 1A from the WT and 57KO BCBL-1 cells with lytic KSHV infection by the CIRI2 pipeline [38, 39].

(XLSX)</p

The sequences and positions of oligos used in this study.

(XLSX)</p

Differential expression of KSHV genes in BCBL-1 single-cell clones carrying a WT B4 or 57KO #6 genome during latent and lytic infection determined by Limma Voom package based on the number of mapped RNA-seq reads in each group (A, B, or C) as described in Fig 1A.

(XLSX)</p

Alternative RNA splicing detected and verified from the K5/K6 locus.

(A) The RNA splice junctions mapped to K5 and K6 locus in the WT genome. Below is the predicted structure of K5, K6, and K6/K5 transcripts with thick black lines representing the exons and dotted lines the introns. The arrows represent the primers used in RT-PCR shown in (B). See other details in Fig 3. (B) Verification of the mapped RNA splice junctions in K5/K6 expression in BCBL-1 cells with lytic KSHV infection. Total RNA extracted from the parental BCBL-1 cells with VA 24 h treatment was examined by RT-PCR. The reaction without reverse transcriptase (RT-) was used as a negative control. The predicted sizes of amplificons from unspliced and spliced transcripts are displayed under each gel electrograph. All spliced products amplified were gel purified and confirmed by sequencing. The number of supporting splice reads from the WT genome in the cells with lytic KSHV induction are in the parentheses.</p

ORF57 regulates K15 RNA alternative splicing.

(A) The splice junctions and RNA-seq coverage of K15 transcripts detected in BCBL-1 cells bearing a WT (orange) or 57KO genome (pink) during KSHV lytic infection. Black arrows mark splice donor (D) and acceptor (A) sites. (B) Structure of the K15 pre-mRNA with the position of mapped pA cleavage site (CS) and predicted transcriptional start site (TSS). The thick black lines represent the exons and the dotted lines the introns. The arrows below show the positions and orientations of the primers used in RT-PCR listed in the S6 Table. (C and D) Gel electrographs of RT-PCR products obtained from total RNA from the WT and 57KO genomes in BCBL-1 cells undergoing lytic replication harvested 24 h after VA treatment, with the primer pairs shown in (B). The samples without reverse transcription (- RT) were used as a negative control. The host GAPDH was used as a loading RNA control. Unspl., unspliced pre-mRNA; Spl., spliced mRNA; PSI, percent spliced-in of the alternative exon (s) or splice site (% inclusion = inclusion/sum of inclusion + exclusion) [76].</p

MOESM2 of Circulating tumor cells capture disease evolution in advanced prostate cancer

Additional file 2. High confidence somatic mutations

Mapping of KSHV RNA splice junctions from the minus strand of viral genome.

The diagrams depicting all splice junctions (colored arches) with ≥10 supporting splice reads mapped to viral transcripts expressed from the minus strand of the KSHV genome, with the splicing events being numbered in the order from the genome 5′ to 3′ direction. The numbered orange arches represent splice junctions detected from the WT genome and pink arches from the 57KO genome in the cells (see Tables 1 and 3). The arch thickness represents a relative abundance of detected splice junction reads. The RNA-seq reads-coverage from one representative sample from the WT and 57KO genome in the cells with lytic infection is shown above the arches, with the reads-coverage depth shown in the upper right corner. See other details in Fig 3. LTc—constitutive latent promoter, LTi—inducible latent promoter, LTd—distal latent promoter, oriL—lytic origin of replication.</p

Linear RNA splicing and production of KSHV circRNAs.

