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.IMPORTANCE 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.

Keywords: KLF15; bovine herpesvirus 1; gene regulation; glucocorticoids; latency; stress response.

FIG 1

KLF15 and the GR are frequently found in the same TG neuron during DEX-induced reactivation from latency. Immunohistochemistry (IHC) of consecutive sections was examined for KLF15 and GR expression in TG neurons when latently infected calves were treated with DEX for 6 h to initiate reactivation from latency, as described in the Materials and Methods section. The numbers denote TG neurons that are positive for KLF15 and the GR. The letters denote GR⁺ KLF15-negative neurons. As a control, TG sections from latently infected calves were examined for GR and KLF15 expression. Since GR and KLF15 were not readily detected in TG neurons during latency, consecutive sections were not examined. These results are representative of TG sections from two different calves.

FIG 2

KLF15 and the GR cooperate to stimulate productive infection. Neuro-2A cells (A) or rabbit skin cells (B) were used for these studies. Twenty-four hours prior to transfection, 2% stripped fetal calf serum was added to the medium. Stripped fetal calf serum was used for these studies because normal serum contains steroid hormones, which activate the GR, as judged by nuclear localization of the GR following incubation with normal fetal calf serum (13). Cells incubated with stripped fetal calf serum for 24 h contain little or no nuclear GR. Cells were then transfected with 1.5 μg of BoHV-1 gCblue and, where indicated, a plasmid that expresses the mouse GR protein (1.0 μg DNA) and KLF15 (0.5 μg DNA). To maintain the same amount of DNA in each sample, empty vector was included in the samples. Designated cultures were then treated with water-soluble DEX (10 μM; Sigma). At 48 h after transfection, the number of β-Gal⁺ cells was counted. The value for the control (gCblue DNA treated with PBS after transfection) was set at 1. The results from DEX-treated cultures (black bars) were compared to those with the controls (white bars) and are an average of three independent studies. An asterisk denotes a significant difference between Neuro-2A cells transfected with BoHV-1 DNA (P < 0.05), using the Student's t test.

FIG 3

KLF15 and the GR cooperate to stimulate IEtu1 promoter activity. (A) Schematic of IEtu1 promoter and location of the TATA box, TAATGARAT motif, and the two GREs. Numbers in parentheses indicate the genomic location of the first nucleotide of each motif. (B) Schematic of the 280-bp fragment that contains the IEtu1 GREs and a KLF-like motif. This fragment was cloned upstream of the minimal SV40 early promoter in the luciferase vector (pGL3-promoter vector; Promega). (C) Nucleotide sequence of motifs in the IEtu1 GREs and mutations that were prepared. The mutations in GRE1 and GRE2 were previously shown to disrupt transactivation by the GR in transient-transfection studies (13). (D) Neuro-2A cells were treated with 2% stripped fetal calf serum 24 h prior to transfection. Neuro-2A cells were then transfected with the designated IEtu1 GRE plasmid (0.5 μg of DNA) and, where indicated, a plasmid that expresses the mouse GR protein (1.0 μg of DNA) and/or KLF15 (0.5 μg of DNA). To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Designated cultures were then treated with water-soluble DEX (10 μM; Sigma) at 24 h after transfection. At 48 h after transfection, cells were harvested, and protein lysate was subjected to a dual-luciferase assay as described in the Materials and Methods section. Levels of promoter activity in the empty luciferase vector (pGL3-promoter vector) were normalized to a value of 1, and fold activation values for other samples are presented. The results are the average of three independent experiments, and error bars denote the standard errors. The double asterisks denote a significant difference (P < 0.05) between results for IEtu1 GREs relative to those for all mutant constructs following transfection with GR plus KLF15 and treatment with DEX, as determined by the Student t test. The single asterisk denotes a significant difference (P < 0.05) between results for the ΔKLF mutant relative to results for the other mutant constructs (ΔGRE1, ΔGRE1ΔKLF, and Δ2×GREΔKLF) when they are cotransfected with GR plus KLF15 and treated with DEX, as determined by the Student t test.

