The Unique IR2 Protein of Equine Herpesvirus 1 Neg
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病菌学杂志 2006年第10期
Department of Microbiology and Immunology, and Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
ABSTRACT
The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.
INTRODUCTION
The equine herpesvirus 1 (EHV-1) genome is comprised of 78 genes that are coordinately regulated and temporally expressed as immediate-early (IE), early (E), and late (L) 1 and true late 2 genes (10, 16, 17). The coordinated transcription of EHV-1 genes is regulated by six regulatory proteins that are expressed as one IE protein (IEP), four early proteins (EICP22P, EICP27P, EICP0P, and IR2P), and the late protein ETIF (5-7, 10, 13, 16-18, 20, 22, 24-26, 34-37). In addition, our ongoing work indicated that the IR3 transcript that is antisense to a portion of the IE mRNA negatively regulates IE gene expression (21; B. C. Ahn, S. K. Kim, and D. J. O'Callaghan, unpublished data).
During a lytic infection, two transcripts arise from the IE open reading frame: a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early IR2 mRNA (20). The IEP (1,487 amino acids [aa]) is the major regulatory protein of EHV-1 and mediates the activation of transcription from E and some L viral promoters (34, 35) and is, thus, essential for replication (15). At the same time, the IEP downregulates its own promoter (34) and represses transcription of the true late glycoprotein K (gK) gene by binding to the transcription initiation site of the gK promoter (24). The IEP binds to the consensus binding sequence 5'-ATCGT-3' that overlaps the transcription initiation site of the IE promoter and to degenerate versions of this cognate cis-acting sequence in E and L promoters (29). The IEP possesses several domains essential for transactivation, including (i) an acidic transactivation domain (TAD; aa 3 to 89) (7, 36), (ii) a serine-rich tract that binds to the cellular EAP (SRT; aa 181 to 220) (27), (iii) a nuclear localization signal (NLS) that is comprised of nonpolar and basic amino acids (NLS; aa 963 to 970) (35), and (iv) a DNA-binding domain (DBD; aa 422 to 597) (29). The IEP aa 407 to 757 directly interact with TFIIB aa 125 to 174 (1, 23).
The IR2 gene lies within the immediate-early (IE) gene and its 4.4-kb transcript encodes the 1,165-aa regulatory IR2 protein (IR2P) (20) that is a truncated form (aa 323 to 1,487) of the IE protein (IEP). Therefore, the IR2P lacks the essential acidic transcriptional activation domain (TAD) and serine-rich tract (SRT) of the IEP but still contains binding domains for DNA and the basal transcription factors TFIIB and TBP, and the NLS (1, 23, 27, 29, 35, 36). It has been shown that the DBD of the IEP and IR2P binds to the IE promoter, the early EICP0, EICP22, EICP27, and thymidine kinase (TK) promoters, and the late IR5 promoter (30). However, the function of the IR2P in EHV-1 gene regulation was not determined.
The EICP0 gene encodes an early nuclear phosphoprotein of 419 aa that transactivates all classes of EHV-1 promoters (5, 6). The EICP0P contains a conserved cysteine-rich zinc RING finger (C3HC4 type) near the N terminus that is essential for activation of the E and L (1 and 2) promoters (5). The regulatory functions of the EICP0P are severely antagonized by the IEP (26), and thus the EHV-1 EICP0P differs from HSV-1 ICP0, which is an IE protein that functions synergistically with ICP4 to activate expression of HSV-1 E and L promoters (11, 14, 19). The EHV-1 early EICP22 protein (EICP22P) functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of E and L viral genes (22), likely due to its ability to enhance sequence-specific DNA-binding activity of the IEP (30). The IEP and EICP22P directly interact and colocalize in the nucleus at early times postinfection to form aggregates of dense nuclear structures within the nucleoplasm (13). The EICP27P cooperates with either the IEP or the EICP0P to synergistically transactivate viral promoters (37). IEP aa 424 to 826 and EICP27P aa 41 to 206 harbor the interactive domains (3), and the interaction of the IE and EICP27 proteins may result in the recruitment of the EICP27P to representative early promoters. The ETIF transactivates only the IE promoter and not the early (EICP0, EICP22, EICP27, and TK) or late IR5 promoters (25).
We present here findings that the IR2P by itself downregulated EHV-1 early promoters and inhibited in a dose-dependent manner the transactivation of viral promoters mediated by the IEP and the EICP27P. In vitro interaction assays indicated that the IR2P interacted with the basal transcription factor TBP (TATA box-binding protein). The overexpression of the IR2P reduced EHV-1 production and viral IE gene expression. These studies suggested that the IR2P negatively regulates EHV-1 gene expression by blocking IEP-binding to viral promoter sequences and/or squelching the limited supplies of the cellular transcription factors.
MATERIALS AND METHODS
Cell culture. Mouse fibroblast L-M, rabbit kidney RK13, equine dermis NBL6, and human cervical carcinoma HeLa cells were maintained at 37°C in complete Eagle minimal essential medium supplemented with 100 U of penicillin/ml, 100 μg of streptomycin/ml, nonessential amino acids, and 5% fetal bovine serum (FBS).
Plasmids. Plasmids were constructed and maintained in Escherichia coli HB101 by standard methods (33).
(i) Mammalian IE and EICP0 expression plasmids. Plasmids pSVIE, pSVIE(323-1487) (pSVIR2), pSVIE(323-1487)AAD, pSVIE(1-951), pSVIE(1-1029), pSVIE(243-1029), pSVIE(539-1029), pSVICP0K (pSVEICP0), pCETIF (34), pcDR4 (pSVEICP22), pSVEICP27, and pSVSPORT1 (Gibco-BRL, Rockville, MD) have been described previously (6, 22, 25, 26, 34-37). To generate plasmid pTri-IR2-His, the 0.3-kb N-terminal DNA fragment of the IR2 gene from pTriExIE(1-1487) (30) was amplified by PCR with the primers IR2 #F1 (5'-CATGCCATGGCTTCTCCGCCGGGCCGGAGC-3') and IR2 #R1 (5'-TCGCACCCTCCCCATCGGTGGTGGATCCGA-3'), which contained NcoI and BamHI sites, respectively, and cloned into the NcoI and BamHI sites of the pTriExIE(1-1487).
(ii) In vitro transcription and translation plasmids. Plasmids pGEM44 and pG3hTBP(1-339) have been described previously (2, 9).
