Varicella-Zoster Virus IE62 Protein Utilizes the Human Mediator Complex in Promoter Activation

Cells and viruses.MeWo cells, a human melanoma cell line that supports VZV replication, were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. VZV strain MSP was propagated in MeWo cell monolayers as described by Lynch et al. (41).

Plasmids.The pGEX-GST62AD and pGEX-GST16AD plasmids expressing glutathione S-transferase (GST) fusions containing the IE62 and VP16 activation domains, respectively, were derived from the pGEX-4T-3 plasmid (Amersham Biosciences, Piscataway, NJ). An N-terminal fragment of the IE62 protein (amino acids [aa] 1 to 107) which exhibited 3- to 10-fold higher levels of transactivation activity in a Gal4 fusion construct than the minimal IE62 TAD (aa 1 to 86) (53) was inserted between the BamHI and the SalI restriction sites in the parental plasmid to create pGEX-GST62AD. A second N-terminal IE62 fragment encoding aa 1 to 226 was inserted into the same sites to create pGEX-62(1-226). The VP16 TAD (aa 410 to 490) was inserted between the BamHI and the XhoI sites in the parental plasmid to create the pGEX-GST16AD plasmid. The pQE-Gal62AD and pQE-Gal16AD plasmids were constructed using the pQE-Tri expression vector (Qiagen, Inc. Valencia, CA). The DNA binding domain of the yeast Gal4 protein (aa 1 to 147) was inserted between the NcoI and the BamHI sites in the pQE-Tri vector followed by insertion of the IE62 TAD-containing N-terminal fragment (aa 1 to 107) yielding the pQE-Gal62AD. The VP16 TAD (aa 410 to 490) was inserted in a similar manner yielding the pQE-Gal16AD expression plasmid. All constructs were verified by sequencing at the Roswell Park Cancer Institute Biopolymer Facility.

The construction of the dual luciferase reporter vector pRFL/WT (where WT is wild type) containing the VZV bidirectional ORF28/29 promoter flanked by Renilla and firefly luciferase reporter genes was previously described by Yang et al. (70). The pUSFTALuc reporter vector described in Yang et al. (71) contains a minimal model IE62-responsive promoter consisting of a typical TATA element and an upstream consensus USF binding site. The cloning of pCMV62 plasmid expressing IE62 under the control of the cytomegalovirus immediate-early (CMV IE) promoter was described previously by Perera et al. (55). The Flag-Med25-expressing plasmid (pCDNA3FlagARC92) was provided by Anders Näär (Harvard University). The hemagglutinin (HA)-Med23-expressing plasmid (pCS2-hSur2-HA) was provided by Arnold Berk (University of California, Los Angeles). The pGalYY1N90 plasmid expressing the GalYY1AD was provided by Techung Lee (State University of New York, Buffalo, NY). The pFRLuc plasmid was purchased from Stratagene (La Jolla, CA).

GST tag protein affinity pull-down assays.Following induction with IPTG (isopropyl-β-d-thiogalactopyranoside), crude lysates of Escherichia coli expressing GST and GST62AD and GST16AD fusions were prepared and clarified as previously described (71). Aliquots (300 to 500 μl) of the bacterial lysates were added to 50 μl of glutathione-Sepharose beads and incubated for 1 h at 4°C. A total of 750 μg of MeWo cell nuclear protein extract (52) in 300 μl of buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) was added to the protein-coupled beads and incubated overnight at 4°C. Following three washes with 0.1% Triton X-100 in buffer D, bound protein was eluted with 60 μl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer by boiling and analyzed by SDS-PAGE and immunoblotting.

Transient transfection and reporter gene assays.Transient transfection assays in MeWo cells were performed in triplicate in 24-well plates as previously described (71). Briefly, cells were transfected at 90% confluence using 2 μl of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) per μg of total plasmid DNA transfected. Specific amounts of the plasmid DNA used in individual experiments are indicated in the figure legends. Cells were lysed at 48 h posttransfection in lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 1% NP-40, 1 mM EDTA), followed by dual luciferase assays using a dual luciferase reporter assay system (Promega, Madison, WI). The pEF1α-RL vector expressing Renilla luciferase under the control of the EF1α promoter (Promega, Madison, WI) was used as an internal normalization control reporter for transfections with reporter vectors expressing only the firefly luciferase reporter. The pCMVβ-GalSport vector (Gibco, Carlsbad, CA) was used as an internal normalization control for assays performed with the dual luciferase pRFL/WT reporter. Each transfection experiment was repeated a minimum of three times. Average values of results from triplicate assays within a typical experiment are presented.

