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CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection.

ABSTRACT: Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites (CTRL2) within the latency-associated transcript (LAT) coding sequences and found that loss of these CTCF-binding sites did not alter lytic replication or levels of establishment of latent infection, but their deletion reduced the ability of the virus to reactivate from latent infection. We also observed increased heterochromatin modifications on viral chromatin over the LAT promoter and intron. We therefore propose that CTCF binding at the CTRL2 sites acts as a chromatin insulator to keep viral chromatin in a form that is poised for reactivation, a state which we call poised latency.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a human pathogen that persists for the lifetime of the host as a result of its ability to establish latent infection within sensory neurons. The mechanism by which HSV-1 transitions from the lytic to latent infection program is largely unknown; however, HSV-1 is able to coopt cellular silencing mechanisms to facilitate the suppression of lytic gene expression. Here, we demonstrate that the cellular CCCTC-binding factor (CTCF)-binding site within the latency associated transcript (LAT) region is critical for the maintenance of a specific local chromatin structure. Additionally, loss of CTCF binding has detrimental effects on the ability to reactivate from latent infection. These results argue that CTCF plays a critical role in epigenetic regulation of viral gene expression to establish and/or maintain a form of latent infection that can reactivate efficiently.

SUBMITTER: Lee JS

PROVIDER: S-EPMC5801469 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection.

Lee Jennifer S JS Raja Priya P Pan Dongli D Pesola Jean M JM Coen Donald M DM Knipe David M DM

mBio 20180206 1

Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites (<i>CTRL2</i>) within the latency-associated transcript (<i>LAT</i>) coding sequences and found that loss of these CTCF-bi ...[more]

PMID: 29437926

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Project description: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.

Project description:The herpes simplex virus (HSV) genome is associated with heterochromatic histone modifications, including trimethylation of the lysine 27 residue of histone H3 (H3K27me3), during latent infection of neurons. Here we have examined the kinetics of general chromatin and H3K27me3 association with the viral genome during establishment of latent infection. Using both wild-type virus and a mutant virus that is unable to undergo replication in neurons, we found that histone H3 associates with viral gene promoters by 7 days postinfection (dpi). Levels of H3K27me3 were low at 7 dpi but increased dramatically by 14 dpi. Hence, general chromatin association and/or other factors may play a key role(s) in the initial silencing of lytic genes, and H3K27me3 may play a role in further suppression of the genome and/or the maintenance of latency. A component of Polycomb repressive complex 2 (PRC2), which mediates the addition of K27me3 to histone H3 (Suz12), was also recruited by 14 dpi. We have shown previously that the levels of H3K27me3 during latent infection are increased in the presence of the latency-associated transcript (LAT). However, the initial targeting of PRC2 was not found to be dependent on the LAT. We found that a component of the PRC1 complex (Bmi1), which binds to H3K27me3, was not enriched at promoters found previously to be enriched for H3K27me3. Our results are consistent with (i) chromatinization of viral DNA or other mechanisms causing the initial silencing of HSV lytic genes and (ii) facultative heterochromatin maintaining that silencing during latent infection of neurons. The human pathogen herpes simplex virus (HSV) hides for the lifetime of the host in peripheral neurons. The mechanism by which HSV is able to shut off its gene expression and persist in neurons is not known. Here we show that the HSV DNA first associates with histone H3, with later recruitment of Polycomb repressor complex 2 (PRC2) and trimethylation of the lysine 27 residue of histone H3 (H3K27me3), a modification associated with heterochromatin. This work indicates that the initial silencing of HSV gene expression is not correlated with enrichment of H3K27me3 and that PRC2 may be recruited to already-silenced genes to further silence gene expression and/or maintain gene silencing. We demonstrate that recruitment of PRC2 is not dependent upon expression of the noncoding HSV latency-associated transcripts, indicating the presence of unknown triggers for PRC2 recruitment during the establishment of latent infection.

Project description:Herpes simplex virus 1 (HSV-1) is a DNA virus belonging to the family Herpesviridae. HSV-1 infection causes severe neurological disease in the central nervous system (CNS), including encephalitis. Ferroptosis is a nonapoptotic form of programmed cell death that contributes to different neurological inflammatory diseases. However, whether HSV-1 induces ferroptosis in the CNS and the role of ferroptosis in viral pathogenesis remain unclear. Here, we demonstrate that HSV-1 induces ferroptosis, as hallmarks of ferroptosis, including Fe2+ overload, reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, and mitochondrion shrinkage, are observed in HSV-1-infected cultured human astrocytes, microglia cells, and murine brains. Moreover, HSV-1 infection enhances the E3 ubiquitin ligase Keap1 (Kelch-like ECH-related protein 1)-mediated ubiquitination and degradation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates the expression of antioxidative genes, thereby disturbing cellular redox homeostasis and promoting ferroptosis. Furthermore, HSV-1-induced ferroptosis is tightly associated with the process of viral encephalitis in a mouse model, and the ferroptosis-activated upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin E2 (PGE2) plays an important role in HSV-1-caused inflammation and encephalitis. Importantly, the inhibition of ferroptosis by a ferroptosis inhibitor or a proteasome inhibitor to suppress Nrf2 degradation effectively alleviated HSV-1 encephalitis. Together, our findings demonstrate the interaction between HSV-1 infection and ferroptosis and provide novel insights into the pathogenesis of HSV-1 encephalitis. IMPORTANCE Ferroptosis is a nonapoptotic form of programmed cell death that contributes to different neurological inflammatory diseases. However, whether HSV-1 induces ferroptosis in the CNS and the role of ferroptosis in viral pathogenesis remain unclear. In the current study, we demonstrate that HSV-1 infection induces ferroptosis, as Fe2+ overload, ROS accumulation, GSH depletion, lipid peroxidation, and mitochondrion shrinkage, all of which are hallmarks of ferroptosis, are observed in human cultured astrocytes, microglia cells, and murine brains infected with HSV-1. Moreover, HSV-1 infection enhances Keap1-dependent Nrf2 ubiquitination and degradation, which results in substantial reductions in the expression levels of antiferroptotic genes downstream of Nrf2, thereby disturbing cellular redox homeostasis and promoting ferroptosis. Furthermore, HSV-1-induced ferroptosis is tightly associated with the process of viral encephalitis in a mouse model, and the ferroptosis-activated upregulation of PTGS2 and PGE2 plays an important role in HSV-1-caused inflammation and encephalitis. Importantly, the inhibition of ferroptosis by either a ferroptosis inhibitor or a proteasome inhibitor to suppress HSV-1-induced Nrf2 degradation effectively alleviates HSV-1-caused neuro-damage and inflammation in infected mice. Overall, our findings uncover the interaction between HSV-1 infection and ferroptosis, shed novel light on the physiological impacts of ferroptosis on the pathogenesis of HSV-1 infection and encephalitis, and provide a promising therapeutic strategy to treat this important infectious disease with a worldwide distribution.

Project description:UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

Dataset Information

CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection.

Publications

CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection.

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