Proteasome-Independent Disruption of PML Oncogenic Domains (PODs), but Not Covalent Modification by SUMO-1, Is Required for Human Cytomegalovirus Immediate-Early Protein IE1 To Inhibit PML-Mediated Transcriptional Repression

Plasmids.

Wild-type HCMV IE1 cDNA expression plasmid pJHA303 and truncation mutant pJHA304 encoding IE1(1-346) were described previously (2). Mutant forms encoding IE1(K163R) (pYX108), -(K197R) (pYX109), -(K331R) (pYX102), -K450R (pYX118), and -(L174P) (pYX145) were generated from pJHA303 with the Stratagene QuikChange site-directed mutagenesis protocol. Expression plasmid pJHA312, encoding Flag-SUMO-1, was described elsewhere (7). The GST-PML-L plasmid was a generous gift from Kun-Sang Chang (University of Texas MD Anderson Cancer Center, Houston, Tex.). Plasmid pYX143 encoding glutathione S-transferase (GST)-PML(1-96) was constructed by placing the NcoI (blunt ended)-SalI fragment from pCMX-PML1 between the SmaI and XhoI sites of pGEX-4T-1. Plasmids pGH250 (pSV₂-GAL4-DB vector), pJHA258 (pSV₂-GAL4-DB/PML1) (2), pJH93 (pSV₂-GAL4-DB/CBF1) (27), (GAL4)₅-TK-CAT (70), and (GAL4)₅-TK-LUC (67) were previously described. A full-length PCR fragment of human Daxx cDNA was isolated from pQE38 (a gift from Jerome F. Strauss III, University of Pennsylvania, Philadelphia) and digested with BamHI. Then the BamHI insert was placed into the BglII site of pGH251 (another version of the pSV₂-GAL4-DB vector) to make the pJHA347 (pSV₂-GAL4-DB/Daxx) expression plasmid.

Cell lines and cell culture.

Permissive human diploid fibroblast (HF) cells, semipermissive U373-MG cells, and nonpermissive 293T and HeLa cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. The U373-AN and U373-A45 cell lines, obtained from Susan Michelson (Institute Pasteur, Paris, France),were described previously (54, 81). The U373-A3 cell line was established by cotransfecting U373-MG cells with pRL103 plasmid DNA containing the entire MIE transcription unit (40). The U373-PML and the control U373-Neo cell lines were described elsewhere (4); the U373-SUMO-1 cell line was established by stable transfection of U373-MG cells with the expression plasmids pJHA312 encoding Flag-SUMO-1 and pSV2-Neo.

Virus preparation and infection.

Virus stocks for wild-type HCMV(Towne) were prepared as previously described (2, 40). Recombinant adenoviruses expressing either wild-type or K450R mutant IE1 proteins [Ad-IE1 and Ad-IE1(K450R)] were generated with the Cre-lox recombination system as described elsewhere (23). Briefly, a BamHI fragment from pJHA303 or pYX118 was subcloned into the BamHI site of recombinant transfer vector pAdlox to make recombinant transfer plasmids pYX137 and pYX138, respectively. pAdlox vector, Ψ5 virus, and CRE8 cells were all generous gifts from Stephen Hardy (Somatix Therapy Corporation, Alameda, Calif.). Recombinant adenoviruses were made by cotransfecting Ψ5 viral DNA with pYX137 or with pYX138 into CRE8 cells. The viruses were passed sequentially through CRE8 cells four times. Viral lysates were harvested by three freeze-thaw cycles and cleared by centrifugation.

For immunoblot analysis, U373-MG cells were seeded into six-well plates at 2 × 10⁵ cells per well. Next day, cells were mock infected or infected with wild-type HCMV(Towne) at an MOI of 5 or with Ad-IE1 at an MOI of 20. Cell lysates were then harvested at different time points after infection. For immunofluorescence experiments, HF cells were infected with HCMV(Towne) at an MOI of 5 and Vero cells were infected with HSV-1(KOS) at an MOI of 20. When MG132 (Calbiochem) was used, the cells were preincubated with medium containing 5 μM MG132 for 30 min before infection and all subsequent incubations were performed in the presence of the drug. Slides for HSV-1-infected Vero cells were fixed at 4 h after infection, and slides for HCMV-infected HF cells were fixed at 6 h after infection.

Yeast two-hybrid interaction assays.

Yeast strain Y190 (MATa gal4Δ gal80Δ his3-200 trp1-901 ade2-101 ura3-52 leu2-3,112 URA3::GAL1-lacZ LYS::GAL-HIS3 cyh^R) was the host for color development filter assays for lacZ expression using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) and for a quantitative assay using o-nitrophenyl-β-d-galactopyranoside (ONPG). Media for yeast growth and the method for yeast transformation were described elsewhere (66). Both the X-Gal filter and ONPG assays were described previously (2, 3).

Transient DNA transfection.

For immunoblot analysis, 293T cells were seeded into six-well plates at 4 × 10⁵ cells per well and DNA mixtures were introduced into subconfluent cells with the N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered saline (BBS) version of the calcium phosphate procedure as described previously (62). For indirect immunofluorescence assays (IFA), HF cells were seeded into two-well slide chambers at 0.4 × 10⁵ cells per well and transfected with Effectene reagent from Qiagen (Chatsworth, Calif.) according to the manufacturer's protocol. For chloramphenicol acetyltransferase (CAT) and luciferase reporter assays, HeLa cells were seeded into six-well plates at 2 × 10⁵ cells per well and transfected by the calcium phosphate procedure.

Antibodies.

