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

Abstract

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 (ΔCTRL2) 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 ΔCTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ΔCTRL2, 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 ΔCTRL2 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 ΔCTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the ΔCTRL2 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.

Regulation of host and viral promoters during human cytomegalovirus latency via US28 and CTCF

“…CTCF has well-established roles in the latencies of the herpesviruses HSV-1, EBV, and KSHV [33][34][35][36][37][38][39][40][41], where particular attention has been paid to control of viral transcription. Many of these studies have used high throughput technologies such as ChIP-Seq and Hi-C to elucidate complex interactions and identify distal chromatin contacts that are important for the virus.…”

“…CTCF can also bind the genomes of DNA viruses and integrated retroviruses, resulting in changes in viral gene expression [29][30][31][32]. CTCF has been shown to be important in the regulation of latency and reactivation of the herpesviruses HSV-1, EBV, and KSHV [33][34][35][36][37][38][39][40][41], and, interestingly, CTCF can also bind within the IE region of the HCMV genome [42]. During lytic infection, CTCF binds to the first intron of the IE1/IE2 locus to repress IE gene expression [42] and, thus, we hypothesised that CTCF binding to this region may be important during latency, when IE transcription must be repressed.…”

Deletion of Herpes Simplex Virus 1 MicroRNAs miR-H1 and miR-H6 Impairs Reactivation

“…Pri-miR-H6/UDG expression was found to be dependent on ICP27 (51), implicating differential regulation of a spliced miR-H6 precursor during acute infection and a Drosha-processed miR-H6 precursor in the absence of ICP27 during the establishment of latency. Importantly, this suggests a possible cis-regulation mechanism of the biallelic LAT miRNAs that would be regulated differently if they were next to UL1 and UL2 (tRL) or in the iRL, where insulator elements were recently shown to influence epigenetic repression during HSV-1 latency (52). In addition, these multiple cis elements in the LAT promoter region make ectopic expression approaches likely to influence the epigenetic repression of an ectopically expressed miR-H1 or miR-H6 in another part of the virus.…”

Chromatin-mediated epigenetic regulation of HSV-1 transcription as a potential target in antiviral therapy

“…During latency, viral transcription is severely restricted, there is no significant expression of any viral proteins, or viral DNA replication, and no infectious virions are produced. The mechanisms that result in the silencing of the viral genomes are likely multifactorial and remain incompletely characterized, but there is a general consensus in that chromatin epigenetics play a major role ( Bloom et al, 2010 ; Cliffe and Wilson, 2017 ; Knipe and Cliffe, 2008 ; Lieberman, 2016 ; Nicoll et al, 2012 ; Singh and Tscharke, 2020 ; Washington et al, 2019 ); recently reviewed in ( Kobiler and Afriat, 2021 ).…”

“…The second hurdle may appear to pose an even bigger challenge. Most models propose that HSV-1 and 2 are epigenetically regulated by the same mechanisms that govern cellular epigenetic regulation ( Bloom et al, 2010 ; Cliffe and Wilson, 2017 ; Knipe et al, 2017 ; Kristie, 2015 ; Lieberman, 2016 ; Nicoll et al, 2012 ; Singh and Tscharke, 2020 ; Washington et al, 2019 ). The obvious expectation from these models is that any epigenetic drug would equally affect viral and cellular chromatin, regardless of continuous or discontinuous use.…”