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Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen of cattle. Like other members of the subfamily Alphaherpesvirinae, BoHV-1 establishes latency in sensory neurons and has the potential to reactivate from latency. Dexamethasone (DEX) treatment of latently infected calves or rabbits consistently leads to reactivation from latency. The BoHV-1 transcript encoding the infected cell protein 0 (bICP0) is consistently detected during reactivation from latency, in part because the bICP0 early promoter is activated by DEX. During DEX-induced reactivation from latency, cyclin expression is stimulated in infected sensory neurons. Cyclin-dependent kinase activity phosphorylates Rb (retinoblastoma tumor suppressor gene product) family proteins and consequently releases the E2F family of transcription factors, suggesting that E2F family members stimulate productive infection and/or reactivation from latency. In this study, we provide evidence that repression of E2F1 by a specific small interfering RNA (siRNA) reduced productive infection approximately 5-fold. E2F1 or E2F2 stimulated bICP0 early promoter activity at least 100-fold in transient transfection assays. Two E2F-responsive regions (ERR) were identified within the early promoter, with one adjacent to the TATA box (ERR1) and one approximately 600 bp upstream from the TATA box (ERR2). Mobility shift assays suggested that E2F interacts with ERR1 and ERR2. E2F1 protein levels were increased at late times after infection, which correlated with enhanced binding to a consensus E2F binding site, ERR1, or ERR2. Collectively, these studies suggest that E2F1 stimulates productive infection and bICP0 early promoter activity, in part because E2F family members interact with ERR1 and ERR2.
Bovine herpesvirus 1 (BoHV-1) is a significant viral pathogen of cattle that can cause conjunctivitis, rhinotracheitis, pneumonia, genital disorders, or abortions. BoHV-1 also initiates shipping fever, a potentially fatal polymicrobial disease (37). Like other members of the Alphaherpesvirinae subfamily, BoHV-1 establishes lifelong latency in trigeminal ganglionic neurons following acute replication in mucosal epithelium. Reactivation from latency occurs periodically, resulting in virus shedding and spread to susceptible cattle. Reactivation from latency can occur after stress or corticosteroid treatment, which mimics stress (30, 34). Dexamethasone (DEX), a synthetic corticosteroid, reproducibly induces expression of BoHV-1 lytic cycle genes and reactivation from latency in calves or rabbits (15, 17-20, 30).
During productive infection of cultured cells, viral gene expression is temporally regulated in three distinct phases: the immediate-early (IE), early (E), and late (L) phases (reviewed in references 17 and 18). IE gene expression is stimulated by a virion component, bTIF, which interacts with a cellular transcription factor (Oct-1) to transactivate IE gene expression (22, 23). Two IE transcription units exist, namely, IE transcription unit 1 (IEtu1) and IEtu2 (44-46). IEtu1 encodes functional homologues of two herpes simplex virus type 1 (HSV-1) proteins, ICP0 and ICP4. IEtu2 encodes a protein that is similar to the HSV-1 IE gene product ICP22 (33). BoHV-1-encoded ICP0 (bICP0) is translated from an IE (IE2.9) or E (E2.6) mRNA, since an IE promoter (IEtu1 promoter) and an E promoter regulate bICP0 RNA expression (7, 44-46). The IE promoter regulates IE expression of bICP4 and bICP0. Expression of the bICP4 protein represses IEtu1 promoter activity, whereas bICP0 activates its own E promoter and all other viral promoters. A recent study demonstrated that during DEX-induced reactivation from latency, bICP0 mRNA, but not bICP4 mRNA, was consistently detected (47). In part, this was due to the fact that the bICP0 early promoter is activated by DEX induction of the cellular transcription factor CAAT-enhancer binding protein alpha (C/EBP-alpha) (47). bICP0 transcription appears to be stimulated during reactivation from latency by cellular transcription factors that transactivate the bICP0 E promoter. Since bICP0 is the major regulatory protein that stimulates productive BoHV-1 infection (7, 44-46), the identification of cellular factors that stimulate the bICP0 E promoter may help us to understand the early stages of reactivation from latency.