Immune capture and coimmunoprecipitation assays.MeWo cells in 10-cm cell culture dishes were transfected with 16 μg of pCMV62 plasmid and/or 8 μg of Flag-Med25 plasmid. In experiments where only one of the expression plasmids was used, pcDNA3.1(+) (Invitrogen, Carlsbad, CA) was added to maintain a constant amount of transfected plasmid DNA. At 48 h posttransfection, cell lysates were collected by adding 1 ml of Flag-lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) supplemented with protease inhibitor cocktail prepared using protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN) to each dish. Clarified cell lysates were stored at −80°C prior to use in coimmunoprecipitation assays.

In the immune capture assays, 50 μl of EZview Red anti-Flag M2 affinity gel monoclonal anti-Flag antibody-coupled agarose beads (Sigma, St. Louis, MO) were incubated with 1 ml of cell lysate overnight. Following three 20-min washes with TBST buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20), bound proteins were eluted by boiling with 50 μl of SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting. Coimmunoprecipitation experiments using the H6 monoclonal anti-IE62 antibody generated by Arvin et al. (3) using VZV-infected cell lysates were performed as described by Peng et al. (52) and Yang et al. (71). The antibodies used for immunoblot detection of the precipitated material included anti-Flag M2 antibody (Sigma, St. Louis, MO) and rabbit polyclonal anti-IE62 antibody raised against full-length IE62 purified from recombinant baculovirus-infected cells as described by Spengler et al. (62).

Med25 RNA interference.Predesigned silencer small interfering RNA (siRNA) targeting Med25 (ARC92) was obtained commercially (Ambion, Inc., Austin, TX) (5′-GGAUGGUCCAGUUCCAUUUTT-3′). A scrambled control RNA (scRNA) (5′-UAUGCUCAGCGUCUAUGUGTT-3′) was generated by Integrated DNA Technologies (Coralville, IA). A 10 nM concentration of Med25 siRNA or scRNA was transfected into MeWo cells at 30% confluence in 24-well plates using Lipofectamine 2000. For reverse transcription-PCR (RT-PCR) assays, cells were harvested 70 h posttransfection, and total cell RNA was isolated by using an RNAqueous kit (Ambion, Inc., Austin, TX) according to the manufacturer's instructions. For reporter gene assays, the pFRLuc reporter plasmids and plasmids expressing Gal4 fusion proteins containing TADs from viral and cellular transactivators were cotransfected into MeWo cells 48 h after the initial siRNA transfection. Cell lysates were collected 30 h after plasmid transfection, and the levels of the firefly luciferase were determined as described above.

RNA isolated from the cells in RNA interference experiments was treated with DNase I (Invitrogen, Carlsbad, CA) and purified using an RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Total RNA (2 μg) was subjected to RT using oligo(dT)₂₀ as the primer and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). The primers used in the PCR to amplify the Med25 gene were 5′-GGTCTACGTGAATCATGGCGAGAAC-3′ (sense) and 5′-AGAGACTCCAGGTCCTTGTTGGTGA-3′ (antisense). Use of these primers generated a PCR product of 159 bp. Quantum RNA β-actin internal standards (Ambion, Inc., Austin, TX) with a 1:9 ratio of primers and competimers were used in the reaction to amplify a 294-bp fragment of the β-actin gene as an internal control. The PCRs were run for 28 cycles, which was predetermined to be in the linear range of detection.

