The reporter plasmid p35_BAS-Luc, which contained the firefly luciferase reporter gene, was constructed by PCR cloning of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) p35 basal promoter into the pGL3-Basic vector (Promega), using the primer pair AcMNPV-p35-F1 and AcMNPV-p35-R1 (Table 1). The other reporter plasmid, G5p35_BAS-Luc, contained five copies of the GAL4 DNA binding site upstream of the AcMNPV p35 basal promoter, and it was constructed by amplifying five copies of the GAL4 DNA binding site from pG5SEAP (Clontech), using primers GAL4bs-F and GAL4bs-R (Table 1), and then cloning them into p35_BAS-Luc vector KpnI and XhoI sites.

The plasmids pGST-IE1_1-224 and pGST-IE1_81-224 were generated by cloning PCR-amplified WSSV ie1 coding region fragments flanked by EcoRI and XhoI restriction sites into the corresponding sites of predigested pGEX-5X-1 vector (Amersham Pharmacia Biotech). pGST-IE1_{81-224 C2-H2mut} was constructed by rolling-circle PCR as described above, using pGST-IE1_81-224 as the template. The plasmid pGST-VP36B was constructed by cloning the WSSV structural protein VP36B into the pGEX-5X-1 vector, using primers VP36B-F and VP36B-R. Primer sequences are listed in Table 3.

PCR cloning was used to insert the WSSV ie1 coding region into the vectors pDHsp/V5-His and pDHsp/FLAG-His, which both contain the heat-inducible Drosophila heat shock protein 70 promoter (28). The resulting plasmids, pDHsp/IE1-V5-His and pDHsp/IE1-FLAG-His, expressed the V5 and FLAG tag fusion proteins, respectively. Another IE1 expression plasmid, pcDNA3/IE1, was constructed by PCR cloning the WSSV ie1 coding region into the commercialized vector pcDNA3 (Invitrogen). Primer sequences are listed in Table 3.

Transfections of Sf9 insect cells were performed using the Cellfectin reagent (Invitrogen). Briefly, the Sf9 insect cells were seeded onto a 24-well plate (1 × 10⁵ cells/well) and grown in Sf-900 II serum-free medium (Invitrogen) overnight at 27°C. Cells were cotransfected with 300 ng of the reporter plasmid containing the firefly luciferase gene, 500 ng of one of the different effector plasmids or the empty vector, and 100 ng of the Renilla luciferase gene plasmid, phRL/AcMNPVie1 (30). The phRL/AcMNPVie1 plasmid contains the AcMNPV ie1 promoter to drive the expression of the Renilla luciferase gene and was used to monitor and normalize transfection efficiency. Cells were collected at 48 h posttransfection, and the cell lysates were prepared according to the Promega instruction manual for the dual-luciferase assay system. Luciferase activities were measured with a luminometer (Labsystems). Firefly luciferase activity values were then normalized against the activities of the Renilla luciferase to correct for transfection efficiency, and data were expressed as relative luciferase activities. Luciferase activities were determined for triplicate transfections in two independent experiments, and the means and standard deviations (SD) were calculated. For the point mutation assays, statistically significant differences from the wild-type TAD expression plasmid were identified using paired Student's t test, with significance set at P values of <0.01.

Total cell lysates were prepared by directly adding 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol [DTT], 4% SDS, 0.2% bromophenol blue, 20% glycerol) to cell pellets and then boiling the samples for 10 min. The samples were separated in 15% polyacrylamide gels, transferred to a polyvinylidene difluoride membrane (MSI), incubated with either anti-V5 antibody (Sigma) or anti-β-actin antibody (Chemicon), and then detected with a secondary peroxidase-conjugated antibody. Detected proteins were visualized using an ECL (Perkin-Elmer) detection system.

