LANA-Dependent Transcription-Replication Conflicts and R-Loops at the Terminal Repeats (TR) Correlate with KSHV Episome Maintenance

Abstract

Transcription-replication conflicts frequently occur at repetitive DNA elements involved in genome maintenance functions. The KSHV terminal repeats (TR) function as the viral episome maintenance element when bound by the viral encoded nuclear antigen LANA. Here, we show that transcription-replication conflicts occur at or near LANA binding sites in the TR. We show by proximity ligation assay (PLA) that PCNA and RNAPII colocalize with LANA-nuclear bodies (LANA-NBs). Using DNA-RNA-IP (DRIP) assays with S9.6 antibody, we demonstrate that R-loops form at the TR. We find that these R-loops are also associated with histone H3pS10 a marker for R-loops associated with transcription-replication conflicts. Inhibitors of RNA polymerase eliminated LANA binding to the TR, along with the loss of R-loops and activation associated histone modifications, and the accumulation of heterochromatic marks. We show that LANA can induce all of these features on a plasmid containing 8, but not 2 copies of the TR, correlating strongly with episome maintenance function. Taken together, our study indicates that LANA induces histone modifications associated with RNA and DNA polymerase activity and the formation of R-loops that correlate with episome maintenance function. These findings provide new insights into mechanisms of KSHV episome maintenance during latency and more generally for genome maintenance of repetitive DNA.

Keywords: DNA virus; H3pS10; KSHV; LANA; R-loops; episome maintenance.

Fig. 1.

Colocalization of transcription and replication machinery with LANA at KSHV TR. A. Schematic of the KSHV genome showing the terminal repeats (TR) relative to the unique region open reading frames (blue) and primer positions for TR, ORF45, and ORF75. B and C. ChIP-qPCR for RNAP II pS5, RNAPII pS2, MCM2, PCNA or control IgG assayed at the TR, ORF45 or ORF75 loci in BCBL1 (B) or iSLK (C) cell lines. ** p<.01, *** p<.001, student 2-tailed t-test, n=3 biological replicates. D. Representative IF microscopy image showing PLA (green) for PCNA+RNAP II-pS2 or PCNA+RNAPII-pS5, RFP-LANA (red), Dapi (blue) and merge. 60X. E. Quantification of IF images showing % colocalization of PLA signal with LANA-NBs. ** p<.01, pairwise Anova.

Fig. 2.

R-loop formation at the KSHV TR. A. DRIP assay with BCBL1 cells using S9.6 (blue) or control IgG (black) assayed with primers for cellular actin, KSHV TR, ORF45 or ORF75. B. Same as in panel A, except for KSHV TR or ORF16 and with specificity control RNase H treatment (pink). *** P<.001, student two-tailed t-test. C. PLA analysis of LANA+S9.6 (red signal) in BCBL1 cells. Dapi (Blue). No antibody control is shown in top panel. D. IGV screen shot of RNA transcripts mapped to KSHV TR region using public data sets for GROseq in BCBL1 in latent or lytic conditions (GSM4414007, GSM4414008). The reference map consists of a small region of the unique region with K15 and 2 copies of the TR. TR transcripts Tr(1) and Tr(2) are indicated above.

Fig. 3.

Cell cycle dependent accumulation of H3pS10 at the TR. A. ChIP-qPCR for LANA, H3pS10 and IgG at the TR, ORF45, ORF50, and ORF75 loci of KSHV in BCBL1 cells. B-C. ChIP-qPCR for RNAPII pS2, MCM, H3pS10 during different stages of the cell cycle using centrifugal elutriation for G1, G1/S, S, Late S, G2, and G2/M. ChIP-qPCR was analyzed at the TR (panel B) or at the ORF16 locus (panel C). D. IF of single cell showing H3pS10 (red), LANA (green), Dapi (blue) and merge. E. PLA for H3pS10+LANA (green), with RFP-LANA (red), Dapi (blue) and merge in RFP-LANA iSLK cells. No-antibody control shown in top panels. F. Quantification of data for representative IF images shown in panel E. **** p<.00001, **p <0.01 using two-tailed t test with Mann-Whitney test and Welch’s correction.

Fig. 4.

IF colocalization of PCNA-H3pS10-LANA in iSLK cells. A. PCNA (green), LANA (red), Dapi (blue). B. Quantification of the percent of LANA-NBs colocalized with PCNA. C. H3pS10 (green), LANA (red), Dapi (blue). D. Quantification of the percentage of LANA-NBs colocalized with H3pS10. E. PCNA (green), H3pS10 (red), Dapi (blue). F. Quantification of the percentage of PCNA foci colocalized with H3pS10. G. Combined IF with PCNA (green), LANA (red), H3pS10 (blue). H. Quantification of the percentage of LANA-NBs colocalized with H3pS10 and PCNA. N=4, total of 35 cells, **p<0.01, student two-tailed t-test.

Fig. 5.

RNA polymerase inhibitors block LANA binding, R-loop formation, and epigenetic programming at the TR. A. Western blot of LANA, Pol2-pS2, Pol2-pS5, and b-Actin in BCBL1 cells treated with flavopiridol (FVP) for 0, 15 min or 2 hrs. B. ChIP-qPCR for IgG, RNAPII pS2 or pS5 at the TR in BCBL1 cells treated with DMSO or FVP for 15’. C. DRIP assay with S9.6 or IgG for BCLB1 cells treated with DMSO or FVP for 15’. D. ChIP-qPCR for H3K4me3, H3K9me3, H3K27ac or IgG in BCBL1 cells treated with DMSO or FVP for 15’. E. ChIP-qPCR for CTCF, RAD21, or IgG at TR in BCLB1 cells treated with DMSO, FVP, or Triptolide for 15’. F. ChIP-qPCR for LANA or IgG at TR in BCLB1 cells treated with DMSO or FVP for 15’ or Triptolide for 15’. ** p<.01, ***p<.001, two tailed student t-test.

Fig. 6.

LANA and TR repeat number dependence of RNA polymerase II activation, epigenetic programming and R-loop formation at the KSHV TR. A. Schematic of the experimental design for co-transfection of either 2xTR or 8xTR episome templates in combination with either empty FLAG-vector (F-Vector) or vector expressing FLAG-LANA (F-LANA) into 293T cells. B. ChIP-qPCR of 8xTR episome template with F-Vector or F-LANA assayed for LANA, H3K4me3, H3K9me3, H3K29ac or IgG. C. ChIP-qPCR for 8xTR episome template with F-Vector of F-LANA assayed for LANA, RNAP II pS2, pS5, or IgG. D. ChIP-qPCR with 2xTR or 8xTR template in the presence of F-LANA assayed for LANA, H3K4me3, H3K27ac, or IgG. E. ChIP-qPCR for 2xTR or 8xTR in present of F-LANA assayed for RNAP II pS2, pS5 or IgG. F. DRIP with S9.6 or IgG assayed at the TR for BCBL1, 8xTR with F-Vector, 2xTR with F-LANA or 8xTR with F-LANA. G. Western blot of 293T cells transfected with F-Vector of F-LANA probed for FLAG or b-actin. * <.05, ** p<.01, p<.001, two tailed student t-test.

Fig. 7.. Model of LANA-dependent R-loops formed by collisions between elongating RNA polymerase II and DNA replication machinery at the TR.

Inhibition of RNA polymerase leads to a loss of LANA binding, R-loops and the formation of heterochromatic H3K9me3.