ChIP assays.ChIP assays were performed by using a ChIP assay kit (Upstate, Temecula, CA) according to the manufacturer's instructions. MeWo cells in 10-cm cell culture dishes (5 × 10⁶ cells per dish) were cotransfected with 4 μg of pUSFTALuc reporter vector and 1 μg of the pCMV62 or pcDNA3.1 vectors using Lipofectamine 2000 (Invitogen, Carlsbad, CA). Cells were fixed 42 h posttransfection with 1% formaldehyde in culture medium for 10 min at 37°C and lysed by addition of 200 μl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1). Cell lysates were sonicated on ice to shear DNA to lengths between 200 and 1,000 bp (four sets of 15-s pulses interspersed with 1-min breaks) using a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) equipped with a 2-mm tip.

The sonicated cell supernatants were diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) and precleared with 75 μl of protein G agarose-salmon sperm DNA (50% slurry) for 2 h at 4°C. Twenty microliters of precleared lysate was saved for use as input controls. Immunoprecipitating antibodies were added to the remainder of the precleared lysate and the mixtures were incubated overnight at 4°C. The antibodies and antiserum used included 5 μg of normal rabbit immunoglobulin G (IgG) as antibody control (Santa Cruz Biotechnology, Santa Cruz, CA), 20 μl of rabbit polyclonal anti-IE62 antiserum (62), 5 μg of rabbit anti-Med23 antibody (Bethyl Laboratories, Inc., Montgomery, TX), and 5 μg of monoclonal anti-CDK8 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

The immune complexes were collected by adding 60 μl of protein G agarose-salmon sperm DNA (50% slurry). The agarose pellet was washed sequentially with 1 ml each of the low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and Tris-EDTA buffer included in the kit. The bound immune complexes were eluted with 2× 250 μl of elution buffer (1% SDS, 0.1 mM NaHCO₃). The eluates were heated at 65°C overnight to reverse the protein-DNA cross-link, and the protein was digested with proteinase K for 1 h at 45°C. The DNA was recovered by phenol-chloroform extraction and ethanol precipitation and subjected to PCR. The primers used to amplify the promoter region within the pUSFTALuc reporter vector were 5′-GCTAACATAACCCGGGAGGTACC-3′ (forward) and 5′-GAGGATAGAATGGCGCCGGCCCT-3′ (reverse). PCR was performed with 22, 24, and 26 amplification cycles to confirm the linear range of the assays. The amplified fragments were resolved on a 2% agarose gel containing 0.5 μg/ml ethidium bromide. Images and data quantification were obtained using Bio-Rad Quantity One software.

Immunofluorescence microscopy.MeWo cells were grown on four-well glass chamber slides (Nalge Nunc International Corp., Naperville, IL). Infections were initiated with VZV MSP strain-infected cell stocks (70). Cells were fixed at 12 and 24 h with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and permeabilized with 1% Triton X-100 in PBS for 2 min. Cells were blocked with 10% goat serum for 10 min and incubated with primary antibody in 10% goat serum for 2 h at room temperature. Primary antibodies used included the monoclonal H6 anti-IE62 antibody (1:1,000 dilution) and rabbit polyclonal anti-Med23 antibody (1:500 dilution) (Bethyl Laboratories, Inc., Montgomery, TX). Fluorescently labeled secondary antibodies in 10% goat serum were added following triplicate washes, and incubation proceeded for 1 h. The secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG(H+L) and Alexa Fluor 568 goat anti-rabbit IgG(H+L) (Molecular Probes, Inc., Eugene, OR), both at a dilution of 1:500. Nuclei were visualized by staining with DAPI ([4′,6′-diamidino-2-phenylindole] 300 nM in PBS) (Invitrogen, Carlsbad, CA) for 3 min at room temperature.

The samples were viewed using a Zeiss Axioimager Z1 fluorescence microscope installed with filter sets suitable for viewing the Alexa Fluor 488 and 568 and DAPI dyes. Images were captured with a Zeiss MrM cooled charge-coupled-device camera controlled by AxioVision Rel, version 4.5, software at ×20 magnification.

