Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle; consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress-induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated that KLF15 and the GR form a stable complex and that these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli.

Following 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. IMPORTANCE The 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.

BAF (Barrier to Autointegration Factor) is a highly conserved DNA binding protein that senses poxviral DNA in the cytoplasm and tightly binds to the viral genome to interfere with DNA replication and transcription. To counteract BAF, a poxviral-encoded protein kinase phosphorylates BAF, which renders BAF unable to bind DNA and allows efficient viral replication to occur. Herein, we examined how BAF phosphorylation is affected by herpes simplex virus type 1 (HSV-1) infection and tested the ability of BAF to interfere with HSV-1 productive infection. Interestingly, we found that BAF phosphorylation decreases markedly following HSV-1 infection. To determine whether dephosphorylated BAF impacts HSV-1 productive infection, we employed cell lines stably expressing a constitutively unphosphorylated form of BAF (BAF-MAAAQ) and cells overexpressing wild type (wt) BAF for comparison. Although HSV-1 production in cells overexpressing wtBAF was similar to that in cells expressing no additional BAF, viral growth was reduced approximately 80% in the presence of BAF-MAAAQ. Experiments were also performed to determine the mechanism of the antiviral activity of BAF with the following results. BAF-MAAAQ was localized to the nucleus, whereas wtBAF was dispersed throughout cells prior to infection. Following infection, wtBAF becomes dephosphorylated and relocalized to the nucleus. Additionally, BAF was associated with the HSV-1 genome during infection, with BAF-MAAAQ associated to a greater extent than wtBAF. Importantly, unphosphorylated BAF inhibited both viral DNA replication and gene expression. For example, expression of two regulatory proteins, ICP0 and VP16, were substantially reduced in cells expressing BAF-MAAAQ. However, other viral genes were not dramatically affected suggesting that expression of certain viral genes can be differentially regulated by unphosphorylated BAF. Collectively, these results suggest that BAF can act in a phosphorylation-regulated manner to impair HSV-1 transcription and/or DNA replication, which is similar to the antiviral activity of BAF during vaccinia infection.

Bovine herpes virus 1 (BoHV-1), an important bovine pathogen, causes conjunctivitis and disorders in the upper respiratory tract. Following acute infection, BoHV1 establishes life-long latency in sensory neurons. Recent studies demonstrated that viral gene products expressed in trigeminal ganglionic neurons during latency stabilize β-catenin levels, an important signaling molecule that interacts with a family of DNA binding proteins (T-cell factors) and subsequently stimulates transcription. In this study, we provide new evidence demonstrating that BoHV-1 transiently increased β-catenin protein levels in bovine kidney (CRIB) cells, but not in rabbit skin cells. β-catenin dependent transcription was also stimulated by infection of CRIB cells. The β-catenin small molecule inhibitor (iCRT14) significantly reduced the levels of BoHV-1 virus during productive infection of CRIB cells and rabbit skin cells. In summary, these studies suggested the ability of β-catenin to stimulate cell survival and cell cycle regulatory factors enhances productive infection in non-neuronal cells.

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.