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.