Members of the E2F family of transcription factors contain a conserved DNA-binding domain, an acidic transcriptional activation domain, and an Rb binding site (13). Functional E2F binding sites are present in the promoters of nearly all genes that control cell cycle progression (3, 24, 27, 32, 41). Several lines of evidence suggest that the E2F family of transcription factors may stimulate productive BoHV-1 infection and reactivation from latency. First, during DEX-induced reactivation from latency, sensory neurons that express abundant levels of lytic cycle genes also express certain cyclins (for example, cyclin E and cyclin A) (43). Phosphorylation of Rb family members by cyclin-dependent kinase-cyclin complexes leads to E2F release, and consequently, certain E2F family members are then able to activate transcription (2, 13, 24, 40). Furthermore, overexpression of E2F4 stimulates productive BoHV-1 infection and E2F1 or E2F2 transactivates IEtu1 promoter activity (9). Finally, the HSV-1 thymidine kinase (TK) promoter is activated by E2F1 by virtue of a GC-rich motif, not a consensus E2F binding site (35).
In this study, we demonstrated that small interfering RNAs (siRNAs) directed against E2F1 reduced productive infection. In transient transfection assays, E2F1 or E2F2 activated bICP0 E promoter activity >100-fold. Two E2F-responsive regions (ERRs) were identified within the bICP0 E promoter. These studies suggest that E2F1 and E2F2 stimulate productive infection, in part by activating bICP0 E promoter activity.
Murine neuroblastoma 2A (neuro-2A) and rabbit skin (RS) cells were grown in Earle's modified Eagle's medium (EMEM) supplemented with 5% fetal calf serum (FCS). Bovine kidney cells (CRIB cells) were grown in EMEM supplemented with 10% FCS. All media contained penicillin (10 U/ml) and streptomycin (100 μg/ml).
The Cooper strain of BoHV-1 (wild-type [wt] virus) was obtained from the National Veterinary Services Laboratory, Animal and Plant Health Inspection Services, Ames, IA. Stock cultures of BoHV-1 were prepared in CRIB cells.
A BoHV-1 mutant containing the LacZ gene in place of the viral gC gene was obtained from S. Chowdury (Baton Rouge, LA) (gCblue virus). The virus grows to titers similar to those of the wild-type parent virus and expresses the LacZ gene as a true late gene.
Plasmids expressing E2F1 and E2F2 (pCMV-E2F1 and pCMV-E2F2, respectively) were obtained from J. R. Nevins (Duke University, Durham, NC). The empty vector pcDNA3.1 was purchased from Invitrogen.
Six bICP0 E promoter constructs were prepared by PCR amplification as previously described (47). The promoter fragments were cloned into the promoterless vector pCAT-Basic (E1871; Promega) at the unique XhoI and KpnI sites to generate plasmids EP-943, EP-638, EP-172, EP-143, EP-133, and EP-71 (see Fig. Fig.2C).2C). EP-50 and EP-42 were synthesized (IDT, IA) to contain XhoI and KpnI restriction sites. Duplex oligonucleotides were digested with XhoI and KpnI and cloned into the promoterless vector pCAT-Basic. The numbers in the plasmid names refer to the length of the bICP0 E promoter fragment inserted into the chloramphenicol acetyltransferase (CAT) vector. The deletions were made from the 5′ terminus of the bICP0 promoter.
Two additional bICP0 E promoter constructs were generated, using the wt BoHV-1 genome as a template and a common 3′ primer (5′-ctcgagCCTGCTGGGCGACACAAACAACAGA-3′) with the following 5′ primers: for EP-398, 5′-ggggtaccAAGACGCAGAACCCCG-3′ ; and for EP-328, 5′-ACCCAGGGGCGGAGC-3′ (lowercase letters indicate restriction sites). The promoter fragments were cloned into the promoterless vector pCAT-Basic as described above. The DNA sequences of the E promoter inserts were confirmed by DNA sequencing (Genomics Core Research Facility-UNL). All plasmids were prepared from bacterial cultures by alkaline lysis and two rounds of cesium chloride centrifugation.
To further localize bICP0 E promoter elements that are responsive to E2F, upstream regions of the bICP0 E promoter (ERRs) were cloned into a minimal-promoter CAT vector (E186A; Promega) containing the simian virus 40 (SV40) early promoter cloned upstream of CAT.