GST fusion proteins were expressed and purified according to the manufacturer's manual. After overnight culture of the GST plasmids in transformed Escherichia coli BL21 Codon Plus cells (Stratagene), the cultures were diluted 1:200 (vol/vol) in Luria-Bertani (LB) medium containing 50 μg/ml of ampicillin and then incubated for another 3 h at 37°C. Expression of the fusion proteins was induced by the addition of IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 1 mM, and the cultures were grown for a further 24 h at 15°C. The soluble GST fusion proteins were resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 1 mM DTT, and 1 mM EDTA) and purified by affinity chromatography with an FF 16/10 GST column (Amersham Biosciences). The fusion proteins were eluted from the beads with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10 mM reduced glutathione, and then the purified proteins were condensed with an Amicon Ultra-30 column (Millipore). To obtain the IE1_1-224 protein, the GST was removed from the GST-IE1_1-224 fusion protein by digestion with factor Xa (10 units of protease/1 mg GST fusion protein; Amersham Biosciences) in 1× phosphate-buffered saline (PBS) containing 1 mM DTT at 22°C for 16 h. The digested GST was removed with a GST column. Purity of the samples was assessed by SDS-PAGE, and the protein concentration was determined using a Bio-Rad protein assay kit.

EMSA was performed as described previously (49), with some modifications. Single-stranded oligonucleotides containing a 25-nucleotide random core sequence flanked on each side by 27 nucleotides [5′-GTCGCTCGAGCGGTATGACGAGATCTA(N)₂₅TAGATCTGCGTCACTAGTCTAGACTAG-3′ (where N can be any of the four deoxyribonucleotides)] were synthesized (9). A double-stranded [α-³²P]dCTP-labeled oligonucleotide library was generated by PCR using the forward primer 5′-GTCGCTCGAGCGGTATGACG-3′ and the reverse primer 5′-CTAGTCTAGACTAGTGACGC-3′. Binding reactions were carried out for 30 min at room temperature in 15-μl reaction mixtures that contained different concentrations of purified recombinant proteins with 10 mM HEPES (pH 7.9), 1 mM DTT, 5 mM MgCl₂, 0.5 mM ZnCl₂, 60 mM KCl, 0.05% NP-40, 200 ng poly(dI-dC), 10% glycerol, and 50 μg/ml bovine serum albumin. The DNA-protein complexes were resolved in 7.5% polyacrylamide gels in 0.5× Tris-glycine buffer (12.5 mM Tris and 100 mM glycine). The gels were dried and visualized by autoradiography. Some EMSA reactions were run with no ZnCl₂ in the binding buffer.

Coupled in vitro transcription-translation reactions were conducted using a TNT kit in accordance with the manufacturer's protocol (Promega). One microgram of plasmid pcDNA3/IE1 DNA and 2 μl of [³⁵S]methionine (1,000 Ci/mmol; 10 mCi/ml) were added to the TNT mixture (50-μl total volume), and reactions were carried out at 30°C for 90 min. To ensure that there was no contamination by nucleic acids, the purified proteins GST and GST-IE1_1-224 and the TNT product [³⁵S]methionine-labeled IE1 were all pretreated with nucleases (1 U DNase I [Invitrogen] and 0.5 μg RNase [Sigma]) for 1 h at 25°C in 50 mM Tris-HCl, pH 8, 5 mM MgCl₂, 2.5 mM CaCl₂, 100 mM NaCl, 5% glycerol, and 1 mM DTT. Subsequently, equal amounts of the TNT product were incubated with GST-IE1_1-224 (10 μg) or GST (10 μg) bound to glutathione-Sepharose beads in 150 μl NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, and a cocktail tablet of protease inhibitors [Roche]) in the presence of ethidium bromide (100 μg/ml) at 4°C for 3 h. After three 10-min washes with NETN buffer, the proteins that bound to the beads were resolved by 15% SDS-PAGE, and the gel was dried and exposed to Kodak Biomax MS film.

