Dataset Information

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 (?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 ?CTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ?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 ?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 ?CTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the ?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.

SUBMITTER: Washington SD

PROVIDER: S-EPMC6580944 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Washington Shannan D SD Singh Pankaj P Johns Richard N RN Edwards Terri G TG Mariani Michael M Frietze Seth S Bloom David C DC Neumann Donna M DM

Journal of virology 20190614 13

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 17<i>syn</i>+ virus. We also showed that CTRL2 was a functional enhancer-blocking insulator in both ...[more]

PMID: 30996085

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Project description:During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Project description:The regulatory functions of 10 individual viral microRNAs (miRNAs) that are abundantly expressed from the herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) region remain largely unknown. Here, we focus on HSV-1 miRNA miR-H8, which is within the LAT 3p exon, antisense to the first intron of ICP0, and has previously been shown to target a host glycosylphosphatidylinositol (GPI)-anchoring pathway. However, the functions of this miRNA have not been assessed in the context of the viral genome during infection. Therefore, we constructed a recombinant virus lacking miR-H8 (17dmiR-H8) and compared it to the parental wild-type and rescue viruses to characterize phenotypic differences. In rabbit skin cells, 17dmiR-H8 exhibited only subtle reductions in viral yields. In contrast, we found significant decreases in both viral yields (8-fold) and DNA replication (9.9-fold) in murine neuroblastoma cells, while 17dmiR-H8 exhibited a 3.6-fold increase in DNA replication in differentiated human neuronal cells (Lund human mesencephalic [LUHMES] cells). These cell culture phenotypes suggested potential host- and/or neuron-specific roles for miR-H8 in acute viral replication. To assess whether miR-H8 plays a role in HSV latency or reactivation, we used a human in vitro reactivation model as well as mouse and rabbit reactivation models. In the LUHMES cell-induced reactivation model, there was no difference in viral yields at 48 h postreactivation. In the murine dorsal root ganglion explant and rabbit ocular adrenergic reactivation models, the deletion of miR-H8 had no detectable effect on genome loads during latency or reactivation. These results indicate that miR-H8 is dispensable for the establishment of HSV-1 latency and reactivation.IMPORTANCE Herpesviruses have a remarkable ability to sustain lifelong infections by evading host immune responses, establishing a latent reservoir, and maintaining the ability to reactivate the lytic cascade to transmit the virus to the next host. The HSV-1 latency-associated transcript region is known to regulate many aspects of HSV-1 latency and reactivation, although the mechanisms for these functions remain unknown. To this end, we characterize an HSV-1 recombinant containing a deletion of a LAT-encoded miRNA, miR-H8, and demonstrate that it plays no detectable role in the establishment of latency or reactivation in differentiated human neurons (LUHMES cells) and mouse and rabbit models. Therefore, this study allows us to exclude miR-H8 from phenotypes previously attributed to the LAT region. Elucidating the genetic elements of HSV-1 responsible for establishment, maintenance, and reactivation from latency may lead to novel strategies for combating persistent herpesvirus infections.

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Dataset Information

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

Publications

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

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