ERR1/40 was created using synthesized duplex sequences (IDT, IA). Duplex oligonucleotides were digested with XhoI and KpnI and cloned into the minimal-promoter CAT vector.
ERR2/254 was generated by a PCR using the forward primer 5′-GCGACGGCGGCAATAAAGACGAGT-3′ and the reverse primer 5′-CGGGGTTCTGCGTCTTGGC-3′. ERR2/180, ERR2/120, ERR2/1-60, and ERR2/61-120 were created using synthesized duplex sequences (IDT, IA). Duplex oligonucleotides were digested with KpnI and XhoI and cloned into the minimal-promoter CAT vector at the unique KpnI and XhoI sites. The identity of each construct was confirmed by DNA sequencing (Genomics Core Research Facility-UNL). Schematics of the ERR1 and ERR2 constructs are shown in Fig. Fig.44 and and5,5, respectively. All plasmids were prepared from bacterial cultures by alkaline lysis and two rounds of cesium chloride centrifugation.
Neuro-2A cells grown in 60-mm dishes were cotransfected with the designated plasmids as indicated in the respective figure legends. Neuro-2A cells were transfected with NeuroTransIt (MIR2145; Mirus) according to the manufacturer's instructions. After 48 h, cell extract was prepared by three freeze-thaw cycles in 0.25 M Tris-HCl, pH 7.4. Cell debris was pelleted by centrifugation, and protein concentrations were determined. CAT activity was measured by incubating the extract with 0.1 μCi [14C]chloramphenicol (CFA754; Amersham Biosciences) and 0.5 mM acetyl-coenzyme A (acetyl-CoA) (A2181; Sigma). The reaction mixture was incubated at 37°C for 15 to 30 min. All forms of chloramphenicol were separated by thin-layer chromatography. CAT activity was quantified using a Bio-Rad FX molecular imager (Molecular Dynamics, CA). Levels of CAT activity are expressed as fold induction relative to the vector control.
Neuro-2A cells were transfected with 100 ng of E2F1 by using NeuroTransIT (Mirus) according to the manufacturer's instructions. Forty-eight hours after transfection, whole-cell lysate was prepared. Cells were washed with phosphate-buffered saline (PBS) and suspended in NP-40 lysis buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and one tablet of Complete protease inhibitor [Roche Molecular Biochemicals] per 10 ml). Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min.
CRIB cells were infected with wt BoHV-1 at a multiplicity of infection (MOI) of 5 and cultured in EMEM containing 10% FCS. At various times after infection, cells were collected and suspended in NP-40 lysis buffer. Cell lysate was incubated on ice for 30 min, sonicated, and then clarified by centrifugation at 10,000 × g at 4°C for 15 min. Protein concentrations were quantified by the Bradford assay.
Twenty micrograms of protein extract was incubated in 16 μl of binding buffer (10 mM Tris-HCl, pH 8, 150 mM KCl, 0.5 mM EDTA, 0.1% Triton X-100, 12.5% glycerol) in the presence of 1 μg poly(dI-dC) (P4929; Sigma) and 0.5 pmol of double-stranded DNA probe labeled with 10 μCi of [γ-32P]ATP. Incubation proceeded for 1 h at room temperature. For competition assays, 500 ng of cold E2F1 consensus probe or a C/EBP-alpha consensus probe was incubated with cell lysate for 20 min prior to addition of radiolabeled probe. DNA-protein complexes were run in a 5% polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE) for 3 h at 100 V. To improve band resolution, 1 M sodium acetate, pH 5.3, was added to the lower buffer chamber during electrophoresis. The gel was exposed to a phosphorimager plate and analyzed using a Bio-Rad FX molecular imager. Probes used for EMSA were as follows: E2F consensus, ATTTAAGTTTCGCGCCCTTTCTCAA; C/EBP-alpha consensus, CGCAATATTGCGCAATATTGCAAT; 40-bp probe of bICP0 E promoter that was used to construct ERR1/40, CGGCGCCCTGCCCCCGCCCCGCCCCCCCGCCCTCGCGGCC; and probe of bICP0 E promoter (bp 1 to 60) that was used to construct ERR2/1-60, CCGGCGCGCGGCGCGCGGGGCGGGCCCCGGGGCGCGAAGCCCGGGAGGGACGCGGGCGTG.
