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.IMPORTANCEBovine 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.

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.

Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause “abortion storms” in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are important sites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, we predict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1 ⁺ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency. IMPORTANCE: Following acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.