(iii) CAT and luciferase reporter plasmids. Plasmids pgK-CAT, pgK(–153/+14)-CAT, pgK(–83/+14)-CAT, pIE-CAT [pIE(–802/+73)-CAT], pTK-CAT, and pEICP0-CAT [pEICP0(–335/-21)-CAT], and pEICP22(E)-CAT have been described previously (22, 26, 28, 34). The IR2, IE, EICP0, EICP22, and EICP27 promoter regions were PCR amplified by using Accuprime Pfx polymerase (Invitrogen, San Diego, CA), template plasmid (pAYC177-XbaI), and proper primers containing KpnI or BglII restriction enzyme sites and cloned into the KpnI and BglII sites of the pGL3-Basic (Promega) or hyg-GL3-Basic that was modified from pGL3-Basic and pREP4 (Invitrogen). To generate plasmid pHyg-GL3-Basic, the hygromycin-resistant gene (3,529 bp) of the pREP4 vector was digested with AseI and SalI and cloned into the AseI and SalI sites of the pGL3-Basic vector. To generate plasmid pIR2(–384/+42)-Luc, the 430-bp DNA fragment of the IR2 promoter from pSVIE was amplified by PCR with primers (forward primer, 5'-AATAGGTACCAGCATCTCCATCTCATCGTCGTCC-3'; reverse primer, 5'-TATAAGATCTCCCCGCGGGCGGTTC-3'), which contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pIR2(–881/+42)-Luc, the 930-bp DNA fragment of the IR2 promoter from pSVIE was amplified by PCR with primers (forward primer, 5'-AATAGGTACCCTTCATCGAGAGCAACGACTTCGG-3'; reverse primer, 5'-TATAAGATCTCCCCGCGGGCGGTTC-3'), which contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pIE(–993/+627)-Luc, the 1,630-bp DNA fragment of the IE promoter from EHV-1 KyA strain clone XbaI B1 (4) was amplified by PCR with primers (forward primer, 5'-AAGGTGGGTACCGGGCATCTCC-3'; reverse primer, 5'-AATAGATCTGGCGTGCTAGCTCCGGC-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pEICP0(–332/–23)-Luc, the 350-bp DNA fragment of the EICP0 promoter from pEICP0(–335/–21)-CAT (28) was amplified by PCR with primers (forward primer, 5'-TAAGGTACCAACCCTTAACTATGCAACC-3'; reverse primer, 5'-TAAAGATCTCCAAATGAAAAGGCTGTATCAG-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic. To generate plasmid pEICP22(–350/+17)-Luc, the 370-bp DNA fragment of the EICP22 promoter from pEICP22(1.0)-CAT (22) was amplified by PCR with primers (forward primer, 5'-AATGGTACCTAAGGGCGGAGACTATGG-3'; reverse primer, 5'-AAAAGATCTGAGCTGGGGTTGCTGG-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic. To generate plasmid pEICP27(–196/+15)-Luc, the 210-bp DNA fragment of the EICP27 promoter from pEICP27-CAT (37) was amplified by PCR with primers (forward primer, 5'-AAAAGATCTGAAAAGGCCAAGAGTGCGG -3'; reverse primer, 5'-AAAGGTACCAGTTGCTGCTCC-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic.
(iv) Glutathione S-transferase (GST) fusion plasmids. Plasmids pGST-IE(1-1487), pGST-IR2 [pGST-IE(323-1487)], pGST-IE(1-88), pGST-IE(1-289), pGST-IE(1-424), pGST-IE(1-960), and pGST-IE(898-1487) have been described previously (20, 23, 29). To generate plasmid pGST-IE(1-88), pGST-IE(1-1487) (29) was digested with NaeI, and a SpeI amber codon linker (5'-CTAGACTAGTCTAG-3') (NEB, Beverly, MA) was inserted.
DNA transfection and CAT assays. L-M cells seeded at 3 x 106 cells per culture dish (60 mm) in Eagle minimum essential medium with 5% FBS were transfected by the liposome-mediated DNA transfection method at 24 h as described elsewhere (7). The reporter and effector plasmids were transfected in the amounts indicated in the figure legends. The total amount of DNA per transfection was adjusted to 8 μg by the addition of pSVSPORT1. After a further 5 h, the cells were washed and refed with fresh medium. At 45 h posttransfection (p.t.), total cell extracts were prepared, and the chloramphenicol acetyltransferase (CAT) activities were assayed as described previously (34).
Luciferase assay. RK13 cells were seeded at 80% confluent in 24-well plates and transfected with 1 pmol of reporter vector and 0.5 pmol of effectors. Then, 6 μl of Lipofectin (Invitrogen, San Diego, CA) was mixed with 330 μl of Opti-MEM medium and incubated for 45 min at room temperature. The effector and reporter vectors were mixed with 330 μl of Opti-MEM medium, and the total amount of DNA was adjusted with pSVSPORT1 DNA. The solutions were combined and incubated at room temperature for 15 min, and one-third volume was transferred into each three wells of RK13 cells. After a further 8 h, the cells were refed with fresh medium. At 48 h p.t., luciferase activity was measured by using a luciferase assay system kit (Promega, Madison, WI) and a Polarstar Optima machine (BMG LABTECH, Durham, NC).
Nucleofection. Batches of 2.0 x 106 cells were used in each transfection experiment with the Nucleofector (Amaxa Biosystems, Germany). NBL6 cells (2 x 106 cells/cuvette) were mixed with 100 μl of Nucleofector solution (Amaxa Biosystems, Germany) at room temperature, followed by the addition of 5 μg of plasmid DNA. The mixture of NBL6 cells, Nucleofector solution, and plasmid DNA was transferred to a cuvette (Amaxa Biosystems) and electroporated using Program T-30. Immediately after transfection, cells were resuspended in 500 μl of Eagle minimum essential medium with 20% FBS and transferred into a 60-mm culture dish. The total amount of DNA per transfection was adjusted to 5 μg by the addition of pSVSPORT1. After 24 h p.t., the cells were harvested and used.
Immunofluorescence assays. RK-13 cell monolayers of 2 x 104 cells per well of two-well chamber slides (Nagle Nunc International, Naperville, IL) were either mock transfected or transfected with pSVIE, pSVIR2, and pTri-IR2-His. At 36 h p.t., the cells were fixed onto the glass slides with 100% ice-cold methanol for 10 min. The slides were washed three times with phosphate-buffered saline (PBS) for 10 min per wash and then incubated with the primary anti-IE polyclonal antibody OC33 (20; antibody diluted 1:500 in PBS) for 1 h. After three washes with PBS, the slides were incubated with the secondary antibody fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody for 1 h. After three washes with PBS, the slides were mounted with a solution of 50% glycerol and 0.1% p-phenylenediamine in PBS. Images were obtained with a Nikon Eclipse TE300 fluorescence microscope.
Western blot analysis. Samples were boiled in 2x Laemmli sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to nitrocellulose filters (Schleicher & Schuell, Inc.) at 100 V for 1 h. Blots were blocked for 30 min in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% Tween 20) containing 10% nonfat powdered milk and then incubated with an IE peptide-specific polyclonal antibody OC33 (20) at a 1:2,000 dilution in TBST for 1 h. The blots were washed three times for 10 min each in TBST and incubated with secondary antibody (anti-rabbit IgG [Fc]-alkaline phosphatase conjugate [Promega]) at a dilution of 1:3,000 for 1 h. The membranes were washed in TBST for three 10-min washes, and the proteins were visualized by incubating the membranes in alkaline phosphatase buffer (0.1 M Tris-HCl [pH 9.5], 0.1 M NaCl, 5.0 mM MgCl2) containing nitroblue tetrazolium (0.33 mg/ml; Life Technologies) and BCIP (5-bromo-4-chloro-3-indolylphosphate; 0.165 mg/ml; Life Technologies).
Expression and purification of GST fusion proteins. The expression and purification of GST fusion proteins has been described elsewhere (29, 30). The bacterial strains E. coli BL21(DE3)pLysE (Novagen, Madison, WI) or trxB(DE3)pLysE (Novagen) were used for the induction of GST fusion protein synthesis. The methods for desalting and for ascertaining the concentration of the purified GST-fusion proteins were described elsewhere (23).
IVTT reactions. In vitro transcription-translation (IVTT) reactions were performed with the TnT Quick-Coupled Rabbit Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's directions. 35S-labeled proteins were synthesized by including [35S]methionine (40 μCi/ml; specific activity, 1,175 Ci/mmol; New England Nuclear Corp., Boston, MA) in the IVTT reactions. The synthesis of the 35S-labeled proteins was verified by SDS-PAGE analysis and autoradiography.
GST-pulldown assays. GST-pulldown assays that used 35S-labeled polypeptides were performed as detailed previously (26). Briefly, 2 μg of either GST alone or the indicated GST fusion protein was mixed with 35S-labeled proteins in 500 μl of NETN buffer. The 35S-labeled proteins precipitated by the GST fusion protein were analyzed by SDS-PAGE, followed by autoradiography and visualization using the Storm PhosphorImager (Molecular Dynamics) or the Molecular Imager FX system (Bio-Rad Laboratories, Hercules, CA).
