The CCCTC Binding Factor, CTRL2, Modulates Heterochromatin Deposition and the Establishment of Herpes Simplex Virus 1 Latency In Vivo

The cellular insulator protein CTCF plays a role in herpes simplex virus 1 (HSV-1) latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the latency-associated transcript (LAT) enhancer was bound by CTCF during latency and underwent CTCF eviction at early times postreactivation in mice latently infected with 17syn+ virus. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (Delta CTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells but not neuronal cells. Following ocular infection, mouse survival decreased in the Delta CTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with Delta CTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the number of ganglia infected with Delta CTRL2 by 31 days postinfection relative to the level with 17syn+ infection, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) analyses of TG from Delta CTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the Delta CTRL2 recombinant genomes was altered compared to that of the wild type, indicating that the CTRL2 site modulates the repression of IE genes during latency. IMPORTANCE It is becoming increasingly clear that chromatin insulators play a key role in the transcriptional control of DNA viruses. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) utilize chromatin insulators to order protein recruitment and dictate the formation of three-dimensional DNA loops that spatially control transcription and latency. The contribution of chromatin insulators in alphaherpesvirus transcriptional control is less well understood. The work presented here begins to bridge that gap in knowledge by showing how one insulator site in HSV-1 modulates lytic gene transcription and heterochromatin deposition as the HSV-1 genome establishes latency.