The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a Delta CTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection. Herpes Simplex Virus 1 (HSV-1) remains a significant lifelong human pathogen. Over 70% of adults worldwide are infected with HSV-1 and following the establishment of a persistent latent infection in the peripheral nervous system, the virus can undergo reactivation. These reactivation episodes can result in recurrence of viral infection in the cornea and repeated episodes of recurrence can result in corneal blindness. HSV-1 remains a leading infectious cause of blindness worldwide. On a molecular level, both the incoming lytic virus and the latent viral genome are chromatinized for the purposes of silencing the virus. Viral gene expression in both lytic and reactivated HSV-1 requires the de-repression of chromatin on the viral genome but it remains unclear whether distinct transcriptional mechanisms regulate viral gene expression during these two stages of the viral life cycle. CCCTC-binding factors, also known as CTCF insulators, are essential regulators of chromatin structure and gene expression in mammalian cells and these cellular insulator elements also play vital regulatory roles in transcriptional control of all classes of herpesviruses as well. Existing research on CTCF and herpesviruses have been done in the context of herpesvirus latency but considering role of CTCF insulators in organizing chromatin architecture, it is logical that these important elements also play a role in either the initial chromatinization of virus, the de-repression of chromatin on incoming viruses so that transcription can occur- or both. Here we show that the CTRL2 insulator is required for de-repression of IE genes during the lytic infection and that the insulator has distinct and divergent functions during the lytic and latent stages of the viral lifecycle. To our knowledge these are the first data that show that a specific CTCF insulator in HSV-1 regulates chromatin accessibility in a site-specific manner. Understanding these mechanisms is particularly important for future drug discovery where cellular elements contributing to the de-repression of viral chromatin might be targeted.