The IE62 acidic TAD targets a Mediator complex lacking CDK8.Based on the evidence that Mediator is a component of the RNAPII transcriptional apparatus and acts as a general coactivator for a variety of cellular and viral transactivators (8, 22, 27, 34, 46, 49, 56, 59, 63, 68), we hypothesized that Mediator interacts with the N-terminal IE62 TAD. The first set of experiments performed was aimed at determining if such an interaction could be demonstrated in protein pull-down assays. The N-terminal 107 amino acids of IE62 containing the acidic TAD (aa 1 to 86) and a second larger IE62 fragment (aa 1 to 226) were fused to the N terminus of GST and expressed in E. Coli. The VP16 acidic TAD (aa 410 to 490 fused to GST) and GST alone were used as positive and negative controls, respectively. The ability of the two TADs to interact with Mediator was assessed using nuclear extracts derived from MeWo cells that had been transfected with a plasmid expressing Flag-tagged Med25. The Med25 subunit interacts directly with the VP16 TAD and Yang et al. (68) showed that ectopically expressed Flag-Med25 is incorporated into the Mediator complex in cells. Eluates containing stably bound proteins were probed for the presence of Flag-Med25 and the endogenous Med23 and CDK8 Mediator subunits.

Both Med25 (Fig. 1A) and Med23 (Fig. 1B) were captured by the IE62 TAD suggesting that the IE62 activation domain, like the VP16 TAD, is capable of interacting with the Mediator complex. However, there was no evidence of CDK8 capture by the IE62 TAD in six repetitions of these experiments, whereas the presence of CDK8 was readily detectable in assays using the VP16 TAD. The VP16 TAD results are consistent with other studies investigating the interaction of this TAD with Mediator (25, 46, 68). The absence of CDK8 binding to the GST62AD fusion protein suggests that the IE62 activation domain targets a form of the Mediator complex lacking CDK8 or possibly causes dissociation of CDK8 upon its interaction with Mediator. The interaction between the Mediator complex and the IE62 TAD also appears to be considerably weaker than that involving the VP16 TAD. As indicated in the lower panels of Fig. 1A and B, despite the fact that the levels of the GST62TAD fusion protein present in the reaction were higher than those of the VP16 TAD fusion, the amounts of Med25 and Med23 captured by the IE62 TAD were lower than the amount captured by the VP16 TAD. Similar levels of capture of Med25 were observed with the GST fusion containing aa 1 of 226 of IE62 compared to that observed with the GST62TAD construct (Fig. 1A). Thus, additional IE62 flanking sequences do not increase this binding, further suggesting that the interaction of Med25 with the IE62 TAD is weaker than that with the VP16 TAD.

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FIG. 1.

In vitro interaction of the IE62 TAD with the Mediator complex. (A) GST pull-down experiments showing that the IE62 TAD can capture ectopically expressed Med25. Bacterially expressed GST, a GST62AD fusion protein containing IE62 aa 1 to 107 which includes the TAD, a GST-fusion protein containing IE62 aa 1 to 226, and a GST16AD fusion protein containing the VP16 TAD (aa 410 to 490) were coupled to glutathione-Sepharose beads and incubated with nuclear extracts of MeWo cells transfected with the Flag-Med25-expressing plasmid. Flag-tagged Med25 was detected by immunoblotting using anti-Flag M2 antibody (upper panel). The lower panel is a Coomassie-stained gel showing the relative amounts of the four GST proteins coupled to the beads. (B) GST pull-down experiments with untransfected MeWo cell nuclear extracts examining the interaction of the IE62 TAD with endogenous Mediator subunits. The presence of Med23 and CDK8 was detected by immunoblotting as shown in the two upper panels. The lower panel is a Coomassie-stained gel showing the relative amounts of the three GST proteins coupled to the beads.

Med25 is a functional target of the IE62 TAD.We next wished to determine if Med25 was a functional target of the IE62 TAD. Med25 has been shown to be a functional target of the VP16 TAD (46, 68). Both of the TADs from these two potent alpha herpesvirus transactivators are composed primarily of acidic and aliphatic amino acids. The two TADs are also similar in length to the IE62 TAD containing 86 amino acids and the VP16 TAD containing 81 residues (15, 54, 64). However, comparison of their sequences (54) indicates that they show very low levels of homology (3.9% identity and 5.9% homology). Further, the acidic activation domain of the cellular transactivator p53 interacts with Med17 as does the VP16 TAD but does not exhibit physical or functional interactions with Med25 (30, 68). Finally, aspects of IE62 function and mechanism are also reminiscent of the adenovirus E1A protein, which interacts with Med23 (4, 8, 13). Thus, there was no a priori reason for Med25 necessarily to be a functional target of the IE62 TAD.

