The CCCTC binding factor, CTRL2, controls heterochromatin deposition and the establishment of HSV-1 latency in vivo

The cellular insulator  CCCTC-binding factor (CTCF) plays a role in HSV-1 latency through the establishment of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the LAT enhancer is bound by CTCF during latency and undergoes CTCF eviction at early times post-reactivation in mice latent with 17Syn+ . It has been previously shown that the CTRL2 binding domain is a functional insulator with both enhancer-blocking and silencing capabilities. Considering these findings, we hypothesized that the CTRL2 site plays a key role in regulating latent HSV-1 gene expression. To test this, we used a mutant virus with a 135 bp deletion spanning only the core CTRL2 domain (DCTRL2) in the 17Syn+ background. Following ocular infection of mice, we found significant differences between DCTRL2 and wild-type virus during the acute infection and latency. Notably, mouse mortalities were significantly higher in the DCTRL2 virus compared to the 17Syn+, while lower viral genomes were found after latency indicating that deletion of CTRL2 site disrupted latency establishment. This fact, coupled with increased LAT intron and VP16 expression indicated that the establishment of latency was disrupted in the DCTRL2 mutant. These data provide evidence that chromatin domains of the latent HSV-1 genome are in part maintained by the CTRL2 binding. 4-6 weeks of age BALB/c mice were anesthetized using intramuscular injections of ketamine and xylazine infected with 2 different strains of HSV1 (WT and DeltaCTRL2) in the corneal epithelium. (ChIP-seq) was performed using trigeminal ganglia (TG) from three mice.