Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 Reactivation In Vivo

Abstract

Herpes Simplex Virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild type virus post-reactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver an siRNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation. Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome post-reactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1 infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

The Role, Mechanism and Transcriptional Regulation of LAT in Herpes Simplex Virus Latency and Reactivation

“…Binding of CTRL sites, the insulators or enhancers, by the CTCF protein activates LAT promoter (LAP) transcription, making the virus latent. Recent works defining the CTCF-insulator's role in lytic gene transcription repression of β and γ herpes viruses further support the possibility that CTCF-insulators also regulate α herpes virus latency through lytic gene repression [1].…”

“…The IE genes encode the protein ICP0. It is important to note that the ICP0 encoding region is flanked by two CTCF binding motifs, the CTRL1 and CTRL2 [1]. Binding of CTRL sites, the insulators or enhancers, by the CTCF protein activates LAT promoter (LAP) transcription, making the virus latent.…”

“…Herpes simplex virus (HSV) can be latent in trigeminal neurons in the host for *Authors contributed equally. several years or establishes a lifelong latent infection [1] [2] [3]. HSV causes a variety of human diseases [4], including infectious corneal blindness [5], gastrointestinal disorders, esophageal disorders and genital herpes infections during both primary and recurrent infections [6] [7] [8].…”

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors

“…These loops serve to insulate domains of transcriptional regulation by disconnecting genes from nearby enhancer elements, and by restricting lateral spread of domains of both inhibitory and activating histone modifications (Hansen et al, 2019;Saldaña-Meyer et al, 2019). Interestingly, CTCF is known to play an important role in regulating transcriptional latency for a number of other viruses, including HSV, KSHV and HPV (Chau et al, 2006;Washington et al, 2018). However, CTCF is not known to have a role in regulation of HIV latency.…”

Strength in diversity: Understanding the pathways to herpes simplex virus reactivation

“…This apparent demarcation in the nature of the chromatin likely arises due to binding sites for the cellular insulator protein CCCTC-binding factor (CTCF) on the viral genome. Interestingly, CTCF eviction coincides with reactivation (Ertel et al, 2012; Washington et al, 2018b), and depletion of CTCF in vivo promotes reactivation (Washington et al, 2018a). Furthermore, one important binding site of CTCF, known as CTRL2, lies downstream of the LAT enhancer and separates it from the nearby lytic ICP0 gene (Amelio et al, 2006b).…”