Virus growth assays. Equine NBL-6 cells were nucleofected with 1 pmol of pSVIE, pSVIR2, or pSVSPORT1. At 24 h p.t., the cells were infected with EHV-1 KyA at a multiplicity of infection (MOI) of 7. Cells were harvested at 1, 24, and 48 h postinfection (p.i.), and virus titers were determined by plaque assay on RK-13 cells.
RESULTS
The EICP0 protein transactivates the early IR2 promoter. To determine the IR2 promoter region, pIR2(–881/+42)-Luc and pIR2(–384/+42)-Luc reporter plasmids were generated and used for luciferase assays (Fig. 1A). The pIR2(–881/+42)-Luc and pIR2(–384/+42)-Luc reporter constructs contain the IR2 promoter region located at nt –881 to +42, and nt –384 to +42, respectively, relative to the transcription initiation site of the IR2 gene. The transcription initiation site of the IR2 mRNA lies 25 bp downstream of a putative TATA-like sequence (20). In luciferase assays the early EICP0P independently transactivated the IR2 promoter (Fig. 1B and C, bar 2). However, the other early regulatory proteins EICP27P, EICP22P, and IR2P failed to transactivate the IR2 promoter (Fig. 1B and C, bars 3, 5, and 6, respectively). The IEP very weakly transactivated the pIR2(–881/+42)-Luc (Fig. 1B, bar 4) but did not transactivate the pIR2(–384/+42)-Luc (Fig. 1C, bar 4), indicating that the IR2 promoter nt –881 to –385 may contain a degenerate version of the IEP-binding consensus sequence ATCGT. Indeed, several putative IEP-binding sequences map in the upstream IR2 promoter region (data not shown). The tegument ETIF protein was not able to transactivate the IR2 promoter (Fig. 1B and C, lane 7), indicating that IR2 may be an early gene. Our previous data showed that the ETIF protein transactivated only the IE promoter, not early (EICP0, EICP22, EICP27, and TK) or late IR5 promoters (25). Figure 1D shows the relative promoter strength of EHV-1 promoters. The IE promoter is strong compared to the promoters of other EHV-1 regulatory genes (Fig. 1D). The IE promoter is stronger than the IR2 promoter by 16-fold.
The IR2P was detected at the early stage of infection in HeLa and NBL6 cells. To detect the IR2P in the EHV-1-infected cells, Western blot analyses were performed with anti-IEP antibody which also detects the IR2P. As a positive control, the IR2P and IR2-His were expressed from the IR2 expression vectors pSVIR2 and pTri-IR2-His, respectively. The IR2-His (Fig. 2B, lane 8, and Fig. 2C) contains a His tag and additional 26 aa residues in the C terminus of the IR2P. A protein identical in size to the 150-kDa IR2P expressed as the positive control appeared at 5 h p.i. and was clearly seen at 7 and 24 h p.i. in EHV-1-infected HeLa cells (Fig. 2A, lanes 4, 5, 6, and 10). Similar results were obtained in the case of EHV-1-infected equine NBL6 cells (Fig. 2B, lanes 4 and 5). Several species of antigenically cross-reactive IEP characterized in previous studies (10, 22, 23) were detected in nuclear extracts of EHV-1-infected and IEP expression vector pSVIE-transfected cells (Fig. 2A, lanes 6 and 8, respectively). The same size IR2P was detected only in EHV-1-infected nuclear extracts, but not in pSVIE-transfected nuclear extracts (Fig. 2, lanes 7 and 8). These results indicated that the 150-kDa IR2P is not a degradation product of the 200-kDa intact IEP. Intracellular distribution of the IR2P within equine NBL6 cells transfected with the IEP and IR2P expression vectors was assessed by indirect immunofluorescence microscopy. The IEP localized to the nuclei of NBL6 cells (Fig. 2C) as expected (35). During a productive lytic infection, the IEP was expressed at the IE stage of infection, dispersed throughout the nucleus, and then later was tightly aggregated in small, dense nuclear structures. IEP aa 963 to 970 are necessary for nuclear localization (35) (Fig. 2C). As expected (9), the IR2P was expressed and also localized to the nuclei of equine NBL6 cells (Fig. 2C) since it harbors the NLS of the IEP.
The TAD of the IEP is not necessary to downregulate its own promoter. Previous results in our laboratory showed that the IEP downregulates its own promoter (34) and represses transcription of the IE gene by binding to an area proximal to the transcription initiation site of the IE promoter (29). The IEP also represses transcription of the true late glycoprotein K (gK) gene by binding to an area proximal to the transcription initiation site of the gK promoter (24). As expected, the IEP downregulated its own promoter in a dose-dependent manner (Fig. 3A, bars 4 and 5). However, the IE promoter was not affected by the empty vector pSVSPORT1 (Fig. 3A, bars 2 and 3). Interestingly, the IR2P, which does not contain the TAD, also downregulated the IE promoter in a dose-dependent manner similar to the IEP (Fig. 3A, bars 6 and 7). These results demonstrated that the IEP TAD is not necessary to downregulate the IE promoter.
The IR2P downregulated the early promoters. To investigate the function of the IR2P in EHV-1 gene expression, CAT assays were performed with early promoter CAT reporter plasmids. The IR2P downregulated the early EICP0 promoter in a dose-dependent manner (Fig. 3B, bars 2 to 4). The IEP alone strongly transactivated the EICP0 promoter (Fig. 3B, bar 5), which is consistent with our previous results (28). When the IEP and IR2P expression vectors were cotransfected, the IR2P abrogated the transactivation mediated by the IEP in a dose-dependent manner (Fig. 3B, bars 6 to 8). The IR2P also abrogated EICP27P-mediated transactivation (Fig. 3B, bars 10 and 11). Similar effects were seen with the early TK and EICP22 promoters (Fig. 3C and D, respectively). These results indicated that the IR2P downregulates early promoters.
The IR2 and IE proteins interact with the basal transcription factor TBP. Our previous results showed that both the IE and the IR2 proteins interact with TFIIB and that IEP aa residues 407 to 757 harbor the TFIIB-binding domain (1, 23). To investigate whether the IR2P interacts with TBP, we carried out GST-pulldown assays in which IEP mutants synthesized as a GST fusion protein (Fig. 4A) were examined for the ability to precipitate radiolabeled TBP (35S-TBP) from an IVTT reaction (Fig. 4B). The GST-IE fusion proteins were stably expressed and purified as described previously (20, 23, 29). Each GST-IE fusion protein migrated at the predicted mass (20, 23, 29). As shown in Fig. 4B, lanes 4 and 5, GST-full-length IE and GST-IR2P precipitated 35S-TBP. However, two N-terminal mutants, GST-IE(1-88) and GST-IE(1-289), failed to precipitate 35S-TBP (Fig. 4B, lanes 6 and 7, respectively). GST-IE(1-424), GST-IE(1-960), and GST-IE(898-1487) also precipitated 35S-TBP (Fig. 4B, lanes 8 to 10, respectively). In Fig. 4C, the IEP and IR2P were produced as radiolabeled proteins by IVTT. GST-TBP precipitated 35S-IEP and 35S-IR2P (Fig. 4C, lanes 4 and 7, respectively), whereas GST alone did not (Fig. 4B, lanes 6 and 7, respectively). Figure 4A shows a summary of the GST-pulldown assays. These results indicated that both the IR2P and IEP interact with TBP and that IR2P aa 1 to 706 (IEP aa 323 to 1,487) harbor more than one domain that mediates an interaction with TBP (Fig. 4A).
