Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection

Abstract

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.

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

“…2A). We also examined KLF family members that are induced during BoHV-1 reactivation from latency (11) because certain KLF family members cooperate with the GR to stimulate the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter (12). The FL ICP0 construct was cotransfected into Neuro-2A or Vero cells with the GR, a KLF family member, and a Renilla luciferase gene driven by a minimal thymidine kinase (TK) promoter.…”

“…Previous studies demonstrated that DEX treatment induced KLF15 expression in many TG neurons (11,12), suggesting that this is part of the general stress response. KLF15, like other KLF family members, resembles the Sp1 transcription factor family, and both families interact with GC-rich motifs (17,34,35).…”

“…Recent studies demonstrated that the GR and KLF15 cooperate to transactivate the BoHV-1 IEtu1 promoter (12). Relative to HSV-1, regulation of BoHV-1 ICP0 and ICP4 coding has several significant differences.…”

“…First, the IEtu1 promoter drives expression of two IE mRNAs, IE/2.9 and IE/4.2, from an alternatively spliced primary mRNA, and these transcripts are subsequently translated into bICP0 and bICP4, respectively (37,38). Second, the IEtu1 promoter contains two consensus "whole" GREs that when mutated inhibit the ability of DEX or DEXϩKLF15 to stimulate promoter activity (12,23). A small fragment encompassing the wt IEtu1 GREs can confer DEX induction to a heterologous promoter; this was not the case for sequences comprising the GR/KLF15 RR (data not shown).…”

“…A subset of these cellular genes encode transcription factors, including four Krüppel-like transcription factors (KLF), including KLF4, KLF6, KLF15, and PZLF (promyelocytic leukemia zinc finger). A recent study demonstrated KLF15 and GR cooperate to transactivate the bovine herpesvirus 1 (BoHV-1) immediate early transcription unit 1 (IEtu1) promoter, which drives IE expression of infected cell protein 0 (ICP0) and ICP4 (12). In response to stress, the GR and KLF15 regulate gene expression dynamics and integrate signals by a positive feed-forward loop (13)(14)(15).…”