Following acute infection, sensory neurons in trigeminal ganglia (TG) are important sites for the life-long latency of human alpha-herpes virus 1 (HSV-1). Acute or chronic stress in humans correlates with increased reactivation from latency, which can lead to recurrent HSV-1 disease, for example, herpes labialis, herpes stromal keratitis, and encephalitis. The glucocorticoid receptor (GR) and the synthetic corticosteroid dexamethasone stimulate key viral transcriptional cis-regulatory modules, viral replication, and explant-induced reactivation from latency. Conversely, a GR-specific antagonist impairs explant-induced reactivation and viral replication. Based on these observations, we hypothesize that GR transcriptional activity enhances reactivation from latency. To test this hypothesis, the HSV-1 latency-reactivation cycle was examined in mice containing a serine 229 to alanine mutation in GR (GR ^S229A ) because phosphorylation of GR serine 229 is crucial for GR-mediated transcription. Virus yields from cornea and conjunctiva of infected GR ^S229A mice ceased before wild-type (wt) mice, consistent with reduced viral replication in kidney cells from GR ^S229A mice. However, viral DNA levels in TG were not significantly different during latency and similar numbers of TG neurons express GR in GR ^S229A and WT mice. Strikingly, HSV-1 viral titers during explant-induced reactivation were significantly reduced in female GR ^S229A mice versus male GR ^S229A mice or wt mice. The number of VP16 + TG neurons in female GR ^S229A mice was significantly lower than in male GR ^S229A or wt mice (males and females) during the early stages of explant-induced reactivation. Collectively, these studies revealed that GR phosphorylation of serine 229 is more important in GR ^S229A female mice versus males during explant-induced reactivation from latency. IMPORTANCE: A correlation exists between stress and increased episodes of human alpha-herpes virus 1 reactivation from latency. Stress increases corticosteroid levels; consequently, the glucocorticoid receptor (GR) is activated. Recent studies concluded that a GR agonist, but not an antagonist, accelerates productive infection and reactivation from latency. Furthermore, GR and certain stress-induced transcription factors cooperatively transactivate promoters that drive the expression of infected cell protein 0 (ICP0), ICP4, and VP16. This study revealed female mice expressing a GR containing a serine to alanine mutation at position 229 (GR ^S229A ) shed significantly lower levels of infectious virus during explant-induced reactivation compared to male GR ^S229A or wild-type parental C57BL/6 mice. Furthermore, female GR ^S229A mice contained fewer VP16 + TG neurons compared to male GR ^S229A mice or wild-type mice during the early stages of explant-induced reactivation from latency. Collectively, these studies revealed that GR transcriptional activity has female-specific effects, whereas male mice can compensate for the loss of GR transcriptional activation.

Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency.

Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Neurons in the central nervous system, including brainstem, also harbor viral genomes during latency. Periodically, certain cellular stressors trigger reactivation from latency, which can lead to recurrent HSV-1 disease: herpes labialis, herpes stromal keratitis, and encephalitis for example. Activation of the glucocorticoid receptor (GR) by stressful stimuli enhances HSV-1 gene expression, replication, and explant-induced reactivation. GR and certain stress-induced Krüppel like factors (KLF) cooperatively transactivate cis-regulatory modules (CRM) that drive expression of viral transcriptional regulatory proteins (ICP0, ICP4, and ICP27). These CRMs lack GR response elements (GRE); however, specificity protein 1 (Sp1) binding sites are crucial for GR and KLF15 or KLF4 mediated transactivation. Hence, we tested whether Sp1 or Sp3 regulate viral replication and transactivation of the ICP0 promoter. During early stages of explant-induced reactivation from latency, the number of Sp3+ TG neurons were significantly higher relative to TG from latently infected mice. Conversely, Sp1+ TG neurons were only increased in females, but not male mice, during explant-induced reactivation. Sp1 siRNA significantly reduced HSV-1 replication in cultured mouse (Neuro-2A) and monkey (CV-1) cells. Mithramycin A, an antibiotic that has anti-tumor activity preferentially interacts with GC-rich DNA, including Sp1 binding sites, significantly reduced HSV-1 replication indicating it has antiviral activity. GR and Sp1 or Sp3 transactivated the HSV-1 ICP0 promoter in Neuro-2A and CV-1 cells confirming these transcription factors enhance viral replication and gene expression.

