Multiple long-range <i>cis</i> interactions generate CTCF insulator-dependent viral chromatin domains in quiescent HSV-1 genomes

In cellular genomes, CCCTC-binding factor (CTCF) insulators impact transcription over small distances in a one-dimensional manner and over much longer distances in a three-dimensional manner by maintaining chromatin loops. We have previously shown that the latent HSV-1 genome contains CTCF insulators that function to regulate lytic transcription of adjacent genes in a one-dimensional manner. Here, we test the hypothesis that HSV-1 CTCF insulators nucleate chromatin loops to regulate the expression of distance-separated gene regions through three-dimensional organization of viral genomes. We used 4C-seq methods to identify multiple long-range <i>cis</i> interactions in HSV-1 genomes that generate viral chromatin domains, including those nucleated by the viral CTCF insulator CTRL2. Deletion of the CTRL2 insulator disrupted these viral chromatin domains. Loop-nucleating interactions were quantitated with a novel approach (UMI-4C-seq) that utilizes unique molecular identifiers to label and count chromatin interactions associated with specific viewpoint primers. <i>Cis</i>-interaction peaks across four different viewpoints were quantified. Viral genomes lacking CTRL2 displayed more <i>cis</i>-interaction peaks and wider ranges of interaction lengths compared to wt virus, suggesting altered chromatin organization. Furthermore, differential looping analysis showed that viral genomes lacking CTRL2 displayed a more transcriptionally permissive chromatin environment. Thus, the CTRL2 insulator functions as a critical regulator of long-range chromatin interactions, and its deletion reshapes the viral chromatin landscape, leading to a more accessible and dynamic regulatory environment that may influence HSV-1 transcriptional programs and latency-associated chromatin states.IMPORTANCEHSV-1 is a significant lifelong human pathogen that infects 70% of adults worldwide. The latent HSV-1 genome is chromatinized and maintained in distinct chromatin structures that silence the virus, while reactivation is facilitated by transient reversal of host factors that maintain those chromatin domains. Understanding how this happens is critical for the development of novel therapeutics. It is becoming clear that CTCF insulators play a key role in the reversal that leads to reactivation. CTCF insulators are essential regulators of chromatin structure and gene expression in mammalian cells and play vital regulatory roles in transcriptional control of DNA viruses by organizing chromatin architecture during both latent and lytic stages of virus lifecycles. Here, we present the first report that latent HSV-1 genomes are organized into 3D structures to support latency yet allow the viral genome to reactivate, opening the door for future therapeutic targets.