Sf9 cells were seeded on six-well plates (8 × 10⁵ cells/well) and cotransfected with 2 μg pDHsp/IE1-V5-His and 2 μg pDHsp/IE1-FLAG-His expression plasmid, using Cellfectin reagent. After transfection for 16 to 18 h, the cells were heat shocked in a 42°C water bath for 30 min and then returned to 27°C. Six hours after being heat shocked, the cells were washed with PBS and lysed in 100 μl of NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) supplemented with a protease inhibitor cocktail tablet. The lysis procedure was carried out on ice for 10 min with occasional shaking. The lysate was centrifuged at 12,000 × g for 5 min, and an aliquot of the supernatant (10 μl) was reserved for immunoblot analysis to confirm the expression of the transfected gene. The remaining supernatant (90 μl) was then incubated with 15 μl of anti-FLAG M2 affinity gel (Sigma) at 4°C overnight with rotation. The gel was then washed five times in 150 μl of NP-40 lysis buffer. Aliquots of the total cell lysates and immunoprecipitates were separated by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. V5-tagged IE1 fusion proteins were detected with rabbit anti-V5 antibody (Sigma) and goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate (Sigma). FLAG-tagged IE1 was detected with mouse anti-FLAG monoclonal antibody (Sigma) and goat anti-mouse immunoglobulin G-horseradish peroxidase conjugate (Sigma).

To evaluate the native molecular size of IE1, purified IE1_1-224 was analyzed using a Superdex 200-pg gel filtration column (Amersham Biosciences) (using buffer comprised of 500 mM NaCl, 1 mM DTT, 1 mM EDTA, and 20 mM sodium acetate, pH 5.5). Gel filtration standard proteins (bovine serum albumin [67 kDa], ovalbumin [43 kDa], chymotrypsinogen A [25 kDa], and RNase A [13.7 kDa]) were used to calibrate the column. For each protein, the logarithm of molecular mass was plotted against K_av, which was calculated as follows: K_av = (V_e − V_o)/(V_t − V_o), where V_e is the elution volume, V_o is the column void volume using blue dextran 2000, and V_t is the total column bed volume (120 ml for Superdex 200-pg gel filtration column).

A PCR fragment representing the coding region of ie1 was amplified using the IE1-NdeI-F/IE1-XhoI-R primer set (Table 3), digested with restriction enzymes, and cloned into pET-28b(+) (Novagen). The resulting pET clone was transformed into BL21 cells. For protein expression and purification, the cells were grown overnight at 37°C in LB medium supplemented with 50 μg of kanamycin/ml and 34 μg of chloramphenicol/ml. The cells were inoculated into new medium at a ratio of 1:50 and grown at 37°C for 2 to 2.5 h. Expression was induced by the addition of 1 mM IPTG, and incubation was continued for another 1.5 to 3 h. The induced bacteria were spun down at 4°C, suspended in ice-cold PBS containing 10% glycerol and a protease inhibitor cocktail tablet, and then sonicated for 3 min on ice. The insoluble debris was collected by centrifugation, suspended in PBS containing 1.5% sodium lauryl sarcosine, and solubilized by shaking at room temperature for 1 h. The supernatant was clarified by centrifugation and mixed with Ni-nitrilotriacetic acid-agarose beads (Qiagen) on a rotating wheel at 4°C for 16 h or overnight. The beads were then washed several times with ice-cold wash buffer (1 M NaCl, 10 mM Tris-HCl, pH 7.5) to remove unbound material. The fusion proteins were eluted directly from the beads with SDS sample buffer and then subjected to SDS-PAGE analysis. The protein bands containing the fusion proteins were sliced from the gel, minced, mixed with Freund's adjuvant, and used for antibody production.