Overexpression of Mediator subunits targeted by the VP16 TAD (Med25) and the adenovirus E1A protein (Med23) resulted in loss of activation in transfection assays due to competition between the free subunits and the Mediator complex for the specific activators (8, 72). Using this approach, both the IE62 and VP16 TADs were fused with the Gal4 DNA binding domain, and their activities were examined in the context of the pFRLuc (Stratagene, La Jolla, CA) reporter vector, which contains five Gal4 binding sites upstream of a consensus TATA element (Fig. 2A). A Gal4 fusion construct incorporating the N-terminal 90 amino acids containing the acidic TAD of the cellular transcription factor, YY1 (11), was also used in the transfection assays as a control since the amino acid composition of the YY1 TAD differs significantly from that of both of the two viral TADs. As shown in Fig. 2B, all three Gal4 fusion proteins exhibited significant and readily detectable levels of activity relative to the pQE-Tri DNA control, with the VP16 TAD being the strongest activator and the YY1 TAD the weakest.

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FIG. 2.

Effects of ectopic overexpression of Med25 and Med23 on transcriptional activation by the IE62TAD. (A) Schematic depicting the pFRLuc reporter vector and the three effector plasmids expressing the Gal62AD, Gal16AD, and GalYY1AD fusion proteins. (B) Comparison of the transactivation activity of the three Gal4 fusion constructs. The plasmid pFRLuc (0.5 μg) and 0.1 μg of each of the plasmids expressing Gal62AD, Gal16AD, and GalYY1AD or the same amount of the parental pQE-Tri plasmid were cotransfected into MeWo cells. The transactivation activities of Gal62AD, Gal16AD, and GalYY1AD are presented as relative induction of the firefly luciferase activity in comparison to that observed with the control pQE-Tri plasmid. Error bars indicate the standard deviations derived from triplicate transfections. (C) Effects of ectopic overexpression of Flag-Med25 and HA-Med23 in transient transfection assays. MeWo cells were transfected with 0.3 μg of pFRLuc reporter vector; 0.1 μg of each of the plasmids expressing Gal62AD, Gal16AD, and GalYY1AD; and 0.4 μg of each of the plasmids expressing pcDNA3.1, Flag-Med25, and HA-Med23. The activity of each transactivator in the absence of the overexpressed Mediator subunits was normalized to 100% (black bars). The levels of transactivation in the presence of overexpressed Flag-Med25 (open bars) and HA-Med23 (gray bars) are presented as the percentage of activation relative to that observed in the absence of ectopic expression of the Mediator subunits. Statistical significance was determined by a one-way analysis of variance followed by Tukey's posthoc test. (**, P < 0.01).

Overexpression of Med25 virtually abolished the activity of the IE62 TAD under our experimental conditions. VP16 TAD activity was decreased to less than 40% of that observed in the absence of exogenously expressed Med25, in agreement with the results of Yang et al. (68). This differential inhibition correlates with the apparently weaker interaction observed with the IE62 TAD in Fig. 1A. Finally, transfection with the equivalent amount of the Med25-expressing plasmid resulted in little or no effect on the activity of the YY1 TAD (Fig. 2C). In contrast, transfection with a Med23-overexpressing plasmid (pCS2-hSur2-HA) whose use resulted in dramatic inhibition of the activity of a Gal4-E1A construct (8) exhibited little, if any, effect on the activities of the three TADs examined here. These results indicate that the Med25 subunit of Mediator is a direct target of the IE62 TAD.

