Stress Induced Transcription Factors Transactivate the Herpes Simplex Virus 1 Infected Cell Protein 27 (ICP27) Transcriptional Enhancer

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons, including sensory neurons within trigeminal ganglia. During latency, lytic cycle viral gene expression is silenced. However, stressful stimuli can trigger reactivation from latency. The viral tegument protein, VP-16, transactivates all immediate early (IE) promoters during productive infection. Conversely, cellular factors are expected to trigger viral gene expression during early stages of reactivation from latency and in non-neuronal cells that do not support high levels of productive infection. The glucocorticoid receptor (GR), synthetic corticosteroid dexamethasone, and certain stress-induced transcription factors cooperatively transactivate infected cell protein 0 (ICP0) and ICP4 promoters. Since ICP27 protein expression is required for productive infection, we hypothesized that the ICP27 promoter is transactivated by stress-induced transcription factors. New studies have demonstrated that ICP27 enhancer sequences were transactivated by GR and Krüppel-like factor 15 (KLF15). Mutation of a consensus Sp1 binding site within ICP27 enhancer sequences impaired transactivation by GR and KLF15. Chromatin immunoprecipitation studies have demonstrated that GR and KLF15 occupy ICP27 promoter sequences during productive infection. Cells transfected with an ICP27 enhancer fragment revealed the GR and KLF15 occupancy of ICP27 enhancer sequences required the intact Sp1 binding site. Notably, GR and KLF15 form a feed-forward transcription loop in response to stress, suggesting these cellular factors promote viral replication following stressful stimuli.

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The bovine herpesvirus 1 gene encoding infected cell protein 0 (bICP0) can inhibit interferon-dependent transcription in the absence of other viral genes

Journal of General Virology ◽

10.1099/vir.0.81109-0 ◽

2005 ◽

Vol 86 (10) ◽

pp. 2697-2702 ◽

Cited By ~ 40

Author(s):

Gail Henderson ◽

Yange Zhang ◽

Clinton Jones

Keyword(s):

Gene Expression ◽

Ring Finger ◽

Viral Gene Expression ◽

Bovine Herpesvirus ◽

Productive Infection ◽

Cell Protein ◽

Viral Gene ◽

Bovine Herpesvirus 1 ◽

Infected Cell Protein ◽

Herpesvirus 1

The infected cell protein 0 (bICP0) encoded by Bovine herpesvirus 1 (BHV-1) stimulates viral gene expression and productive infection. As bICP0 is expressed constitutively during productive infection, it is considered to be the major viral regulatory protein. Like other alphaherpesvirus ICP0 homologues, bICP0 contains a zinc RING finger near its N terminus that activates transcription and regulates subcellular localization. In this study, evidence is provided that bICP0 represses the human beta interferon (IFN-β) promoter and a simple promoter with consensus IFN-stimulated response elements following stimulation with double-stranded RNA (polyinosinic–polycytidylic acid), IFN regulatory factor 3 (IRF3) or IRF7. bICP0 also inhibits the ability of two protein kinases (TBK1 and IKKε) to activate IFN-β promoter activity. The zinc RING finger is necessary for inhibiting IFN-dependent transcription in certain cell types. Collectively, these studies suggest that bICP0 activates productive infection by stimulating viral gene expression and inhibiting IFN-dependent transcription.

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Simple and Rapid High-Throughput Assay to Identify HSV-1 ICP0 Transactivation Inhibitors

10.1101/2021.01.13.426532 ◽

2021 ◽

Author(s):

Cindy Y. Ly ◽

Chunmiao Yu ◽

Peter McDonald ◽

Anuradha Roy ◽

David Johnson ◽

...

Keyword(s):

Gene Expression ◽

High Throughput ◽

Viral Gene Expression ◽

Lytic Replication ◽

Cell Protein ◽

Viral Gene ◽

Herpes Simplex Virus 1 ◽

Cellular Factors ◽

In Cells ◽

Hsv 1

AbstractHerpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to it’s ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Mechanisms by which ICP0 activates viral gene expression and the cellular factors involved are largely unknown. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6β (wt) and dlx3.1-6β (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter::lacz expression cassette. A ≥10-fold difference in β-galactosidase activity was observed in cells infected with KOS6β compared to dlx3.1-6β, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z′- factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.

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RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

Journal of Virology ◽

10.1128/jvi.00943-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Daniel J. Kim ◽

William Khoury-Hanold ◽

Priyanka Caroline Jain ◽

Jonathan Klein ◽

Yong Kong ◽

...