FIG 4

Location of GREs on BoHV-1 and intergenic regions analyzed for stress-induced transcriptional activity. (A) Diagram of the linear BoHV-1 genome (horizontal line) with predicted GREs denoted by vertical lines. Lines above the genome represent GREs on the positive/forward DNA strand, while lines below indicate GREs on the negative strand. The terminal repeats are denoted by gray rectangles. Numbers denote genomic coordinates. Genomic regions that contain at least two putative GREs and potential KLF binding sites and are 400 bp or less are denoted by the gene in which they are located. LRG refers to the latency-related gene (10). The Circ gene encodes an IE transcript from the left end of the genome and spans covalently joined genome ends. These sequences were synthesized (Genescript), cloned into the pGL3-promoter vector, and then used for the studies described below. U_L, unique long region. (B) Expanded genomic region that includes bICP0, bICP4, and bICP22. Genomic sequences located between bICP4 and bICP22 contain the origin of replication (ORI_s, denoted by a black diamond) and IEtu1 and IEtu2 to the left and right of the ORI_s, respectively (not highlighted). Black boxes denote enlarged GREs on positive (above) or negative (below) strands. GRE1 and GRE2 within the IEtu1 promoter are indicated, and the genomic region for the beginning and end of the sequences is given. (C) Neuro-2A cells were cotransfected with a plasmid containing the firefly luciferase gene downstream of the designated plasmid constructs (1 μg of DNA) and a plasmid encoding Renilla luciferase (0.05 μg of DNA) using Lipofectamine 3000. The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation for other constructs is presented as enhancer activity. An asterisk denotes a significant difference between luciferase activities of the designated construct relative to the activity of the empty vector that contains only the SV40 early promoter (pGL3-promoter vector), using the Student's t test.

FIG 5

Identification of BoHV-1 intergenic regions that are regulated by stress-induced transcription factors. (A) Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene that contains the BoHV-1 sequences, a plasmid that expresses the GR plasmid (1 μg of DNA), and a plasmid that expresses Renilla luciferase (0.05 μg of DNA). Following transfection, Neuro-2A cells were cultured in 2% stripped fetal calf serum after transfection. Twenty-four hours after transfection the designated Neuro-2A cultures were treated with water-soluble DEX (10 μM; Sigma). (B) Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene and viral sequences, a plasmid encoding Renilla luciferase (0.05 μg of DNA), and a KLF transcription factor (KLF4, KLF15, or promyelocytic leukemia zinc finger [PLZF]) or a plasmid expressing Slug-1 (0.5 μg of DNA). The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation values for other samples are presented. At 48 h after transfection, cells were harvested, and protein extracts were subjected to a dual-luciferase assay as described in the Materials and Methods section. The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation values for other samples are presented. The results are the average of three independent experiments, and error bars denote the standard errors.

FIG 6

GR and KLF15 cooperate to transactivate certain BoHV-1 intergenic regions. Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene downstream, a plasmid encoding Renilla luciferase (0.05 μg of DNA), KLF15 (A) or KLF4 (B) (0.5 μg of DNA), and the GR expression plasmid (1 μg of DNA). To maintain equal plasmid amounts in the transfection mixtures, the empty expression vector was added as needed. For these studies, Neuro-2A cells were cultured in 2% stripped fetal calf serum after transfection. Twenty-four hours after transfection the designated Neuro-2A cultures were treated with water-soluble DEX (10 μM; Sigma). At 48 h after transfection, cells were harvested, and protein extracts were subjected to a dual-luciferase assay as described in the Materials and Methods section. The results are the average of three independent experiments, and error bars denote the standard errors. An asterisk denotes a significant difference (P < 0.05) between the value for the empty control (pGL3-promoter vector), as determined by the Student t test. It should be noted that for UL52 the transactivation levels obtained with KLF4 plus GR were not significantly different when DEX was added.

FIG 7

Identification of UL52 intergenic sequences that are important for transactivation by KLF15 and the GR. (A) Location of the GRE half-binding sites (GRE1 1/2 and GRE2 1/2) and potential KLF binding sites in the 294-bp UL52 fragment. (B) UL52 sequences contain three closely linked and partially overlapping Sp1 binding sites that are underlined. The ΔSp1 binding site mutant replaced the Sp1 binding site with an EcoRI restriction enzyme site (GAATTC). The consensus GRE binding site is shown (small letters above consensus are nucleotides that can be part of a functional GRE). The GRE1 half-binding site is shown, and the underlined nucleotides match the consensus. The ΔGRE1 half-binding site contains an EcoRI restriction enzyme site (GAATTC) that replaced the GRE sequences. The GRE2 half-binding site is shown, and the underlined nucleotides match the consensus. The ΔGRE2 half-binding site contains an SmaI restriction enzyme site (CCCGGG) that replaced GRE sequence. The KLF-like binding site is shown, and the ΔKLF mutant contains a HindIII restriction enzyme site (AAGCTT) that replaced the KLF-like binding motif. (C) Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene downstream, a plasmid encoding Renilla luciferase (0.05 μg of DNA), KLF15 (0.5 μg DNA), and the GR-expressing plasmid (1 μg of DNA). To maintain equal plasmid amounts in the transfection mixtures, the empty expression vector was added as needed. For these studies, Neuro-2A cells were cultured in 2% stripped fetal calf serum after transfection. Twenty-four hours after transfection the designated Neuro-2A cultures were treated with water-soluble DEX (10 μM; Sigma). At 48 h after transfection, cells were harvested, and protein extracts were subjected to a dual-luciferase assay as described in the Materials and Methods section. The results are the average of three independent experiments, and error bars denote the standard errors. The asterisk denotes a significant difference (P < 0.05) between results with UL52 and the mutant constructs when they are cotransfected with GR plus KLF15 and treated with DEX, as determined by a Student t test.