Since the above experiments were performed with model promoters and Gal4 fusion proteins incorporating only the IE62 TAD, we wished to determine the effect of overexpression of Med25 on the activity of full-length IE62 in the context of a native viral regulatory element. The element chosen was the VZV ORF28/29 regulatory element, expression from which is activated by IE62. This element consists of two divergent overlapping promoters sharing an essential USF site and regulates expression of the viral DNA polymerase (ORF28) and the VZV major DNA-binding protein (ORF29) (70). The effect of ectopic expression of Med25 on this regulatory element was assessed using a dual luciferase reporter construct with Renilla luciferase in the position of the ORF28 gene and firefly luciferase in the presence of the ORF28 gene (Fig. 3A). The results from transfection assays in the presence of increasing amounts of the Med25 expression plasmid are shown in Fig. 3. Exogenous Med25 inhibited IE62-mediated activation of the ORF28/29 promoter in a dose-dependent manner (Fig. 3B). In contrast, the basal promoter activity in the absence of IE62 was not altered in the presence of the highest amount of the Med25-expressing plasmid used in the presence of IE62 (Fig. 3C). Taken together, these results suggest that Med25 is a bona fide target of IE62 via the IE62 TAD and that this interaction is a key step in the mechanism of IE62 transactivation.

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FIG. 3.

Functional and physical interaction between Med25 and the full-length IE62 protein. (A) Schematic of the pRFL/WT dual luciferase reporter construct containing the VZV ORF28/29 regulatory element with important cis-acting elements indicated. The arrows indicate the transcription start sites and the direction of transcription for the ORF28 (left) and ORF29 (right) genes. RL, Renilla luciferase; FL, firefly luciferase. (B). Effect of ectopic overexpression of Flag-Med25 on IE62-mediated activation of the ORF28/29 regulatory element. MeWo cells were transfected with 0.5 μg of pRFL/WT vector, 0.01 of μg pCMV62 plasmid, and increasing amounts of the Flag-Med25-expressing plasmid. The solid bar represents the Renilla luciferase activity controlled by the ORF28 promoter (28RL). The open bar represents the firefly luciferase activity controlled by the ORF29 promoter (29FL). The promoter activities in the absence of Flag-Med25 were normalized to 100%. (C) Effect of ectopic overexpression of Flag-Med25 on the basal activities of the ORF28/29 regulatory element. MeWo cells were transfected with 0.5 μg of p28RL/29FL vector and 0.1 μg of Flag-Med25-expressing plasmid or the same amount of pcDNA3.1 plasmid as control. (D) Immune capture experiments using anti-Flag M2 antibody. Whole-cell lysates of MeWo cells transfected with plasmids expressing IE62 alone or both Flag-Med25 and IE62 were incubated with anti-Flag M2 antibody-coupled agarose beads. Bead-bound proteins were resolved by SDS-PAGE and detected by immunoblotting. Lanes 1 and 2, 2% of the input whole-cell lysates, respectively; lanes 3 and 4, immunoprecipitates from the whole-cell lysates probed with anti-Flag M2 antibody (upper panel) and rabbit polyclonal anti-IE62 antibody (lower panel), respectively. (E) Whole-cell lysates of MeWo cells expressing Flag-Med25 alone or both IE62 and Flag-Med25 were incubated with monoclonal anti-IE62 antibody-coupled protein G-Sepharose (51, 70). Lanes 1 and 2, 1.5% of the whole-cell lysates, respectively; lanes 3 and 4, immunoprecipitates from the whole-cell lysates probed with rabbit polyclonal anti-IE62 antibody (upper panel) and anti-Flag M2 antibody (lower panel).

IE62 associates with Med25 in situ.We next wanted to confirm that the full-length IE62 protein associates with Med25 in situ. Immune capture and coimmunoprecipitation experiments were performed using nuclear extracts derived from MeWo cells in which IE62 and Flag-Med25 were coexpressed via transient transfection. IE62 was captured along with Flag-Med25 using anti-Flag antibody-coupled agarose beads, indicating that the two exogenously expressed proteins were associated in these extracts (Fig. 3D, lanes 3 and 4). In a reciprocal experiment using the monoclonal anti-IE62 antibody, Flag-Med25 was coprecipitated only from extracts containing the IE62 protein (Fig. 3E, lanes 3 and 4). As indicated above, it has been shown that ectopically expressed Med25 can be incorporated into Mediator (68). Thus, the Med25 coprecipitated in these assays in association with IE62 likely represents a mixture of free Med25 and Med25 in the Mediator complex. The relatively small amounts of the coimmunoprecipitated proteins suggest, as did the data in Fig. 1 and 2, that the interaction between IE62 and Med25 is weak under our experimental conditions.