Keyword(s):

Transcription Factors ◽

Binding Sites ◽

Viral Gene Expression ◽

Viral Gene ◽

Drg Neurons ◽

Herpes Simplex Virus 1 ◽

Viral Genes ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2 can efficiently establish lifelong, transcriptionally silent latency states in sensory neurons to escape host detection. While host factors have previously been associated with long-range insulators in the viral genome, it is still unknown whether host transcription factors can repress viral genes more proximately to promote latency in dorsal root ganglion (DRG) neurons. Here, we assessed whether RUNX (runt-related transcription factor) transcription factors, which are critical in the development of sensory neurons, could be binding HSV-1 genome directly to suppress viral gene expression and lytic infection. Using previously published transcriptome sequencing data, we confirmed that mouse DRG neurons highly express Runx1 mRNA. Through computational analysis of HSV-1 and HSV-2 genomes, we observed that putative RUNX consensus binding sites (CBSs) were more enriched and more closely located to viral gene transcription start sites than would be expected by chance. We further found that RUNX CBSs were significantly more enriched among genomes of herpesviruses compared to those of nonherpesviruses. Utilizing an in vitro model of HSV-1 infection, we found that overexpressed RUNX1 could bind putative binding sites in the HSV-1 genome, repress numerous viral genes spanning all three kinetic classes, and suppress productive infection. In contrast, knockdown of RUNX1 in neuroblastoma cells induced viral gene expression and increased HSV-1 infection in vitro. In sum, these data support a novel role for RUNX1 in directly binding herpesvirus genome, silencing the transcription of numerous viral genes, and ultimately limiting overall infection. IMPORTANCE Infecting 90% of the global population, HSV-1 and HSV-2 represent some of the most prevalent viruses in the world. Much of their success can be attributed to their ability to establish lifelong latent infections in the dorsal root ganglia (DRG). It is still largely unknown, however, how host transcription factors are involved in establishing this latency. Here, we report that RUNX1, expressed highly in DRG, binds HSV-1 genome, represses transcription of numerous viral genes, and suppresses productive in vitro infection. Our computational work further suggests this strategy may be used by other herpesviruses to reinforce latency in a cell-specific manner.

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Transactivation of HSV-1 Infected Cell Protein 4 (ICP4) Enhancer by Glucocorticoid Receptor and Stress-Induced Transcription Factors Requires Overlapping Krüppel like Transcription Factor 4/Sp1 Binding Sites.

Journal of Virology ◽

10.1128/jvi.01776-20 ◽

2020 ◽

Author(s):

Jeffery B. Ostler ◽

Prasanth Thunuguntla ◽

Bailey Y. Hendrickson ◽

Clinton Jones

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Glucocorticoid Receptor ◽

Binding Sites ◽

Zinc Finger Protein ◽

Productive Infection ◽

Cell Protein ◽

Simplex Virus ◽

Enhancer Sequences ◽

Hsv 1

Following acute infection, Herpes simplex virus 1 (HSV-1) lytic cycle viral gene expression is silenced: consequently, lifelong latency in neurons is established. Certain external stimuli that trigger reactivation from latency also activate the glucocorticoid receptor (GR). The synthetic corticosteroid dexamethasone, but not a GR specific antagonist, increases the frequency of explant-induced reactivation from latency and stimulates productive infection. Furthermore, dexamethasone increases expression of cellular transcription factors in trigeminal ganglionic neurons: for example, Slug and three Krüppel-like transcription factor (KLF) family members, KLF4, KLF15 and promyelocytic leukemia zinc finger protein (PLZF). Consequently, we hypothesized that stress-induced transcription factors stimulate ICP4 expression, a viral transcriptional regulator required for productive infection. New studies demonstrated GR and KLF4, PLZF, or SLUG cooperatively transactivate the ICP4 enhancer upstream of a minimal promoter in monkey kidney (Vero), and mouse neuroblastoma cells (Neuro-2A). Strikingly, mutagenesis of two KLF4/Sp1 binding sites reduced GR plus KLF4, PLZF, or SLUG mediated transactivation to basal levels. A consensus enhancer (E)-box adjacent to a KLF4/Sp1 binding site was also required for GR and SLUG, but not KLF family member, mediated transactivation of the ICP4 promoter. Chromatin immunoprecipitation studies (ChIP) revealed GR and stress induced transcription factors occupy ICP4 enhancer sequences. Conversely, specific binding was generally reduced in the KLF4/Sp1 mutant. Furthermore, GR and Slug occupancy of ICP4 enhancer sequences was reduced in the E-Box mutant. Based on these studies. we suggest stressful stimuli can trigger productive infection because GR and specific stress-induced transcription factors activate ICP4 expression. IMPORTANCE Certain stressful stimuli activate the glucocorticoid receptor (GR) and increase the incidence of herpes simplex virus type I (HSV-1) reactivation from latency. For example, a corticosteroid antagonist impairs productive infection and virus shedding following explant of trigeminal ganglia from latently infected mice. Infected cell protein 4 (ICP4) is the only immediate early viral transcriptional regulator required for productive infection suggesting stressful stimuli stimulate ICP4 expression. New studies revealed GR and stress-induced transcription factors identified during reactivation from latency, SLUG and three Krüppel-like transcription factor family members (KLF4, KLF15 and promyelocytic leukemia zinc finger protein) cooperatively transactivate the ICP4 enhancer. Two KLF4 consensus binding sites were crucial for cooperative transactivation of the ICP4 enhancer. A consensus enhancer-box also mediated cooperative transactivation of the ICP4 enhancer by GR and SLUG. The ability of GR and stress-induced transcription factors to transactivate ICP4 enhancer activity is predicted to trigger productive infection following stressful stimuli.