RS cells were transfected with 1 μg of pcDNA3.1 empty vector or 100 mM E2F1 siRNA (sc-35247; Santa Cruz Biotechnology) or control siRNA (44-2926; Invitrogen) by use of Lipofectamine 2000 according to the manufacturer's specifications (Invitrogen). Block-iT-Fluorescent oligonucleotide was used as a control siRNA (44-2926; Invitrogen). This oligonucleotide is a fluorescently conjugated control containing a scrambled sequence that does not reduce the level of any known mammalian gene. Forty-eight hours after transfection, whole-cell lysate was prepared as previously described (47). Protein concentrations were quantified by the Bradford assay. Standard 12% SDS gels were prepared and used for these studies. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), blocked for 4 h in 5% nonfat dry milk with Tris-buffered saline-0.1% Tween 20 (TBS-T), and incubated with primary antibody overnight at 4°C. The E2F1 antibody (sc-193X; Santa Cruz Biotechnology) was diluted 1:10,000 in blocking solution. The E2F2 antibody (sc-633X; Santa Cruz Biotechnology) was diluted 1:10,000 in blocking solution. Antiserum directed against cleaved caspase 3 (catalogue no. 9661; Cell Signaling) was also used for these studies, at a 1:1,000 dilution in blocking solution. After 45 min of washing with TBS-T, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham Biosciences), which was diluted 1:2,000 in 5% nonfat milk in TBS-T. Blots were washed for 45 min with TBS-T and exposed to Amersham ECL reagents, and then autoradiography was performed. The β-actin protein was used as a loading control, and this protein was detected using a polyclonal antiserum (Santa Cruz Biotechnology, Santa Cruz, CA).
RS cells grown in 60-mm dishes were transfected with 1 μg of blank pcDNA3.1 vector, 100 nM E2F1 siRNA, or 100 nM control siRNA by use of Lipofectamine 2000 according to the manufacturer's specifications (Invitrogen). Twenty-four hours after transfection, cells were transfected with 1 μg of the gCBlue virus genome. After 24 h, cells were fixed (2% formaldehyde, 0.2% glutaraldehyde in PBS) and stained (1% Bluo-Gal, 5 mM ferric potassium, 5 mM ferrous potassium, 0.5 M MgCl2 in PBS), and the number of blue cells was counted as described previously (8-10). The number of blue cells in cultures expressing the blank vector was set to 100. To calculate percent plaque formation, the number of blue cells in cultures transfected with the E2F1 or control siRNA was divided by the number of blue cells in cultures transfected with the blank vector. The results are averages for three independent experiments.
CRIB cells were infected with wt BoHV-1 at an MOI of 5. Sixteen hours after infection, cells were fixed in 4% paraformaldehyde for 10 min, followed by three washes with PBS. Cells were permeabilized by incubation with 100% ethanol at −20°C for 5 min. Coverslips were then washed three times and blocked in 3% bovine serum albumin (BSA) in PBS for 1 h to reduce nonspecific binding. The E2F1 primary antibody (sc-193X; Santa Cruz Biotechnology) was diluted 1:3,000 in PBS with 0.05% Tween 20 and 1% BSA and incubated on coverslips for 2 h at room temperature. After three washes, coverslips were incubated with Cy5-conjugated donkey anti-rabbit antibody (A-31573; Invitrogen) at a dilution of 1:400 for 1 h in the dark. After slides were washed, DAPI (4′,6-diamidino-2-phenylindole) staining was performed to visualize the nucleus. Coverslips were then mounted on slides by use of Gelmount aqueous mounting medium (Electron Microscopy Sciences). Images were obtained with a Bio-Rad confocal laser scanning microscope (MRC-1024ES).