Med25 is required for full activation by the IE62 TAD in situ.Since all of the above data had been gathered using ectopically expressed Flag-tagged Med25, experiments were performed to examine the effects of knockdown of the expression of endogenous Med25 using siRNA. At 70 h posttransfection of Med25-specific siRNAs and scRNAs, total cellular RNA was isolated and examined by RT-PCR to assess the levels of Med25 message. Typical results are presented in Fig. 4A and show that Med25 message was significantly reduced in the presence of the Med25-specific siRNA but not in cells transfected with the scrambled control or in untransfected cells. The effect of the presence of Med25-specific siRNA on the activities of the Gal62TAD and Gal16TAD activator constructs was examined in parallel experiments using identical transfection conditions. As shown in Fig. 4B, concomitant with the depletion of Med25 message in the siRNA-transfected cells, Gal16AD and Gal62AD activities were both reduced. This result indicates that Med25 is required for full activation by the IE62 activation domain in vivo. The observed level of reduction of activity is similar to that reported by Yang et al. (68) in their characterization of the VP16 TAD-Med25 interaction.

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FIG. 4.

Requirement of endogenous Med25 for transactivation by the IE62 TAD. (A) siRNA-mediated depletion of Med25 mRNA in MeWo cells. Total RNA from untransfected (−) MeWo cells and MeWo cells transfected with Med25 specific siRNA and scRNA oligonucleotides was isolated, and the mRNA levels of Med25 and β-actin were examined by RT-PCR. (B) Transactivation activity of the IE62 TAD in Med25-depleted MeWo cells. The pFRLuc reporter vector (0.5 μg), pEFRL control vector (0.01 μg), and 0.1 μg of plasmid expressing Gal16AD or Gal62AD were cotransfected into untreated MeWo cells (WT), Med25 siRNA-transfected MeWo cells (siRNA), and scRNA-transfected MeWo cells (scRNA). Luciferase activities resulting from the presence of the plasmids expressing Gal16AD and Gal62AD in WT MeWo cells were normalized to 100%. Statistical significance was determined by a one-way analysis of variance followed by Tukey's post hoc test. (**, P < 0.01).

IE62 stimulates recruitment of Mediator to a model promoter.Activator-dependent recruitment of the Mediator complex has been observed in studies in both yeast and mammalian cells (7, 9, 17, 38, 46, 66). The possibility of IE62-dependent recruitment of Mediator to promoters was examined via ChIP assays. Our previous experiments demonstrated that Med23 was present as well as Med25 in the IE62-associated Mediator complex lacking CDK8 (Fig. 1). In the absence of commercially available anti-Med25 antibody, we targeted Med23 and CDK8 in examining the recruitment of Mediator complex in our ChIP assays. These experiments were performed using an IE62-responsive model promoter, the USFTA promoter, driving expression of a firefly luciferase reporter gene. This promoter contains a consensus USF binding site 25 bp upstream of the adenovirus major late promoter TATA element. The response of this promoter to the presence of IE62 was previously characterized in our laboratory (71).

The pUSFTALuc reporter vector was cotransfected with pCMV62 or the equivalent amount of the empty plasmid pcDNA3.1 into MeWo cells. The cells were harvested 42 h posttransfection, and ChIP experiments were performed as described in Materials and Methods. The presence of IE62, Med23, and CDK8 at the promoter was monitored using antibodies specifically directed against these proteins. Normal rabbit IgG was used as the antibody control. The primer pair used for PCR amplified a 159-bp fragment spanning the USF binding site and the core promoter elements within the promoter region (Fig. 5A). Each PCR was performed in triplicate for 24, 26, and 28 cycles to ensure proper linear range of the amplification. The ChIP experiments were repeated three times with samples from independent transfections. Results of a typical experiment are shown in Fig. 5C. The presence of IE62 resulted in an obvious and significant increase of Med23 at the promoter whereas there was either no change or frequently even a slight decrease in the low levels of CDK8 detected in the absence of IE62. These data correlate with the results in Fig. 1 and strongly suggest that IE62 activation involves recruitment of a Mediator complex lacking CDK8.