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Two Pioneer Transcription Factors, Krüppel-Like Transcription Factor 4 and Glucocorticoid Receptor, Cooperatively Transactivate the Bovine Herpesvirus 1 ICP0 Early Promoter and Stimulate Productive Infection

Journal of Virology ◽

10.1128/jvi.01670-19 ◽

2019 ◽

Vol 94 (4) ◽

Author(s):

Fouad S. El-mayet ◽

Laximan Sawant ◽

Prasanth Thunuguntla ◽

Jing Zhao ◽

Clinton Jones

Keyword(s):

Transcription Factors ◽

Glucocorticoid Receptor ◽

Viral Gene Expression ◽

Bovine Herpesvirus ◽

Transcription Unit ◽

Productive Infection ◽

Cell Protein ◽

Viral Gene ◽

Bovine Herpesvirus 1 ◽

Herpesvirus 1

ABSTRACT An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), i.e., KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives bovine infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR and KLF family members transactivate the bICP0 E promoter. GR and KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity in a ligand-independent manner in mouse neuroblastoma cells (Neuro-2A). Furthermore, GR and KLF4 stimulated productive infection. Mutating both half GR binding sites did not significantly reduce GR- and KLF4-mediated transactivation of the bICP0 E promoter, suggesting that a novel mechanism exists for transactivation. GR and KLF15 cooperatively stimulated bICP0 activity less efficiently than GR and KL4: however, KLF6, PLZF, and GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest that cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict that increased corticosteroid levels activate the glucocorticoid receptor (GR). Consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, bovine infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Krüppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR and KLF15 cooperate to stimulate bICP0 E promoter activity but significantly less than GR and KLF4. The bICP0 E promoter contains enhancer-like domains necessary for GR- and KLF4-mediated transactivation that are distinct from those for GR and KLF15. Stress-induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.

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Stress-Induced Cellular Transcription Factors Expressed in Trigeminal Ganglionic Neurons Stimulate the Herpes Simplex Virus 1 ICP0 Promoter

Journal of Virology ◽

10.1128/jvi.02476-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 13042-13047 ◽

Cited By ~ 10

Author(s):

Devis Sinani ◽

Ethan Cordes ◽

Aspen Workman ◽

Prasanth Thunuguntia ◽

Clinton Jones

Keyword(s):

Transcription Factors ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Protein ◽

Herpes Simplex Virus 1 ◽

Viral Promoters ◽

Infected Cell Protein ◽

Simplex Virus ◽

Cellular Transcription

Alphaherpesvirinaefamily members can reactivate from latency following stress. The synthetic corticosteroid dexamethasone induces certain cellular transcription factors in murine and bovine trigeminal ganglionic neurons. Three dexamethasone-induced transcription factors, Krüppel-like factor 15, Slug, and SPDEF, stimulated the herpes simplex virus type 1-infected cell protein 0 (ICP0) promoter more than 150-fold. Conversely, other viral promoters (VP16 and ICP4) were not strongly stimulated, suggesting that the ICP0 promoter is preferentially activated by dexamethasone-simulated stress.