Our previous studies indicated that overexpression of E2F4, but not E2F1 or E2F2, stimulated productive BoHV-1 infection (9). In contrast, the same study demonstrated that E2F1 and E2F2, but not E2F4, stimulated the IEtu1 promoter. Since E2F1 and E2F2, but not E2F4, are potent transactivators (2, 13, 35), this result was surprising. Overexpression of E2F1 can induce apoptosis (2, 25), suggesting that E2F1 and E2F2 were unable to stimulate productive infection because they were toxic to transfected cells.
To test whether E2F1-specific siRNA affected productive infection, RS cells were transfected with E2F1 siRNA and BoHV-1 genomic DNA. RS cells were used for these studies because they are permissive for BoHV-1 and can be transfected efficiently. For this study, we used the BoHV-1 strain gCblue, which contains the LacZ gene inserted downstream of the gC promoter. Twenty-four hours after transfection, cells were fixed and the number of β-galactosidase-positive (β-Gal+) cells identified. This time point was used to minimize the number of virus-positive cells resulting from virus spread. At later times, many β-Gal+ cells lift off the dish, making it difficult to count virus-positive cells (10, 16). The number of β-Gal+ cells directly correlates with the number of plaques produced following transfection with the gCblue virus (9, 10, 16). Relative to the case with a control siRNA, cotransfection of E2F1 siRNA with BoHV-1 genomic DNA reduced the number of β-Gal+ cells approximately 4-fold (Fig. (Fig.1A).1A). The control siRNA reduced the number of β-Gal+ cells approximately 20% compared to results obtained when RS cells were transfected with just BoHV-1 DNA. Western blot analysis demonstrated that the E2F1 siRNA, but not the control siRNA, reduced E2F1 steady-state protein levels (Fig. (Fig.1B).1B). Conversely, the E2F1-specific siRNA had no obvious effect on E2F2 or β-actin protein levels. The E2F1 siRNA reduced the percentage of cells in S phase 24 h after transfection (data not shown), suggesting that in RS cells there is a correlation between reducing E2F1 protein levels and cell cycle progression.
As with HSV-1 and HSV-2, there are two copies of the bICP0 and bICP4 genes in the BoHV-1 repeats (Fig. (Fig.2A).2A). However, the organization of the BoHV-1 ICP4 and ICP0 genes is unique because a common IE promoter (IEtu1 promoter) drives expression of bICP0 and bICP4 mRNAs (44) (Fig. (Fig.2B).2B). bICP0 also contains an E promoter located near the 5′ end of the bICP0 coding exon (e2). Recent evidence indicated that the bICP0 E promoter, but not the IEtu1 promoter, is stimulated by DEX treatment (47). Expression of the cellular transcription factor C/EBP-alpha is stimulated by DEX, thus stimulating bICP0 E promoter activity. Since repression of E2F1 protein levels by specific siRNAs reduced productive infection, we hypothesized that E2F1 may transactivate the bICP0 early promoter.
To test this hypothesis, neuro-2A cells were cotransfected with a bICP0 E promoter construct (Fig. (Fig.2C)2C) plus an E2F1 or E2F2 expression plasmid, and CAT activity was measured. Neuro-2A cells were use for this study because these cells are neuron-like and it was of interest to begin to understand whether E2F regulates gene expression in neurons. E2F1 and E2F2 are potent stimulators of E2F-responsive promoters (2, 13, 24, 40) and thus were used for these studies. E2F1 transactivated the EP-943 and EP-638 promoter constructs approximately 200-fold, and E2F2 transactivated the same promoter constructs >100-fold (Fig. (Fig.2C).2C). EP-172, EP-143, and EP-133 were transactivated >40-fold by E2F1 and at least 11-fold by E2F2. EP-72, EP-50, EP-42, and the promoter-lacking CAT vector (pCAT-Basic) were not transactivated by E2F1 or E2F2.
