LANA-dependent transcription-replication conflicts and R-loops at the terminal repeats (TR) correlate with KSHV episome maintenance

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 RNAPII eliminated R-loop formation at TR and reduced active histone modifications H3K4me3 and H3K27ac, with a corresponding increase in heterochromatic H3K9me3. RNAPII inhibitors also disrupted LANA binding to the TR, but did not eliminate LANA-NBs. We show that LANA can induce R-loops on a plasmid containing 8, but not 2 copies of the TR, and that the N-terminal histone binding function of LANA is required for this activity. RNaseH treatment eliminated R-loops and reduced LANA binding to the TR. 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.