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FIG. 5.

Stimulation of Mediator recruitment to a model promoter by IE62. (A) Schematic presentation of the IE62-responsive model promoter contained within the pUSFTALuc reporter plasmid used in the ChIP analysis. The relative position of each cis-acting element was marked in reference to the start site of the coding region of the luciferase gene (Luc). The sequences of the two cis-acting elements are given under each box. The arrows indicate the primer pair used to amplify the promoter region in the ChIP analysis. (B) Activation of the USFTALuc promoter in the presence of IE62. The pUSFTALuc plasmid (0.5 μg) with or without 0.01 μg of the pCMV62 plasmid was transfected into MeWo cells in 24-well plates. The level of firefly luciferase expression in the absence of the pCMV62 expression plasmid was normalized to 1. (C) Typical results from ChIP analysis of IE62-dependent recruitment of Mediator. The pUSFTALuc vector was cotransfected with or without the pCMV62 plasmid into MeWo cells. The presence of IE62, Med23, and CDK8 at the promoter was examined by using antibodies directed against the individual proteins. Normal rabbit IgG was used as the antibody control. Promoter fragments amplified from 1% of the input and immunoprecipitates (IP) are shown. These experiments were repeated three times and yielded very similar results. Ab, antibody.

Intranuclear translocation of Mediator in VZV-infected cells.IE62 is predominantly nuclear during early infection and localizes to viral replication compartments. At late times of infection in MeWo cells, IE62 is phosphorylated by a viral kinase, resulting in its translocation from the nucleus to the cytoplasm (19, 35, 36, 61). Confocal microscopy was employed to examine the cellular localization of Mediator in relation to that of IE62 in VZV-infected MeWo cells. Med23 was employed as the marker for Mediator-associated with IE62 based on the results obtained from the protein capture and ChIP assays.

In uninfected cells, Med23 appeared to be present exclusively in the nuclei and exhibited a diffuse punctate pattern throughout the nucleoplasm (Fig. 6, upper panel). This pattern is similar to that observed with TRAP220/Med1 in COS cells (51). In early-phase infected cells, however, the intranuclear distribution of Med23 was significantly altered, with the majority of the signal observed in large tight foci which completely overlapped with the presence of IE62 in viral replication compartments. These replication compartments are the major sites of viral transcription and DNA replication during alphaherpesvirus infection (40).

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FIG. 6.

Intranuclear redistribution of Mediator in VZV-infected MeWo cells during the early phase of infection. Uninfected and VZV-infected MeWo cells were immunostained for Med23 (red) and IE62 (green) with rabbit polyclonal antibody and the H6 mouse monoclonal antibodies, respectively. Images show virtually complete overlap of the two signals in large foci in infected cells. The MeWo cell nuclei were counterstained with DAPI (blue). The cells were visualized using a Zeiss Axioimager Z1 fluorescence microscope. Images were recorded with a 20× objective. Bar, 10 μm.

At late times during infection, transcription of the VZV genome begins to wane, IE62 is redistributed from the nucleus to the cytoplasm, and infected MeWo cells form large multinucleate syncytia (26, 35, 36, 41). Observation of late-phase infected cells (Fig. 7) showed the redistribution of the majority of IE62 from nuclear replication compartments to the cytoplasm, which now surrounds multiple nuclei within a syncytium. Med23, in contrast, returned to its diffuse punctuate distribution within the nuclei. These results demonstrate that the subnuclear localization of Mediator is dynamically regulated in VZV-infected cells at different stages of infection and correlates with the presence of IE62 in viral replication compartments during the early phase of infection.

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FIG. 7.

Redistribution of Mediator and IE62 at late times during infection. Infected MeWo cells are shown forming large syncytia characteristic of the late phase of VZV infection. The loss of colocalization of the intracellular distribution of Med23 (red) and IE62 (green) and the redistribution of Med23 resulting in the return to a more diffuse intranuclear signal are shown. The MeWo cell nuclei were counterstained with DAPI (blue). Bar, 10 μm.