Glucocorticoid receptor (GR) activation enhances Human alpha-herpes virus 1 (HSV-1) replication and explant-induced reactivation from latency. Furthermore, GR and Krüppel-like factor 15 (KLF15) cooperatively transactivate cis-regulatory modules (CRMs) that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. KLF and specificity protein (Sp) family members bind GC-rich or C-rich sequences and belong to the same super-family of transcription factors. Based on these observations, we hypothesized CRMs spanning the ICP0 promoter are transactivated by GR and Sp1 or Sp3. CRM-A (-800 to -635), CRM-B (-485 to -635), and CRM-D (-232 to -24), but not CRM-C, were significantly transactivated by GR, DEX, and Sp1 or Sp3 in mouse neuroblastoma cells (Neuro-2A). Mutagenesis of Sp1/Sp3 binding sites were important for transactivation of CRM-A and CRM-B. Chromatin immunoprecipitation studies revealed significantly higher levels of GR occupied ICP0 promoter sequences when Sp1 or Sp3 was over-expressed suggesting these transcriptions factors recruit GR to ICP0 CRM sequences. Mithramycin A, an antibiotic that preferentially binds GC-rich DNA and impairs Sp1/Sp3 dependent transactivation and reduced virus shedding during reactivation from latency in mice latently infected with HSV-1. These studies indicate GR and certain stress-induced cellular transcription factors preferentially bind GC rich DNA, which stimulates HSV-1 gene expression and reactivation from latency in trigeminal ganglia of latently infected mice.

Human alpha-herpesvirus 1 (HSV-1) acute infection culminates in life-long latency in sensory neurons in trigeminal ganglia and certain neurons in the central nervous system. Previously, E2F family members and glucocorticoid receptor (GR) were shown to stimulate HSV-1 and bovine herpesvirus 1 (BoHV-1) replication. Consequently, we hypothesized GR and E2F family members activate certain HSV-l promoters. To test this hypothesis, we determined if four HSV-1 ICP0 cis-regulatory modules (CRM) upstream of the ICP0 promoter were activated by E2F. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate the ICP0 CRM-C, but not CRM-A, -B, or -D fragments upstream of a minimal promoter in a luciferase reporter construct. CRM-C sequences contain two E2F consensus binding sites, a GC-rich motif that E2F2 can bind, and a consensus ½ GR response element (GRE) adjacent to the consensus E2F #2 binding site. Mutating the ½ GRE or the 3 E2F binding sites significantly reduced GR- and E2F2-mediated transactivation. Chromatin immunoprecipitation studies revealed E2F2 occupied ICP0 CRM-C sequences during productive infection and mutating the E2F binding sites prevented E2F2 binding. These studies reveal GR and E2F2 transactivate ICP0-promoter activity, which may enhance viral replication in certain cell types.

Following herpes simplex virus-1 (HSV-1) acute infection, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Acute or chronic stress increases the incidence of HSV-1 reactivation from latency, in part because the glucocorticoid receptor (GR) is activated. GR antagonists impair HSV-1 replication and explant-induced reactivation from latency. A mouse strain expressing GR with a serine 229 mutated to alanine (GR^S229A) previously revealed that female GR^S229A mice latently infected with HSV-1 shed significantly lower virus in TG during explant-induced reactivation when compared to male GR^S229A mice and wild-type (wt) C57BL/6J mice regardless of sex. A new study demonstrated that heat-stress-induced HSV-1 reactivation from latency is significantly reduced in female GR^S229A mice but not male GR^S229A mice or wt C57BL/6J mice. A mineralocorticoid receptor (MR)-specific antagonist (Esaxerenone) increased explant-induced or heat-stress-increased reactivation from latency in female GR^S229A mice but not male GR^S229A mice or wt C57BL/6J mice regardless of sex. Additional studies revealed Esaxerenone increased virus shedding in primary kidney cells prepared from GR^S229A males or females and neuroblastoma cells (Neuro-2A). In conclusion, this study revealed virus shedding was reduced in the TG of GR^S229A female mice when reactivation from latency was induced by explant or heat stress; but, Esaxerenone rescued reactivation in GR^S229A female mice.