IR2P residues that mediate negative regulation. To determine the essential domain of the IR2P that mediates negative regulation, luciferase assays were performed with a panel of IEP deletion mutants. The IEP mutants were previously used to determine domains essential for the antagonism between the IE and EICP0 proteins (26). As expected, the IEP and IR2P downregulated the IE promoter (Fig. 5B, bars 2 and 3, respectively). IR2-AAD encodes a protein in which the TAD of the IEP is deleted and the acidic activation domain (AAD) of the strong transactivator HSV-1 VP16 is inserted at the C terminus of the IR2P. The IR2-AAD fusion protein also downregulated the IE promoter (Fig. 5B, bar 4), confirming results in Fig. 3A, which showed that the IEP TAD is not necessary to downregulate its own promoter. IE(1-1029) and IE(243-1029) also downregulated the IE promoter (Fig. 5B, bars 5 and 6, respectively). However, IE(1-951) and IE(539-1029) did not (Fig. 5B, bars 7 and 8, respectively). IE(1-951) and IE(539-1029) lack the NLS and the DNA-binding activity, respectively (28, 34), indicating that the DBD and NLS of the IR2P may be important for the downregulation. The NLS of the IR2P is required for entry to the nucleus of infected cells. Luciferase assays were performed with the EICP0 promoter luciferase reporter plasmid pEICP0-Luc (Fig. 5C). The IEP strongly transactivated the EICP0 promoter (Fig. 5C, bar 2); however, the IR2P downregulated the EICP0 promoter (Fig. 5C, bar 3). Both the IR2-AAD and the IE(1-1029) contain an activation domain and were able to transactivate the EICP0 promoter (Fig. 5C, bars 4 and 5, respectively). The IE(243-1029) that lacks the TAD downregulated the EICP0 promoter (Fig. 5C, bar 5). The IE(1-951) and IE(539-1029) which lack the NLS and the DBD, respectively, failed to downregulate the EICP0 promoter (Fig. 5C, bars 7 and 8, respectively). When luciferase assays were performed with the early EICP22 promoter reporter plasmid pEICP22-Luc, similar results were observed (Fig. 5D). These results demonstrated that the IR2P domain(s) essential for negative regulation lies within aa 1 to 706 (IEP aa 323 to 1,029) which contain DBD, NLS, and domains that interacts with TFIIB, TBP, and EICP27P (Fig. 5A).
The overexpression of the IR2P reduces EHV-1 production and viral IE gene expression. To investigate whether the IR2P inhibits EHV-1 replication and virus production in NBL-6 cells, transient-transfection and virus growth assays were performed to measure IR2P-mediated decreases in virus titers. Ectopic expression of the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector (Fig. 6A). In contrast, transfection with the IEP expression vector increased virus production by eightfold compared to virus titers achieved in cells transfected with the empty vector (Fig. 6A). The virus reduction by the IR2P was dependent on the transfection efficiency (data not shown).
To investigate whether ectopic expression of the IR2P affected viral gene expression, NBL-6 cells were nucleofected with 1 pmol of empty vector pSVSPORT1 or with the IR2 or IR2-His expression vector, and the production of the IEP after infection was ascertained. The cells were infected with EHV-1 KyA at an MOI of 7 at 24 h p.t. and were harvested at 26 h. after infection for Western blot analysis. The IEP was expressed to a high level in the cells transfected with empty vector (Fig. 6B, lane 5). However, the expression of the IEP was greatly reduced in the cells transfected with either the IR2 or IR2-His expression vector (Fig. 6B, lanes 7 and 9, respectively). These results indicated that the IR2P inhibits the viral gene expression and virus production.
DISCUSSION
The studies presented in this study revealed that the early regulatory IR2P is a negative regulator of EHV-1 gene expression. The IR2P, a truncated form (aa 323 to 1,487) of the IEP, lacks the transcriptional activation domain (TAD) and serine-rich tract (SRT) of the IEP but harbors binding domains for DNA, EICP27P, NLS, and the cellular transcription factors TFIIB and TBP (1, 23, 27, 28, 35, 36) (Fig. 5).
The IR2P alone downregulated early promoters in a dose-dependent manner and abrogated transactivation of viral promoters mediated by the IEP and the EICP27P. Of the many possibilities that may explain how the IR2P abrogates the transactivation function of the IE and EICP27 proteins, one possible mechanism would be a physical interaction with TFIIB and TBP, such that these general transcription factors would not be available to assemble preinitiation complexes for viral transcription (Fig. 7). Indeed, both the IEP and the IR2P are DNA-binding proteins (29) and interact with factors in the basal transcriptional machinery, such as TFIIB and TBP (1, 2, 23) and other EHV-1 regulatory proteins (3, 12, 13, 26). The EICP27P enhances gene expression via a direct interaction with TBP (2) and does not bind to any EHV-1 promoter tested to date (3). The EICP27P cooperates with the IEP to synergistically transactivate viral promoters (37) and is recruited to viral promoters by its interaction with the IEP (3). The IR2P also contains the EICP27P-binding domain, suggesting that the IR2P may interact with the EICP27P. A second possible mechanism of the downregulation mediated by the IR2P is a steric hindrance effect. The IEP binds to its consensus sequence ATCGT (29) and interacts with TFIIB (1, 22) and TBP (Fig. 5) to activate transcription (Fig. 7A). We know that the IR2P also binds to the ATCGT consensus sequence (29) and interacts with TFIIB (1, 23) and TBP (Fig. 5). From these facts, we speculate that the IR2P may block the formation of preinitiation complexes by steric hindrance because the IR2P does not contain the TAD. This possibility will be explored by future gel shift and DNase I footprint assays.
The overexpression of the IR2P reduced IE gene expression and virus production (Fig. 6). These results indicated that the IR2P may inhibit the viral IE and E gene expression, which will lead to virus attenuation by direct interferences with viral replication. To define more precisely the effect of the IR2P on virus replication, a Vero cell line that expresses the IR2P was generated and is being used to assess the levels of reduction in EHV-1 replication. The IR2P domain(s) essential for negative regulation lies within residues 1 to 706, which contain the DBD, the NLS, and domains that interact with TFIIB and TBP. Our previous studies with IE mutant viruses demonstrated that the IEP TAD, SRT, DBD, and NLS are essential for virus replication (8). Further, we demonstrated that the C terminus of the IEP, while not required for maximal transactivation activity, contributes to the efficiency of viral gene expression and replication during the course of a productive infection (8). The C terminus (aa 707 to 1,165) of the IR2P is not necessary for the protein to downregulate EHV-1 promoters.
The exact role of the IR2P in the EHV-1 gene program is unknown, but the protein may contribute to the efficient switch from early to late gene expression. The genes of EHV-1 are coordinately expressed and temporally regulated in an immediate-early (IE), early (E), and late (L) fashion (7, 16, 17), and the sole IEP activates the expression of the E genes (22, 31, 32, 34, 37) but by itself is not able to activate the expression of some of the L genes (22, 34). However, the IEP did not transactivate the early IR2 promoter, which is transactivated only by the EICP0P (Fig. 1). The IR2P downregulated the IE promoter and representative E promoters but did not repress its own promoter (Fig. 1B and C). Both the IE and the IR2 proteins antagonize the transactivation ability of the EICP0P (26). The IR2P by itself downregulated early promoters and abrogated the transactivation of early promoters mediated by the IEP and the EICP27P. This negative regulatory function of the IR2P may help govern the temporal regulation of gene expression. At late times, the IR2P may inhibit the production of the IE and early regulatory proteins and thereby favor the expression of late viral proteins.
To determine whether the IR2P interacts with basal transcription factors such as TFIIB, TBP, and TAF to downregulate EHV-1 promoters, in vitro interaction and gel shift assays will be performed with the IR2P and components that assemble a functional transcriptional preinitiation complexes. In vitro and in vivo binding assays with the IR2P and these cellular factors may give insight into the mechanism by which this unique regulatory protein negatively regulates EHV-1 promoters and contributes to the gene program of this alphaherpesvirus.
ACKNOWLEDGMENTS
We thank Suzanne Zavecz for excellent technical assistance.
Support for this investigation was obtained from Public Health Service research grant AI-22001 from the National Institutes of Health and by NIH grant P20-RR018724 from the National Center for Research Resources.