FIG 8

The GR interacts with KLF15. Neuro-2A cells were grown to 80% confluence on 100-mm dishes. Cells were cotransfected with plasmids that express KLF15 (1.5 μg) and the GR (2 μg). Cultures were treated with DEX (10 μM) in 2% stripped-serum medium for 4 h (A) before cell lysate was harvested or treated with DEX (B). Whole-cell lysate was prepared, and co-IP studies were performed using the GR or KLF15 antibody as described in Materials and Methods. Following IP with the designated antibody, the GR or KLF15 was detected in the immunoprecipitate by Western blotting (WB). Input lysate (50 μg of protein) was used as a positive control. (C) Neuro-2A cells were cotransfected with plasmids that express KLF15 (0.5 μg), KLF4 (0.5 μg), and the GR (1 μg) as indicated in the figure. Whole-cell lysate was prepared using RIPA buffer, proteins were separated by SDS-PAGE (50 μg in each lane), and GR expression was detected by Western blotting. Cell lysate from nontransfected Neuro-2A cells was used to examine endogenous GR expression. (D) Neuro-2A cells were cotransfected with plasmids that express KLF15 (0.5 μg), KLF4 (0.5 μg), and the GR (1 μg) as indicated on the figure. Whole-cell lysate was prepared using RIPA buffer, proteins (50 μg in each lane) were separated by SDS-PAGE, and Western blot analysis was performed using anti-KLF15. Cell lysate from nontransfected Neuro-2A cells was used to examine endogenous KLF15 expression. (E) As a loading control, tubulin levels were examined. For each lane, 50 μg was loaded. Values on the sides of the blots indicate molecular mass in kilodaltons.

FIG 9

Interaction between GR and KLF15 with IEtu1 GREs and UL52 sequences. Neuro-2A cells were cotransfected with the IEtu1 GRE construct (A; 4 μg of DNA), the Δ2×GREΔKLF (B; 4 μg of DNA), or the UL52 plasmid (C; 4 μg of DNA) and KLF15 and/or the GR plasmid (1 μg of DNA). Empty vector was added to maintain the same concentration of DNA in each transfection assay. Designated cultures were treated with DEX as described above. (A) Neuro-2A cells were transfected with no plasmid (lanes 1), with the IEtu1 GRE construct (lanes 2), with IEtu1 GREs, KLF15, and the GR (no DEX treatment) (lanes 3), and with IEtu1 GREs, KLF15, and the GR (lanes 4). Cultures were treated with DEX (10 μM) in 2% stripped-serum medium for 4 h before cells were harvested. (B) Neuro-2A cells were transfected with no plasmid (lanes 1), with the Δ2×GREΔKLF construct (lanes 2), with Δ2×GREΔKLF, KLF15, and the GR (no DEX treatment) (lanes 3), and with Δ2×GREΔKLF, KLF15, and the GR (lanes 4). Cultures were treated with DEX (10 μM) in 2% stripped-serum medium for 4 h before cells were harvested. (C) Neuro-2A cells were transfected with no plasmid (lanes 1), with a UL52 construct (lanes 2), with UL52, KLF15, and the GR (no DEX treatment) (lanes 3), and with UL52, KLF15, and the GR (lanes 4). Cultures were treated with DEX (10 μM) in 2% stripped-serum medium for 4 h before cells were harvested. Transfected cells were processed for ChIP as described in the Materials and Methods section, and immunoprecipitation (IP) was conducted using the GR antibody, KLF15 antibody, or isotype control antibody. Input was 10% of the total DNA-protein complexes that were used for IP, and then PCR was performed using PCR primers described in the Materials and Methods section. The filled circle denotes the specific PCR fragment of 107 bp for the IEtu1 GREs, 107 bp for Δ2×GREΔKLF, or 132 bp for UL52, and the open circle denotes the position of primer dimers. These results are representative of five independent studies for the GR ChIP and of two for the KLF15 ChIP and Δ2×GREΔKLF. M, molecular mass marker.