(A) Identification of KSHV circRNAs from RNA-seq in BCBL-1 cells with a WT or 57KO genome during lytic replication induced by 24 h valproic acid treatment using a circular RNA prediction software CIRI2 [38, 39]. (B) Distribution and coverage of RNA-seq reads on PAN, detected linear PAN RNA splice junctions (red and blue arches) with minimum 2 splice junction reads and predicted viral PAN circRNAs (thick blue lines, see panel A for details) in WT and 57KO BCBL-1 cells with lytic replication. MRE-I represents the Mta or ORF57-responsive element I in PAN RNA identified by anti-ORF57 CLIP [26]. (C) The RNA-seq reads mapped to KSHV vIRF4 (K10) locus in WT (orange) and 57KO (pink) cells with reads-coverage depth shown in the upper right corner. The arches show a direction and number of splice reads detected for linear canonical splicing (orange and pink arches) and back-splicing (grey arches). Below is a diagram of KSHV vIRF4 locus with detected exons (orange boxes), introns (numbered orange arches) with the corresponding splice sites (D = splice donor and A = splice acceptor). The open triangle represents a predicted transcriptional start site (TSS). Predicted unspliced and spliced forms of WT-circRNA-6 are shown in grey. The primers used to amplify linear and circular vIRF4 RNAs are shown as arrows. (D) The effect of 57KO on the production of vIRF4 linear and circRNAs. The total RNA from WT or 57KO cells isolated 24 h after induction of viral lytic cycle with or without RNase R digestion was used as a template to amplify circular vIRF4 RNA using a primer pair of oVM490 and oVM516 and linear spliced vIRF4 RNA using a primer pair of oVM489 and oVM491 as shown in the panel C. Host GAPDH RNA was amplified as an RNA loading control with the primer pair shown in the S6 Table. The percentage of individual RNA was calculated based on band signal density using ethidium bromide-stained agarose gels using ImageLab software (BioRad) with the amount of PCR products obtained from WT RNA without RNase R treatment set as 100%. The asterisk (*) indicates a heteroduplex band derived from two RT-PCR products as described [75].</p

Ectopic ORF57 inhibits alternative splicing of bicistronic ORF70/K3 RNAs to regulate the expression of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3 in HEK293T cells.

(A) A number of the amino acid residues of full length (FL), spliced (SP), truncated (TR) ORF70 and K3 proteins expressed from the corresponding RNA splicing isoforms are shown on the right (see details in Fig 10B). (B) Diagram of an ORF70/K3 minigene with a FLAG-tag on the ORF70 N-terminus and a c-myc-tag on the K3 C-terminus. The minigene under control by a CMV IE promoter contains the viral genomic DNA fragment covering the K3 and ORF70 coding regions and their intergenic region to ensure RNA splicing of this bicistronic RNA transcript. (C and D) Western (C, WB) and Northern blot (D, NB) analyses of HEK293T cells transfected with various ratios of the ORF70/K3 minigene over the ORF57 expression (+) or empty (-) vectors. (C) Expression of ORF70 protein was detected by WB using an anti-FLAG and K3 protein using an anti-c-myc antibody. ?, unknown protein band. (D) The changes in ORF70/K3 RNA splicing from the minigene vector in HEK293T cells in the absence (-) or presence (+) of ectopic ORF57 expression were monitored by Northern blot analysis on total cell RNA with two separate oligoprobes shown in Fig 10B. The 18S rRNA level detected by ethidium bromide (EtBr) staining was used as a loading control. (E) A diagram depicting the role of ORF57 in the regulation of ORF70/K3 RNA alternative splicing to mediate a switch from K3 to ORF70 protein expression during KSHV lytic replication.</p

Mapping of 57KO-specific splice junctions in intronless and antisense viral transcripts.

(A) The RNA splice junctions supported with ≥10 splice reads from the 57KO genome were mapped to intronless viral transcripts. See Figs 3 and 4 for a detailed description. (B) RT-PCR validation of selected 57KO-specific RNA splicing events using total RNA from BCBL-1 57KO cells treated with VA for 24 h. The sizes of expected amplicons with indicated primer pairs in the presence of reverse transcriptase (+RT) are shown below, together with the chromatographs from Sanger sequencing confirming the mapped splice junctions. The asterisks (*) mark the non-specific RT-PCR amplicons. SJ-61+6 means a RT-PCR product from the double RNA splicing first from the splice junction SJ-61 and then SJ-6 (see Fig 3).</p