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ABSTRACT
The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.
INTRODUCTION
The equine herpesvirus 1 (EHV-1) genome is comprised of 78 genes that are coordinately regulated and temporally expressed as immediate-early (IE), early (E), and late (L) 1 and true late 2 genes (10, 16, 17). The coordinated transcription of EHV-1 genes is regulated by six regulatory proteins that are expressed as one IE protein (IEP), four early proteins (EICP22P, EICP27P, EICP0P, and IR2P), and the late protein ETIF (5-7, 10, 13, 16-18, 20, 22, 24-26, 34-37). In addition, our ongoing work indicated that the IR3 transcript that is antisense to a portion of the IE mRNA negatively regulates IE gene expression (21; B. C. Ahn, S. K. Kim, and D. J. O'Callaghan, unpublished data).
During a lytic infection, two transcripts arise from the IE open reading frame: a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early IR2 mRNA (20). The IEP (1,487 amino acids [aa]) is the major regulatory protein of EHV-1 and mediates the activation of transcription from E and some L viral promoters (34, 35) and is, thus, essential for replication (15). At the same time, the IEP downregulates its own promoter (34) and represses transcription of the true late glycoprotein K (gK) gene by binding to the transcription initiation site of the gK promoter (24). The IEP binds to the consensus binding sequence 5'-ATCGT-3' that overlaps the transcription initiation site of the IE promoter and to degenerate versions of this cognate cis-acting sequence in E and L promoters (29). The IEP possesses several domains essential for transactivation, including (i) an acidic transactivation domain (TAD; aa 3 to 89) (7, 36), (ii) a serine-rich tract that binds to the cellular EAP (SRT; aa 181 to 220) (27), (iii) a nuclear localization signal (NLS) that is comprised of nonpolar and basic amino acids (NLS; aa 963 to 970) (35), and (iv) a DNA-binding domain (DBD; aa 422 to 597) (29). The IEP aa 407 to 757 directly interact with TFIIB aa 125 to 174 (1, 23).
The IR2 gene lies within the immediate-early (IE) gene and its 4.4-kb transcript encodes the 1,165-aa regulatory IR2 protein (IR2P) (20) that is a truncated form (aa 323 to 1,487) of the IE protein (IEP). Therefore, the IR2P lacks the essential acidic transcriptional activation domain (TAD) and serine-rich tract (SRT) of the IEP but still contains binding domains for DNA and the basal transcription factors TFIIB and TBP, and the NLS (1, 23, 27, 29, 35, 36). It has been shown that the DBD of the IEP and IR2P binds to the IE promoter, the early EICP0, EICP22, EICP27, and thymidine kinase (TK) promoters, and the late IR5 promoter (30). However, the function of the IR2P in EHV-1 gene regulation was not determined.
The EICP0 gene encodes an early nuclear phosphoprotein of 419 aa that transactivates all classes of EHV-1 promoters (5, 6). The EICP0P contains a conserved cysteine-rich zinc RING finger (C3HC4 type) near the N terminus that is essential for activation of the E and L (1 and 2) promoters (5). The regulatory functions of the EICP0P are severely antagonized by the IEP (26), and thus the EHV-1 EICP0P differs from HSV-1 ICP0, which is an IE protein that functions synergistically with ICP4 to activate expression of HSV-1 E and L promoters (11, 14, 19). The EHV-1 early EICP22 protein (EICP22P) functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of E and L viral genes (22), likely due to its ability to enhance sequence-specific DNA-binding activity of the IEP (30). The IEP and EICP22P directly interact and colocalize in the nucleus at early times postinfection to form aggregates of dense nuclear structures within the nucleoplasm (13). The EICP27P cooperates with either the IEP or the EICP0P to synergistically transactivate viral promoters (37). IEP aa 424 to 826 and EICP27P aa 41 to 206 harbor the interactive domains (3), and the interaction of the IE and EICP27 proteins may result in the recruitment of the EICP27P to representative early promoters. The ETIF transactivates only the IE promoter and not the early (EICP0, EICP22, EICP27, and TK) or late IR5 promoters (25).
We present here findings that the IR2P by itself downregulated EHV-1 early promoters and inhibited in a dose-dependent manner the transactivation of viral promoters mediated by the IEP and the EICP27P. In vitro interaction assays indicated that the IR2P interacted with the basal transcription factor TBP (TATA box-binding protein). The overexpression of the IR2P reduced EHV-1 production and viral IE gene expression. These studies suggested that the IR2P negatively regulates EHV-1 gene expression by blocking IEP-binding to viral promoter sequences and/or squelching the limited supplies of the cellular transcription factors.
MATERIALS AND METHODS
Cell culture. Mouse fibroblast L-M, rabbit kidney RK13, equine dermis NBL6, and human cervical carcinoma HeLa cells were maintained at 37°C in complete Eagle minimal essential medium supplemented with 100 U of penicillin/ml, 100 μg of streptomycin/ml, nonessential amino acids, and 5% fetal bovine serum (FBS).
Plasmids. Plasmids were constructed and maintained in Escherichia coli HB101 by standard methods (33).
(i) Mammalian IE and EICP0 expression plasmids. Plasmids pSVIE, pSVIE(323-1487) (pSVIR2), pSVIE(323-1487)AAD, pSVIE(1-951), pSVIE(1-1029), pSVIE(243-1029), pSVIE(539-1029), pSVICP0K (pSVEICP0), pCETIF (34), pcDR4 (pSVEICP22), pSVEICP27, and pSVSPORT1 (Gibco-BRL, Rockville, MD) have been described previously (6, 22, 25, 26, 34-37). To generate plasmid pTri-IR2-His, the 0.3-kb N-terminal DNA fragment of the IR2 gene from pTriExIE(1-1487) (30) was amplified by PCR with the primers IR2 #F1 (5'-CATGCCATGGCTTCTCCGCCGGGCCGGAGC-3') and IR2 #R1 (5'-TCGCACCCTCCCCATCGGTGGTGGATCCGA-3'), which contained NcoI and BamHI sites, respectively, and cloned into the NcoI and BamHI sites of the pTriExIE(1-1487).
(ii) In vitro transcription and translation plasmids. Plasmids pGEM44 and pG3hTBP(1-339) have been described previously (2, 9).