Transfection of neuro-2A cells with increasing concentrations of a plasmid expressing E2F1 led to increased levels of cleaved caspase 3 in neuro-2A cells (Fig. (Fig.2D).2D). Although low levels of cleaved caspase 3 were detected in mock-transfected cells, increasing levels of E2F1 correlated with increased levels of cleaved caspase 3, which is considered to be the point of “no return” during apoptosis (39, 42). We believe that low levels of apoptosis in mock-transfected cells were due to the fact that these cells have a tendency to lift off the plate if they are not subcultured every 3 to 4 days and due to the stress of transfection. Cultures that were transfected with the highest levels of E2F1 contained cells that were rounded up and appeared to be dead. When neuro-2A cells were cotransfected with 0.1 or 1 μg E2F1, many cells had a similar morphology to that of mock-transfected cells (data not shown). Although E2F1 can induce apoptosis (2, 25), we do not believe that merely inducing apoptosis accounts for transactivation of the bICP0 E promoter by E2F1, as we used a low level of E2F1 (0.1 μg) for the transactivation studies and this amount of E2F1 did not alter the morphology of neuro-2A cells or dramatically increase cleaved caspase 3 levels relative to those in mock-transfected cells (Fig. (Fig.2D).2D). Furthermore, Bax, a known apoptotic gene (39, 42), did not transactivate the bICP0 E promoter (data not shown).
The ability of E2F1 or E2F2 to transactivate the IEtu1 promoter was also examined because a previous study demonstrated that E2F1 transactivated the IEtu1 promoter 15- to 20-fold in bovine testicle cells (9). However, in neuro-2A cells, E2F1 and E2F2 transactivated the three IEtu1 promoters (Fig. (Fig.3A)3A) only 3- to 5-fold (Fig. (Fig.3B).3B). In summary, this study demonstrates that E2F1 and E2F2 efficiently transactivate the bICP0 E promoter, but not the IEtu1 promoter, in neuro-2A cells.
The results in Fig. Fig.22 suggest that the bICP0 E promoter contains two separate E2F-responsive regions: (i) ERR1, spanning the 5′-terminal 60 bases of EP-133; and (ii) ERR2, located at or near the 5′ terminus of EP-638.
To test whether ERR1 conferred E2F responsiveness to a heterologous promoter, a 40-bp segment spanning the 5′ terminus of EP-133 to EP-71 was synthesized and cloned upstream of a CAT reporter construct containing a minimal SV40 E promoter construct (see Fig. Fig.4A4A for a schematic of the predicted ERR1 region). This construct (ERR1/40) was transactivated approximately 5-fold by E2F1 (Fig. (Fig.4B).4B). A 60-bp fragment between EP-133 and EP-71 was not transactivated more efficiently than ERR1/40 (data not shown). In contrast, the SV40 E promoter (SV40 minCAT) was not efficiently transactivated by E2F1.
Sequences spanning the 5′ terminus of EP-638 and adjacent regions within EP-943 (ERR2) were also synthesized and then cloned into the SV40 minCAT construct (see Fig. Fig.5A5A for a schematic of these constructs). A construct containing 254 bp of the bICP0 E promoter (ERR2/254), 180 bp of the E promoter (ERR2/180), or 120 bp of the E promoter (ERR2/120) was transactivated approximately 20-fold by E2F1 (Fig. (Fig.5B).5B). The 120-bp fragment within ERR2 was further divided into two equal pieces (ERR2/1-60 and ERR2/61-120) and then tested for transactivation by E2F1. ERR2/1-60, but not ERR2/61-120, was transactivated >15-fold by E2F1 (Fig. (Fig.5B).5B). In summary, these studies identified a 60-bp fragment within ERR2 that was transactivated by E2F1 when cloned upstream of a minimal SV40 E promoter.
In Fig. 6A and B, the DNA sequences of ERR1 and ERR2 are shown. ERR1 is located within sequences that are present in EP-133 but lacking in EP-72. Within this region, there are two regions of overlapping Sp1 binding sites (Fig. (Fig.6A).6A). The E2F transcriptional activator is a heterodimer consisting of an E2F family member and a Dp family member (2, 13, 24). The core DNA binding site of an E2F-Dp heterodimer is G/CGCGCC/G (49). Within ERR1, there is one E2F-Dp core consensus sequence. Two binding sites for LSF (late SV40 transcription factor), a transcription factor that interacts with the SV40 21-base repeats and activates late transcription (21), and binding sites for a transcription factor that preferentially interacts with CAC sequences were also identified. Finally, a Yi binding site, which is present in the mouse thymidine kinase promoter, was detected (5). Proteins that interact with the Yi binding site exhibit G1/S-phase-inducible binding. Within sequences of ERR1/40, binding sites for E2F/Dp, Sp1 Yi, LSF, and CAC are clustered.