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The Glucocorticoid Receptor (GR) Stimulates Herpes Simplex Virus 1 Productive Infection, in Part Because the Infected Cell Protein 0 (ICP0) Promoter Is Cooperatively Transactivated by the GR and Krüppel-Like Transcription Factor 15

Journal of Virology ◽

10.1128/jvi.02063-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 6

Author(s):

Jeffery B. Ostler ◽

Kelly S. Harrison ◽

Kayla Schroeder ◽

Prasanth Thunuguntla ◽

Clinton Jones

Keyword(s):

Transcription Factor ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Infected Cell ◽

Promoter Activity ◽

Productive Infection ◽

Cell Protein ◽

Herpes Simplex Virus 1 ◽

Infected Cell Protein ◽

Infected Cell Protein 0

ABSTRACTFollowing acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. Physical, emotional, and chemical stresses are linked to increasing the incidence of reactivation from latency, but the mechanism of action is not well understood. In general, stress increases corticosteroid levels, leading to activation of the glucocorticoid receptor (GR), a pioneer transcription factor. Consequently, we hypothesized that stress-mediated activation of the GR can stimulate productive infection and viral gene expression. New studies demonstrated that the GR-specific antagonist (CORT-108297) significantly reduced HSV-1 productive infection in mouse neuroblastoma cells (Neuro-2A). Additional studies demonstrated that the activated GR and Krüppel-like transcription factor 15 (KLF15) cooperatively transactivated the infected cell protein 0 (ICP0) promoter, a crucial viral regulatory protein. Interestingly, the synthetic corticosteroid dexamethasone and GR or KLF15 alone had little effect on ICP0 promoter activity in transfected Neuro-2A or Vero cells. Chromatin immunoprecipitation (ChIP) studies revealed that the GR and KLF15 occupied ICP0 promoter sequences important for transactivation at 2 and 4 h after infection; however, binding was not readily detected at 6 h after infection. Similar results were obtained for cells transfected with the full-length ICP0 promoter. ICP0 promoter sequences lack a consensus “whole” GR response element (GRE) but contain putative half-GREs that were important for dexamethasone induced promoter activity. The activated GR stimulates expression of, and interacts with, KLF15; consequently, these data suggest KLF15 and the GR form a feed-forward loop that activates viral gene expression and productive infection following stressful stimuli.IMPORTANCEThe ability of herpes simplex virus 1 (HSV-1) to periodically reactivate from latency results in virus transmission and recurrent disease. The incidence of reactivation from latency is increased by chronic or acute stress. Stress increases the levels of corticosteroids, which bind and activate the glucocorticoid receptor (GR). Since GR activation is an immediate early response to stress, we tested whether the GR influences productive infection and the promoter that drives infected cell protein 0 (ICP0) expression. Pretreatment of cells with a GR-specific antagonist (CORT-108297) significantly reduced virus replication. Although the GR had little effect on ICP0 promoter activity alone, the Krüppel-like transcription factor 15 (KLF15) cooperated with the GR to stimulate promoter activity in transfected cells. In transfected or infected cells, the GR and KLF15 occupied ICP0 sequences important for transactivation. Collectively, these studies provide insight into how stress can directly stimulate productive infection and viral gene expression.

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Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422657112 ◽

2014 ◽

Vol 112 (1) ◽

pp. E49-E55 ◽

Cited By ~ 34

Author(s):

Te Du ◽

Zhiyuan Han ◽

Guoying Zhou ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cultured Cells ◽

Viral Gene Expression ◽

The Body ◽

Productive Infection ◽

Viral Gene ◽

Infected Cells ◽

Simplex Virus ◽

Portal Of Entry

The key events in herpes simplex virus (HSV) infections are (i) replication at a portal of entry into the body modeled by infection of cultured cells; (ii) establishment of a latent state characterized by a sole latency-associated transcript and microRNAs (miRNAs) modeled in murine peripheral ganglia 30 d after inoculation; and (iii) reactivation from the latent state modeled by excision and incubation of ganglia in medium containing anti-NGF antibody for a timespan of a single viral replicative cycle. In this report, we examine the pattern of synthesis and accumulation of 18 HSV-1 miRNAs in the three models. We report the following: (i) H2-3P, H3-3P, H4-3P, H5-3P, H6-3P, and H7-5P accumulated in ganglia harboring latent virus. All but H4-3P were readily detected in productively infected cells, and most likely they originate from three transcriptional units. (ii) H8-5P, H15, H17, H18, H26, and H27 accumulated during reactivation. Of this group, only H26 and H27 could be detected in productively infected cells. (iii) Of the 18 we have examined, only 10 miRNAs were found to accumulate above background levels in productively infected cells. The disparity in the accumulation of miRNAs in cell culture and during reactivation may reflect differences in the patterns of regulation of viral gene expression during productive infection and during reactivation from the latent state.

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