In order to determine the essential domains for transactivation, three series of deletion mutants of WSSV IE1 were generated and constructed as fusion proteins with the GAL4 DBD at the N terminus (Fig. (Fig.2A,2A, B, and C). Additional V5 tags were attached to the C termini of these fusion proteins for Western blot analysis (Fig. (Fig.2D).2D). The various constructs encoding the IE1 deletion mutants were transfected into Sf9 cells together with the GAL4-responsive reporter plasmid G5p35_BAS-Luc. Surprisingly, coarse mapping (Fig. (Fig.2A)2A) showed that the transactivation activity of most of the IE1 mutants was almost completely abolished, except for that of GAL4-IE1_1-92, which exhibited an activity that was 2 times higher than that of the full-length IE1 fusion protein and 5.4 times higher than that of the GAL4 DBD construct. These initial results suggested that WSSV IE1 residues 1 to 92 function as a TAD, that residues 50 to 92 may be critical for this function (compare GAL4-IE1_1-49 to GAL4-IE1_1-92), and that residues 93 to 137 may inhibit this transactivation activity (compare GAL4-IE1_1-92 to GAL4-IE1_1-137). Next, the series of N-terminal and C-terminal truncation constructs, shown in Fig. 2B and C, respectively, were designed to more finely delineate the boundaries of the N-terminal activation domain. Transfection with these series showed that residues 1 to 80 exhibited an even stronger transactivation activity than residues 1 to 92 did (Fig. (Fig.2C),2C), which is consistent with the results for GAL4-IE1_81-224 versus GAL4-IE1_92-224 (Fig. (Fig.2A).2A). These results suggest that the minimal IE1 TAD may be located within aa 1 to 80. Two additional potential inhibitory domains were also identified. The results of deletions made in the region of aa 1 to 49 suggested that amino acid residues 41 to 49 might act as an inhibitory domain (Fig. (Fig.2B,2B, compare GAL4-IE1_50-92 to GAL4-IE1_20-92, GAL4-IE1_30-92, and GAL4-IE1_40-92). The other possible inhibitory domain was identified at residues 81 to 92 (Fig. (Fig.2C,2C, compare GAL4-IE1_1-92 to GAL4-IE1_1-80), although we note that the different activation levels might have been due, at least in part, to the larger quantity of expressed GAL4-IE1_1-80 fusion protein. The Western blots in Fig. Fig.2D2D confirmed that all of these constructs were successfully expressed. Note that every deletion mutant had a higher expression level than that of the wild-type IE1 fusion protein (compare Fig. Fig.2D2D to Fig. Fig.1D).1D). These higher expression levels were probably due to the shorter lengths of the transcripts and encoded proteins, which would lead to increased transcriptional and translational efficiencies.

Sequence analysis shows that there are 12 acidic amino acids and 6 basic residues in the IE1 minimal TAD (aa 1 to 80), giving the TAD a net negative charge of −6 and a pI of 4.3. Since most TADs can be classified as either acidic activators (46), glutamine-rich activators (11), aromatic and hydrophobic activators (44), or proline-rich activators (37), the acidity of the WSSV IE1 TAD suggested that it might fall within the acidic class of activation domains. We investigated this possibility by constructing mutants (Table 2) in which alanine (A) was used to replace the wild-type aspartate (D), glutamate (E), or glycine (G) residues. GAL4 transactivation assays showed that almost all of these alanine substitutions significantly reduced G5p35_BAS-Luc activation (P < 0.01). Replacement of increasing numbers of acidic residues led to a further decrease in transactivation activity (Fig. (Fig.3A),3A), while substitutions of nonacidic residues (G20A and G41A) (Fig. (Fig.3A)3A) had no significant effect on transactivation. We therefore concluded that the negatively charged residues are critical for WSSV IE1 transactivation activity. An immunoblot analysis of the mutated IE1 proteins after a typical transfection showed that most of the alanine substitution mutants produced proteins at levels close to that of the wild-type construct (Fig. (Fig.3B).3B). Only the E71A/D72A mutant exhibited a very low level of protein expression. This may have been due to instability and rapid degradation of the expressed protein, which might in turn explain why this protein exhibited the lowest transactivation activity in Fig. Fig.3B.3B. Meanwhile, the results for all other mutants indicate that in the case of IE1, as with other virus immediate-early proteins, specific acidic amino acids are particularly critical to transcriptional function (6, 15).