Within the first 60 bp of ERR2 (Fig. (Fig.6B),6B), 5 Sp1 binding sites were identified: 3 match those of the mouse Erk1 gene (29), 1 is present in the SV40 E promoter (11), and 1 is a target for binding to SP1 (36). In addition, 5 core E2F-Dp binding sites were present. Four of these were overlapping and located near the 5′ terminus of this fragment. Conversely, DNA sequences spanning nucleotides 61 to 120 of ERR2, which were not efficiently transactivated by E2F1, contained just 2 E2F-Dp consensus binding sites and one Sp1 binding site present in the human heat shock binding protein 70 promoter (12). In summary, these studies suggested that a cluster of E2F-Dp core binding sites and Sp1 binding sites located within the 60 bp of ERR2 may be important for E2F-mediated transactivation.
To test whether E2F interacts with ERR1 or ERR2, EMSAs were performed with oligonucleotides derived from ERR1, ERR2, or a consensus E2F binding site and with whole-cell extracts prepared from neuro-2A cells. Shifted bands were readily detected when the respective probes were incubated with neuro-2A cell extracts (Fig. (Fig.7A,7A, shifted bands are denoted by brackets). E2F interacted with the E2F consensus probe as well as with the ERR1 and ERR2 probes, since an oligonucleotide containing a consensus E2F binding site, but not a C/EBP-alpha binding site, competed for binding of nuclear factors (Fig. (Fig.7A7A).
Additional studies were performed to test whether productive infection stimulated binding to the E2F consensus binding site, ERR1, or ERR2. For these studies, we used bovine kidney cells (CRIB cells) because they are permissive for BoHV-1 infection, whereas neuro-2A cells are not. Sixteen or 24 h after infection, increased binding to the E2F consensus, ERR1, and ERR2 was observed (Fig. (Fig.7B,7B, enhanced binding is denoted by closed circles). A consensus E2F sequence, but not a C/EBP-alpha binding site, reduced the intensity of certain bands when samples were incubated with the respective radioactive probes (Fig. (Fig.7B).7B). Although these studies indicated that E2F interacted with ERR1 and ERR2, this was not readily detectable until late stages of productive infection, suggesting that E2F family members are not the major activators of bICP0 E promoter activity.
Western blot analysis determined that E2F1 protein levels, but not those of E2F2, increased during the course of productive infection (Fig. (Fig.8).8). Sixteen and 24 h after infection, E2F1 protein levels were dramatically higher, which correlated with enhanced binding to the E2F consensus, ERR1, or ERR2 oligonucleotide. Confocal microscopy was performed to determine if E2F1 was detected in the nuclei of infected cells 16 h after infection (Fig. (Fig.8B).8B). Higher levels of E2F1 were detected in infected cells, and the E2F1 protein was localized in the nucleus, which was in agreement with the Western blot studies shown in Fig. Fig.8A.8A. Previous studies have demonstrated that BoHV-1 E transcripts (thymidine kinase and ribonucleotide reductase) are readily detected 2 h after infection (MOI of 0.02) by use of poly(dT) as a primer for reverse transcription-PCR (RT-PCR) (31). Using an MOI of 5, most CRIB cells were rounded up and some were beginning to detach from the dish between 16 and 24 h after infection (Fig. (Fig.8B8B).