(iii) CAT and luciferase reporter plasmids. Plasmids pgK-CAT, pgK(–153/+14)-CAT, pgK(–83/+14)-CAT, pIE-CAT [pIE(–802/+73)-CAT], pTK-CAT, and pEICP0-CAT [pEICP0(–335/-21)-CAT], and pEICP22(E)-CAT have been described previously (22, 26, 28, 34). The IR2, IE, EICP0, EICP22, and EICP27 promoter regions were PCR amplified by using Accuprime Pfx polymerase (Invitrogen, San Diego, CA), template plasmid (pAYC177-XbaI), and proper primers containing KpnI or BglII restriction enzyme sites and cloned into the KpnI and BglII sites of the pGL3-Basic (Promega) or hyg-GL3-Basic that was modified from pGL3-Basic and pREP4 (Invitrogen). To generate plasmid pHyg-GL3-Basic, the hygromycin-resistant gene (3,529 bp) of the pREP4 vector was digested with AseI and SalI and cloned into the AseI and SalI sites of the pGL3-Basic vector. To generate plasmid pIR2(–384/+42)-Luc, the 430-bp DNA fragment of the IR2 promoter from pSVIE was amplified by PCR with primers (forward primer, 5'-AATAGGTACCAGCATCTCCATCTCATCGTCGTCC-3'; reverse primer, 5'-TATAAGATCTCCCCGCGGGCGGTTC-3'), which contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pIR2(–881/+42)-Luc, the 930-bp DNA fragment of the IR2 promoter from pSVIE was amplified by PCR with primers (forward primer, 5'-AATAGGTACCCTTCATCGAGAGCAACGACTTCGG-3'; reverse primer, 5'-TATAAGATCTCCCCGCGGGCGGTTC-3'), which contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pIE(–993/+627)-Luc, the 1,630-bp DNA fragment of the IE promoter from EHV-1 KyA strain clone XbaI B1 (4) was amplified by PCR with primers (forward primer, 5'-AAGGTGGGTACCGGGCATCTCC-3'; reverse primer, 5'-AATAGATCTGGCGTGCTAGCTCCGGC-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pGL3-Basic. To generate plasmid pEICP0(–332/–23)-Luc, the 350-bp DNA fragment of the EICP0 promoter from pEICP0(–335/–21)-CAT (28) was amplified by PCR with primers (forward primer, 5'-TAAGGTACCAACCCTTAACTATGCAACC-3'; reverse primer, 5'-TAAAGATCTCCAAATGAAAAGGCTGTATCAG-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic. To generate plasmid pEICP22(–350/+17)-Luc, the 370-bp DNA fragment of the EICP22 promoter from pEICP22(1.0)-CAT (22) was amplified by PCR with primers (forward primer, 5'-AATGGTACCTAAGGGCGGAGACTATGG-3'; reverse primer, 5'-AAAAGATCTGAGCTGGGGTTGCTGG-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic. To generate plasmid pEICP27(–196/+15)-Luc, the 210-bp DNA fragment of the EICP27 promoter from pEICP27-CAT (37) was amplified by PCR with primers (forward primer, 5'-AAAAGATCTGAAAAGGCCAAGAGTGCGG -3'; reverse primer, 5'-AAAGGTACCAGTTGCTGCTCC-3') that contain KpnI or BglII sites, respectively, and the fragment was cloned into the KpnI and BglII sites of the pHyg-GL3-Basic.
(iv) Glutathione S-transferase (GST) fusion plasmids. Plasmids pGST-IE(1-1487), pGST-IR2 [pGST-IE(323-1487)], pGST-IE(1-88), pGST-IE(1-289), pGST-IE(1-424), pGST-IE(1-960), and pGST-IE(898-1487) have been described previously (20, 23, 29). To generate plasmid pGST-IE(1-88), pGST-IE(1-1487) (29) was digested with NaeI, and a SpeI amber codon linker (5'-CTAGACTAGTCTAG-3') (NEB, Beverly, MA) was inserted.
DNA transfection and CAT assays. L-M cells seeded at 3 x 106 cells per culture dish (60 mm) in Eagle minimum essential medium with 5% FBS were transfected by the liposome-mediated DNA transfection method at 24 h as described elsewhere (7). The reporter and effector plasmids were transfected in the amounts indicated in the figure legends. The total amount of DNA per transfection was adjusted to 8 μg by the addition of pSVSPORT1. After a further 5 h, the cells were washed and refed with fresh medium. At 45 h posttransfection (p.t.), total cell extracts were prepared, and the chloramphenicol acetyltransferase (CAT) activities were assayed as described previously (34).
Luciferase assay. RK13 cells were seeded at 80% confluent in 24-well plates and transfected with 1 pmol of reporter vector and 0.5 pmol of effectors. Then, 6 μl of Lipofectin (Invitrogen, San Diego, CA) was mixed with 330 μl of Opti-MEM medium and incubated for 45 min at room temperature. The effector and reporter vectors were mixed with 330 μl of Opti-MEM medium, and the total amount of DNA was adjusted with pSVSPORT1 DNA. The solutions were combined and incubated at room temperature for 15 min, and one-third volume was transferred into each three wells of RK13 cells. After a further 8 h, the cells were refed with fresh medium. At 48 h p.t., luciferase activity was measured by using a luciferase assay system kit (Promega, Madison, WI) and a Polarstar Optima machine (BMG LABTECH, Durham, NC).
Nucleofection. Batches of 2.0 x 106 cells were used in each transfection experiment with the Nucleofector (Amaxa Biosystems, Germany). NBL6 cells (2 x 106 cells/cuvette) were mixed with 100 μl of Nucleofector solution (Amaxa Biosystems, Germany) at room temperature, followed by the addition of 5 μg of plasmid DNA. The mixture of NBL6 cells, Nucleofector solution, and plasmid DNA was transferred to a cuvette (Amaxa Biosystems) and electroporated using Program T-30. Immediately after transfection, cells were resuspended in 500 μl of Eagle minimum essential medium with 20% FBS and transferred into a 60-mm culture dish. The total amount of DNA per transfection was adjusted to 5 μg by the addition of pSVSPORT1. After 24 h p.t., the cells were harvested and used.
Immunofluorescence assays. RK-13 cell monolayers of 2 x 104 cells per well of two-well chamber slides (Nagle Nunc International, Naperville, IL) were either mock transfected or transfected with pSVIE, pSVIR2, and pTri-IR2-His. At 36 h p.t., the cells were fixed onto the glass slides with 100% ice-cold methanol for 10 min. The slides were washed three times with phosphate-buffered saline (PBS) for 10 min per wash and then incubated with the primary anti-IE polyclonal antibody OC33 (20; antibody diluted 1:500 in PBS) for 1 h. After three washes with PBS, the slides were incubated with the secondary antibody fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody for 1 h. After three washes with PBS, the slides were mounted with a solution of 50% glycerol and 0.1% p-phenylenediamine in PBS. Images were obtained with a Nikon Eclipse TE300 fluorescence microscope.
Western blot analysis. Samples were boiled in 2x Laemmli sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to nitrocellulose filters (Schleicher & Schuell, Inc.) at 100 V for 1 h. Blots were blocked for 30 min in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% Tween 20) containing 10% nonfat powdered milk and then incubated with an IE peptide-specific polyclonal antibody OC33 (20) at a 1:2,000 dilution in TBST for 1 h. The blots were washed three times for 10 min each in TBST and incubated with secondary antibody (anti-rabbit IgG [Fc]-alkaline phosphatase conjugate [Promega]) at a dilution of 1:3,000 for 1 h. The membranes were washed in TBST for three 10-min washes, and the proteins were visualized by incubating the membranes in alkaline phosphatase buffer (0.1 M Tris-HCl [pH 9.5], 0.1 M NaCl, 5.0 mM MgCl2) containing nitroblue tetrazolium (0.33 mg/ml; Life Technologies) and BCIP (5-bromo-4-chloro-3-indolylphosphate; 0.165 mg/ml; Life Technologies).
Expression and purification of GST fusion proteins. The expression and purification of GST fusion proteins has been described elsewhere (29, 30). The bacterial strains E. coli BL21(DE3)pLysE (Novagen, Madison, WI) or trxB(DE3)pLysE (Novagen) were used for the induction of GST fusion protein synthesis. The methods for desalting and for ascertaining the concentration of the purified GST-fusion proteins were described elsewhere (23).
IVTT reactions. In vitro transcription-translation (IVTT) reactions were performed with the TnT Quick-Coupled Rabbit Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's directions. 35S-labeled proteins were synthesized by including [35S]methionine (40 μCi/ml; specific activity, 1,175 Ci/mmol; New England Nuclear Corp., Boston, MA) in the IVTT reactions. The synthesis of the 35S-labeled proteins was verified by SDS-PAGE analysis and autoradiography.
GST-pulldown assays. GST-pulldown assays that used 35S-labeled polypeptides were performed as detailed previously (26). Briefly, 2 μg of either GST alone or the indicated GST fusion protein was mixed with 35S-labeled proteins in 500 μl of NETN buffer. The 35S-labeled proteins precipitated by the GST fusion protein were analyzed by SDS-PAGE, followed by autoradiography and visualization using the Storm PhosphorImager (Molecular Dynamics) or the Molecular Imager FX system (Bio-Rad Laboratories, Hercules, CA).