The DNA binding activity of WSSV IE1 was investigated by gel mobility shift assays using a 79-bp double-stranded DNA oligonucleotide containing a central 25-bp randomized sequence. GST-tagged versions of IE1 and VP36B (a WSSV structural protein which served as a negative control) were expressed in E. coli to produce either GST-IE1_1-224, GST-VP36B, or IE1_1-224 alone. These soluble, well-expressed proteins were then purified with glutathione-Sepharose beads (Fig. (Fig.4A).4A). The entire purification process was conducted in the presence of EDTA to ensure that the expressed proteins were free of Zn²⁺ contamination. Purified IE1_1-224 and GST-IE1_1-224 proteins both retarded the migration of the oligonucleotide and formed major discrete bands by 7.5% native PAGE (Fig. (Fig.4B,4B, lanes 4 to 6 and 7 to 9, respectively). Furthermore, the intensities of these bands increased with increasing amounts of purified protein. In contrast, no complex was formed with the GST or with the purified WSSV structural protein control GST-VP36B (Fig. (Fig.4B,4B, lanes 1 to 3 and 11). The last three lanes in Fig. Fig.4B4B show that Zn²⁺ is not required for the DNA binding activity of IE1_1-224. These preliminary data suggested that even though the ie1 coding region contains a Cys₂/His₂-type zinc finger motif (X₃-Cys-X_3-4-Cys-X₁₂-His-X_3-4-His-X₄) (31), WSSV IE1 may not in fact be a zinc finger protein. This was further investigated in the following experiments.

Since DBDs and TADs usually do not overlap, we hypothesized that the DBD of IE1 was located in the C-terminal region. To test this hypothesis, we expressed and purified two GST-IE1 proteins: GST-IE1_81-224 was a deletion without the N-terminal TAD, and GST-IE1_{81-224C2-H2mut} was identical except for a mutated zinc finger motif (Fig. (Fig.5A).5A). An SDS-PAGE gel of these two fusion proteins is shown in Fig. Fig.5B.5B. The lower band, at ~26 kDa, was confirmed by Western blot analysis to be nonfused GST (data not shown). The EMSA showed that when the TAD was deleted, IE1 was still able to bind DNA in either the presence or absence of Zn²⁺ (Fig. (Fig.5C,5C, lanes 2 to 4 and 5 to 7). When the deletion's zinc finger domain was point mutated, DNA binding activity was not markedly affected in either the presence or absence of Zn²⁺ (Fig. (Fig.5C,5C, lanes 8 to 10 and 11 to 13). The binding activity of the mutant was not affected by the presence of Zn²⁺ ions.

Many virus immediate-early proteins are in dimeric form when they bind to DNA (8, 16, 40, 57). We therefore performed an in vitro biochemical binding assay to determine whether IE1 can also self-interact directly. For this assay, the GST-IE1_1-224 fusion protein was bound to glutathione-Sepharose beads and incubated with in vitro-translated, [³⁵S]methionine-labeled IE1. SDS-PAGE analysis showed that ³⁵S-labeled IE1 bound to the GST-IE1_1-224 fusion protein but not to GST, indicating that IE1 can interact directly with itself (Fig. (Fig.6A).6A). We also studied the homotypic interaction between IE1 proteins by a coimmunoprecipitation assay using V5-tagged and FLAG-tagged versions of IE1 expressed in Sf9 insect cells. Complexes consisting of IE1-V5 plus IE1-FLAG were coimmunoprecipitated by anti-FLAG antiserum and detected by Western blotting using anti-V5 antibody (Fig. (Fig.6B).6B). Both sets of results indicated that IE1 can undergo specific self-interaction directly.