In this study, we demonstrated that the bICP0 E promoter, but not the IEtu1 promoter, was transactivated >100-fold by E2F1 or E2F2 in neuro-2A cells. Silencing of E2F1 reduced virus infection approximately 4-fold, indicating that E2F1 has the potential to stimulate productive infection. Previous studies also suggested that E2F family members stimulated productive BoHV-1 infection (9, 43). With respect to productive HSV-1 infection, the level of E2F that is not associated with Rb family members increases following infection of human cells (C33-A) (14). Relocalization of E2F4 to the nucleus occurs in human C33-A and U2-OS cells following HSV-1 infection (28). Further support for E2F4 playing a role in HSV-1 replication comes from the finding that infection of p107−/− p130−/− mouse cells leads to a reduced level of infectious virus (6). In primary human fibroblasts or HeLa cells, the subcellular distribution of E2F4 is altered following HSV-1 infection, which is assumed to inactivate E2F4 activity (1). The same study also concluded that HSV-1 infection leads to posttranslational modification of E2F1 and E2F5, translocation of E2F family members from the nucleus to the cytoplasm, and reduced E2F binding to consensus E2F binding sites. In contrast, enhanced binding to a consensus E2F binding site was detected at late times after infection with BoHV-1 (Fig. (Fig.7B).7B). Many DNA synthetic genes are activated by E2F family members (13), suggesting that transient induction of E2F family members may stimulate viral synthesis in highly differentiated cells. It is also possible that induction of E2F binding activity occurs because BoHV-1 induces p53-dependent apoptosis during productive infection (4).
The bICP0 E promoter contains two separate regions that are transactivated by E2F: an upstream region localized to a 60-bp fragment (ERR2) and sequences located near the 5′ terminus of the EP-133 construct (ERR1). Since ERR2 was transactivated approximately 20-fold by E2F1 but EP-638 was transactivated approximately 200-fold by E2F1, we suggest that other sequences within the bICP0 E promoter play a role in transactivation by E2F. EMSAs suggested that E2F interacted with ERR1 and ERR2, because a consensus E2F sequence, but not a C/EBP-alpha binding site, competed for binding of nuclear factors (Fig. (Fig.7).7). ERR2 contains 5 core E2F-Dp binding sites (Fig. (Fig.6B)6B) (49), suggesting that these elements may be important for E2F-mediated transactivation. The 5 Sp1 binding sites located in ERR2 may also be crucial for transactivation, because E2F family members can interact with and transactivate certain promoters containing GC-rich motifs that resemble Sp1 binding sites. For example, E2F1 can transactivate GC-rich motifs in the HSV-1 thymidine kinase promoter (35) and in the human ASK (activator of S-phase kinase) gene, encoding the regulatory subunit for human cdc7-related kinase (48). In addition, a subunit of the mouse DNA polymerase alpha promoter contains a GC-rich element that is crucial for cell cycle regulation (26). Finally, 3 GC-rich motifs in the human thymidine kinase promoter are bound by E2F and are crucial for cell cycle-dependent expression (38). Not all GC-rich promoters are transactivated by E2F, as we previously demonstrated that the BoHV-1 IEtu2 promoter was not efficiently transactivated by E2F1 or E2F2 (9).
In trigeminal ganglia of latently infected calves, bICP0 transcription is stimulated from the E promoter during DEX-induced reactivation from latency, regardless of whether infectious virus is detected (47). In addition, the bICP0 E promoter is stimulated by DEX, in part because of the cellular transcription factor C/EBP-alpha. Conversely, the IEtu1 promoter does not appear to be as active during reactivation from latency, since bICP4 is not consistently detected. This may be important because activating bICP0, but not bICP4, during the early stages of reactivation may allow BoHV-1 to “test the waters” and determine whether important cellular factors are present for production of infectious virus without extensive viral gene expression occurring, which could lead to neuronal death. If bICP4 and bICP0 are expressed equally, then extensive viral gene expression may occur in too many neurons that cannot support production of infectious virus. The ability of E2F1 or E2F2 to strongly transactivate the bICP0 E promoter, but not the IEtu1 promoter, may play a role in this process. It will be of interest to test whether E2F1 or E2F2 can cooperate with bICP0 or bICP4 to stimulate E or L genes, since they also contain GC-rich promoters.
This research was supported by grants from the USDA and the Agriculture and Food Research Initiative Competitive Grants program (08-00891 and 09-01653). A grant to the Nebraska Center for Virology (1P20RR15635) also supported certain aspects of these studies. Aspen Workman was partially supported by a fellowship from a Ruth L. Kirschstein National Research Service Award (1 T32 AIO60547; National Institute of Allergy and Infectious Diseases).
Published ahead of print on 21 April 2010.