Virus growth assays. Equine NBL-6 cells were nucleofected with 1 pmol of pSVIE, pSVIR2, or pSVSPORT1. At 24 h p.t., the cells were infected with EHV-1 KyA at a multiplicity of infection (MOI) of 7. Cells were harvested at 1, 24, and 48 h postinfection (p.i.), and virus titers were determined by plaque assay on RK-13 cells.
RESULTS
The EICP0 protein transactivates the early IR2 promoter. To determine the IR2 promoter region, pIR2(–881/+42)-Luc and pIR2(–384/+42)-Luc reporter plasmids were generated and used for luciferase assays (Fig. 1A). The pIR2(–881/+42)-Luc and pIR2(–384/+42)-Luc reporter constructs contain the IR2 promoter region located at nt –881 to +42, and nt –384 to +42, respectively, relative to the transcription initiation site of the IR2 gene. The transcription initiation site of the IR2 mRNA lies 25 bp downstream of a putative TATA-like sequence (20). In luciferase assays the early EICP0P independently transactivated the IR2 promoter (Fig. 1B and C, bar 2). However, the other early regulatory proteins EICP27P, EICP22P, and IR2P failed to transactivate the IR2 promoter (Fig. 1B and C, bars 3, 5, and 6, respectively). The IEP very weakly transactivated the pIR2(–881/+42)-Luc (Fig. 1B, bar 4) but did not transactivate the pIR2(–384/+42)-Luc (Fig. 1C, bar 4), indicating that the IR2 promoter nt –881 to –385 may contain a degenerate version of the IEP-binding consensus sequence ATCGT. Indeed, several putative IEP-binding sequences map in the upstream IR2 promoter region (data not shown). The tegument ETIF protein was not able to transactivate the IR2 promoter (Fig. 1B and C, lane 7), indicating that IR2 may be an early gene. Our previous data showed that the ETIF protein transactivated only the IE promoter, not early (EICP0, EICP22, EICP27, and TK) or late IR5 promoters (25). Figure 1D shows the relative promoter strength of EHV-1 promoters. The IE promoter is strong compared to the promoters of other EHV-1 regulatory genes (Fig. 1D). The IE promoter is stronger than the IR2 promoter by 16-fold.
The IR2P was detected at the early stage of infection in HeLa and NBL6 cells. To detect the IR2P in the EHV-1-infected cells, Western blot analyses were performed with anti-IEP antibody which also detects the IR2P. As a positive control, the IR2P and IR2-His were expressed from the IR2 expression vectors pSVIR2 and pTri-IR2-His, respectively. The IR2-His (Fig. 2B, lane 8, and Fig. 2C) contains a His tag and additional 26 aa residues in the C terminus of the IR2P. A protein identical in size to the 150-kDa IR2P expressed as the positive control appeared at 5 h p.i. and was clearly seen at 7 and 24 h p.i. in EHV-1-infected HeLa cells (Fig. 2A, lanes 4, 5, 6, and 10). Similar results were obtained in the case of EHV-1-infected equine NBL6 cells (Fig. 2B, lanes 4 and 5). Several species of antigenically cross-reactive IEP characterized in previous studies (10, 22, 23) were detected in nuclear extracts of EHV-1-infected and IEP expression vector pSVIE-transfected cells (Fig. 2A, lanes 6 and 8, respectively). The same size IR2P was detected only in EHV-1-infected nuclear extracts, but not in pSVIE-transfected nuclear extracts (Fig. 2, lanes 7 and 8). These results indicated that the 150-kDa IR2P is not a degradation product of the 200-kDa intact IEP. Intracellular distribution of the IR2P within equine NBL6 cells transfected with the IEP and IR2P expression vectors was assessed by indirect immunofluorescence microscopy. The IEP localized to the nuclei of NBL6 cells (Fig. 2C) as expected (35). During a productive lytic infection, the IEP was expressed at the IE stage of infection, dispersed throughout the nucleus, and then later was tightly aggregated in small, dense nuclear structures. IEP aa 963 to 970 are necessary for nuclear localization (35) (Fig. 2C). As expected (9), the IR2P was expressed and also localized to the nuclei of equine NBL6 cells (Fig. 2C) since it harbors the NLS of the IEP.
The TAD of the IEP is not necessary to downregulate its own promoter. Previous results in our laboratory showed that the IEP downregulates its own promoter (34) and represses transcription of the IE gene by binding to an area proximal to the transcription initiation site of the IE promoter (29). The IEP also represses transcription of the true late glycoprotein K (gK) gene by binding to an area proximal to the transcription initiation site of the gK promoter (24). As expected, the IEP downregulated its own promoter in a dose-dependent manner (Fig. 3A, bars 4 and 5). However, the IE promoter was not affected by the empty vector pSVSPORT1 (Fig. 3A, bars 2 and 3). Interestingly, the IR2P, which does not contain the TAD, also downregulated the IE promoter in a dose-dependent manner similar to the IEP (Fig. 3A, bars 6 and 7). These results demonstrated that the IEP TAD is not necessary to downregulate the IE promoter.
The IR2P downregulated the early promoters. To investigate the function of the IR2P in EHV-1 gene expression, CAT assays were performed with early promoter CAT reporter plasmids. The IR2P downregulated the early EICP0 promoter in a dose-dependent manner (Fig. 3B, bars 2 to 4). The IEP alone strongly transactivated the EICP0 promoter (Fig. 3B, bar 5), which is consistent with our previous results (28). When the IEP and IR2P expression vectors were cotransfected, the IR2P abrogated the transactivation mediated by the IEP in a dose-dependent manner (Fig. 3B, bars 6 to 8). The IR2P also abrogated EICP27P-mediated transactivation (Fig. 3B, bars 10 and 11). Similar effects were seen with the early TK and EICP22 promoters (Fig. 3C and D, respectively). These results indicated that the IR2P downregulates early promoters.
The IR2 and IE proteins interact with the basal transcription factor TBP. Our previous results showed that both the IE and the IR2 proteins interact with TFIIB and that IEP aa residues 407 to 757 harbor the TFIIB-binding domain (1, 23). To investigate whether the IR2P interacts with TBP, we carried out GST-pulldown assays in which IEP mutants synthesized as a GST fusion protein (Fig. 4A) were examined for the ability to precipitate radiolabeled TBP (35S-TBP) from an IVTT reaction (Fig. 4B). The GST-IE fusion proteins were stably expressed and purified as described previously (20, 23, 29). Each GST-IE fusion protein migrated at the predicted mass (20, 23, 29). As shown in Fig. 4B, lanes 4 and 5, GST-full-length IE and GST-IR2P precipitated 35S-TBP. However, two N-terminal mutants, GST-IE(1-88) and GST-IE(1-289), failed to precipitate 35S-TBP (Fig. 4B, lanes 6 and 7, respectively). GST-IE(1-424), GST-IE(1-960), and GST-IE(898-1487) also precipitated 35S-TBP (Fig. 4B, lanes 8 to 10, respectively). In Fig. 4C, the IEP and IR2P were produced as radiolabeled proteins by IVTT. GST-TBP precipitated 35S-IEP and 35S-IR2P (Fig. 4C, lanes 4 and 7, respectively), whereas GST alone did not (Fig. 4B, lanes 6 and 7, respectively). Figure 4A shows a summary of the GST-pulldown assays. These results indicated that both the IR2P and IEP interact with TBP and that IR2P aa 1 to 706 (IEP aa 323 to 1,487) harbor more than one domain that mediates an interaction with TBP (Fig. 4A).
