Increased RNA Polymerase Activity and Pausing at CTCF binding sites on the Epstein Barr Virus Genome During Reactivation from Latency

The ability of Epstein-Barr Virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent to lytic switch we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in latently infected Mutu-I cells at 1, 4 and 12 h post-reactivation. During latency, Pol activity was primarily limited to the EBNA1 transcript initiating at the Qp promoter, the EBER and RPMS1/BART regions and the BHLF1 transcript. Unexpectedly at early time-points post-reactivation, the EBV transcripts with the largest increase in Pol activity were LMP-2A, EBER-1 and RPMS1. Closer analysis of the PRO-Seq data at these regions revealed a distinct pattern of high Pol activity with bidirectional transcription and strong peaks indicative of Pol pausing. Alignment to ChIP-Seq data revealed a strong correlation with CTCF binding sites on the EBV genome. In addition, alignment to ATAC-Seq data indicated that many of these transcription regulatory regions were sites of accessible chromatin. Similar features were observed in Akata cells activated from latency with anti-IgG. Overall, these data suggest that during reactivation, EBV recruits RNA polymerase to CTCF binding sites where it transcribes short distances and pauses. These activities likely help open chromatin on the viral genome to initiate productive replication. Author summary The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B-cells and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remains unknown. In this study, we were able to precisely map RNA polymerase positioning and activity during latency and reactivation. Unexpectedly, RNA polymerase activity was not enriched at the IE genes during early reactivation but accumulated at distinct regions, characteristic of enhancers, on the EBV genome previously shown to be associated with CTCF and open chromatin. We speculate that Pol accumulation at these sites helps maintain open chromatin locally, eventually leading to changes in the quaternary structure of the EBV genome to promote lytic replication. ### Competing Interest Statement The authors have declared no competing interest.