IR2P residues that mediate negative regulation. To determine the essential domain of the IR2P that mediates negative regulation, luciferase assays were performed with a panel of IEP deletion mutants. The IEP mutants were previously used to determine domains essential for the antagonism between the IE and EICP0 proteins (26). As expected, the IEP and IR2P downregulated the IE promoter (Fig. 5B, bars 2 and 3, respectively). IR2-AAD encodes a protein in which the TAD of the IEP is deleted and the acidic activation domain (AAD) of the strong transactivator HSV-1 VP16 is inserted at the C terminus of the IR2P. The IR2-AAD fusion protein also downregulated the IE promoter (Fig. 5B, bar 4), confirming results in Fig. 3A, which showed that the IEP TAD is not necessary to downregulate its own promoter. IE(1-1029) and IE(243-1029) also downregulated the IE promoter (Fig. 5B, bars 5 and 6, respectively). However, IE(1-951) and IE(539-1029) did not (Fig. 5B, bars 7 and 8, respectively). IE(1-951) and IE(539-1029) lack the NLS and the DNA-binding activity, respectively (28, 34), indicating that the DBD and NLS of the IR2P may be important for the downregulation. The NLS of the IR2P is required for entry to the nucleus of infected cells. Luciferase assays were performed with the EICP0 promoter luciferase reporter plasmid pEICP0-Luc (Fig. 5C). The IEP strongly transactivated the EICP0 promoter (Fig. 5C, bar 2); however, the IR2P downregulated the EICP0 promoter (Fig. 5C, bar 3). Both the IR2-AAD and the IE(1-1029) contain an activation domain and were able to transactivate the EICP0 promoter (Fig. 5C, bars 4 and 5, respectively). The IE(243-1029) that lacks the TAD downregulated the EICP0 promoter (Fig. 5C, bar 5). The IE(1-951) and IE(539-1029) which lack the NLS and the DBD, respectively, failed to downregulate the EICP0 promoter (Fig. 5C, bars 7 and 8, respectively). When luciferase assays were performed with the early EICP22 promoter reporter plasmid pEICP22-Luc, similar results were observed (Fig. 5D). These results demonstrated that the IR2P domain(s) essential for negative regulation lies within aa 1 to 706 (IEP aa 323 to 1,029) which contain DBD, NLS, and domains that interacts with TFIIB, TBP, and EICP27P (Fig. 5A).
The overexpression of the IR2P reduces EHV-1 production and viral IE gene expression. To investigate whether the IR2P inhibits EHV-1 replication and virus production in NBL-6 cells, transient-transfection and virus growth assays were performed to measure IR2P-mediated decreases in virus titers. Ectopic expression of the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector (Fig. 6A). In contrast, transfection with the IEP expression vector increased virus production by eightfold compared to virus titers achieved in cells transfected with the empty vector (Fig. 6A). The virus reduction by the IR2P was dependent on the transfection efficiency (data not shown).
To investigate whether ectopic expression of the IR2P affected viral gene expression, NBL-6 cells were nucleofected with 1 pmol of empty vector pSVSPORT1 or with the IR2 or IR2-His expression vector, and the production of the IEP after infection was ascertained. The cells were infected with EHV-1 KyA at an MOI of 7 at 24 h p.t. and were harvested at 26 h. after infection for Western blot analysis. The IEP was expressed to a high level in the cells transfected with empty vector (Fig. 6B, lane 5). However, the expression of the IEP was greatly reduced in the cells transfected with either the IR2 or IR2-His expression vector (Fig. 6B, lanes 7 and 9, respectively). These results indicated that the IR2P inhibits the viral gene expression and virus production.
DISCUSSION
The studies presented in this study revealed that the early regulatory IR2P is a negative regulator of EHV-1 gene expression. The IR2P, a truncated form (aa 323 to 1,487) of the IEP, lacks the transcriptional activation domain (TAD) and serine-rich tract (SRT) of the IEP but harbors binding domains for DNA, EICP27P, NLS, and the cellular transcription factors TFIIB and TBP (1, 23, 27, 28, 35, 36) (Fig. 5).
The IR2P alone downregulated early promoters in a dose-dependent manner and abrogated transactivation of viral promoters mediated by the IEP and the EICP27P. Of the many possibilities that may explain how the IR2P abrogates the transactivation function of the IE and EICP27 proteins, one possible mechanism would be a physical interaction with TFIIB and TBP, such that these general transcription factors would not be available to assemble preinitiation complexes for viral transcription (Fig. 7). Indeed, both the IEP and the IR2P are DNA-binding proteins (29) and interact with factors in the basal transcriptional machinery, such as TFIIB and TBP (1, 2, 23) and other EHV-1 regulatory proteins (3, 12, 13, 26). The EICP27P enhances gene expression via a direct interaction with TBP (2) and does not bind to any EHV-1 promoter tested to date (3). The EICP27P cooperates with the IEP to synergistically transactivate viral promoters (37) and is recruited to viral promoters by its interaction with the IEP (3). The IR2P also contains the EICP27P-binding domain, suggesting that the IR2P may interact with the EICP27P. A second possible mechanism of the downregulation mediated by the IR2P is a steric hindrance effect. The IEP binds to its consensus sequence ATCGT (29) and interacts with TFIIB (1, 22) and TBP (Fig. 5) to activate transcription (Fig. 7A). We know that the IR2P also binds to the ATCGT consensus sequence (29) and interacts with TFIIB (1, 23) and TBP (Fig. 5). From these facts, we speculate that the IR2P may block the formation of preinitiation complexes by steric hindrance because the IR2P does not contain the TAD. This possibility will be explored by future gel shift and DNase I footprint assays.
The overexpression of the IR2P reduced IE gene expression and virus production (Fig. 6). These results indicated that the IR2P may inhibit the viral IE and E gene expression, which will lead to virus attenuation by direct interferences with viral replication. To define more precisely the effect of the IR2P on virus replication, a Vero cell line that expresses the IR2P was generated and is being used to assess the levels of reduction in EHV-1 replication. The IR2P domain(s) essential for negative regulation lies within residues 1 to 706, which contain the DBD, the NLS, and domains that interact with TFIIB and TBP. Our previous studies with IE mutant viruses demonstrated that the IEP TAD, SRT, DBD, and NLS are essential for virus replication (8). Further, we demonstrated that the C terminus of the IEP, while not required for maximal transactivation activity, contributes to the efficiency of viral gene expression and replication during the course of a productive infection (8). The C terminus (aa 707 to 1,165) of the IR2P is not necessary for the protein to downregulate EHV-1 promoters.
The exact role of the IR2P in the EHV-1 gene program is unknown, but the protein may contribute to the efficient switch from early to late gene expression. The genes of EHV-1 are coordinately expressed and temporally regulated in an immediate-early (IE), early (E), and late (L) fashion (7, 16, 17), and the sole IEP activates the expression of the E genes (22, 31, 32, 34, 37) but by itself is not able to activate the expression of some of the L genes (22, 34). However, the IEP did not transactivate the early IR2 promoter, which is transactivated only by the EICP0P (Fig. 1). The IR2P downregulated the IE promoter and representative E promoters but did not repress its own promoter (Fig. 1B and C). Both the IE and the IR2 proteins antagonize the transactivation ability of the EICP0P (26). The IR2P by itself downregulated early promoters and abrogated the transactivation of early promoters mediated by the IEP and the EICP27P. This negative regulatory function of the IR2P may help govern the temporal regulation of gene expression. At late times, the IR2P may inhibit the production of the IE and early regulatory proteins and thereby favor the expression of late viral proteins.
To determine whether the IR2P interacts with basal transcription factors such as TFIIB, TBP, and TAF to downregulate EHV-1 promoters, in vitro interaction and gel shift assays will be performed with the IR2P and components that assemble a functional transcriptional preinitiation complexes. In vitro and in vivo binding assays with the IR2P and these cellular factors may give insight into the mechanism by which this unique regulatory protein negatively regulates EHV-1 promoters and contributes to the gene program of this alphaherpesvirus.
ACKNOWLEDGMENTS
We thank Suzanne Zavecz for excellent technical assistance.
Support for this investigation was obtained from Public Health Service research grant AI-22001 from the National Institutes of Health and by NIH grant P20-RR018724 from the National Center for Research Resources.
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