Mouse monoclonal antibody (MAb) 6E1 against HCMV IE1 (exon 4) was purchased from Vancouver Biotech (Vancouver, British Columbia, Canada). MAb CH810, which detects epitopes present in both IE1 and IE2 (exons 2 and 3), was purchased from Chemicon (Temecula, Calif.). Rabbit anti-SUMO-1 polyclonal antibody (PAb) FL101 was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Rabbit antipeptide PAbs PML(C) and IE110(N) were described elsewhere (5, 56).

Immunoprecipitation.

U373-MG cells in 10-cm-diameter dishes were infected with HCMV(Towne) at an MOI of 5 for 72 h, harvested cell pellets were lysed by boiling with two volumes of 2% sodium dodecyl sulfate (SDS) in Tris-buffered saline (TBS; 10 mM Tris-Cl [pH 8.0] and 150 mM NaCl) supplemented with 5 mM N-ethylmaleimide (NEM; Sigma; E-1271) for 10 min. Eight volumes of 1% Triton X-100 in TBS with 5 mM NEM were added, and lysates were sonicated for 2 min. For immunoprecipitation, cell lysates were incubated with 5 μg of anti-SUMO-1 PAb FL101 at 4°C overnight before 30 μl of protein A- and G-Sepharose beads was added. The remaining steps were the same as those described by Buschmann et al. (10), and the immunoblot was first probed with MAb CH810 and then stripped and reprobed with MAb 6E1.

Immunoblot analysis.

Transfected 293T or infected U373 (U373-MG, U373-Neo, and U373-PML) cells were washed with phosphate-buffered saline and lysed with 200 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (Santa Cruz Biotechnology) and 5 mM NEM. Clarified cell extracts from the equivalent of 10⁴ cells were separated on SDS–8% polyacrylamide gel, followed by standard immunoblot procedures with the enhanced chemiluminescence system (Amersham). Densitometry quantitation of protein band intensity was carried out with a Fluorchem 8000 digital imaging system (Alpha Innotech, San Leandro, Calif.) according to the user's manual.

In vitro SUMO-1 conjugation assay.

[³⁵S]Met-labeled wild-type and mutant IE1 proteins were in vitro translated from pJHA303 and pYX118. The conjugation assay procedures were slightly modified from those described by Desterro et al. (15) and described in detail elsewhere (7). The GST-PML-L protein was prepared by standard procedures. In reaction mixtures containing GST-PML-L, its approximate final concentration was 1 μg/μl.

IFA.

Transfected or virus-infected cells were fixed in methanol or with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100; all subsequent procedures were described previously (2).

CAT and luciferase reporter assays.

Transfected HeLa cells were lysed 48 h after transfection in six-well plates by three freeze-thaw cycles in 200 μl of 0.25 M Tris-HCl (pH 7.9) plus 1 mM dithiothreitol. Both the CAT assay (62) and luciferase assay (4) procedures were described previously.

The HCMV IE1 protein is covalently modified by SUMO-1 in DNA-transfected cells.

When we examined the relative amounts and levels of intactness of both the IE1 and IE2 proteins in two U373 cell lines stably expressing either IE1 (U373-A3) or IE1 plus IE2 (U373-A45), the cell lines were found to include aberrantly high-molecular-weight forms. For example, a 90-kDa IE1 isoform in both cell lines (Fig. 1A, lanes 3 and 2) and two novel IE2 isoforms in U373-A45 cells with apparent sizes of 105 and 120 kDa (Fig. 1A, lane 2) were detected with MAb CH810, in addition to the expected and more-abundant regular 72-kDa IE1 and 86-kDa IE2 protein bands. We have shown elsewhere that the two unusual IE2 isoforms represent conjugated mono- and disumoylated IE2 (7). Earlier studies by Muller and Dejean indicated that IE1 could also be a substrate for SUMO-1 modification in transiently transfected HeLa cells (58). Therefore, we were interested to examine whether the 90-kDa IE1 isoform detected in stable cell lines could be reproduced in transient cotransfections with SUMO-1. 293T cells were transfected with an IE1 expression plasmid in the presence or absence of Flag-tagged SUMO-1 plasmid. In this experiment, no 90-kDa IE1 isoform was detected by either MAb 6E1 or CH810 in cells transfected with IE1 alone (Fig. 1B and C, lanes 2), but when exogenous Flag-SUMO-1 was cotransfected, a small fraction of the 90-kDa isoform was detected by both antibodies (Fig. 1B and C, lanes 3), although the amount was not as great as the amount of the 105-kDa form of IE2 in parallel positive-control samples (Fig. 1C, lane 5).

Because the cell lysates here were prepared with radioimmunoprecipitation assay buffer (without the supplementation of 5 mM NEM), these results are probably underestimates because of the known propensity for sumoylated proteins to be readily deconjugated by isopeptidases upon cell lysis in nondenaturing buffers (53). Overexpression of SUMO-1 increases the level of sumoylation and was necessary to detect the modification of IE1 in this experiment. However, when we used alternative experimental conditions that were expected to improve the identification of SUMO-1 modifications by lysing the cells under denaturing conditions or in the presence of 5 mM NEM to inhibit desumoylating isopeptidases (51, 58), both the 72- and the 90-kDa isoforms were detected in cells transfected with IE1 alone (see Fig. 3A, lane 2). Therefore, all subsequent immunoblot and immunoprecipitation experiments to study IE1 modification by endogenous SUMO were carried out in buffers supplemented with 5 mM NEM.

Covalent modification of IE1 by SUMO-1 in virus-infected cells.

We then asked whether this posttranslational modification of IE1 also occurs during HCMV infection. We first examined U373-MG cell lysates infected with HCMV(Towne) for 24, 48, and 72 h by immunoblot analysis with MAb 6E1. An increasing amount of both the unmodified 72-kDa band and the 90-kDa SUMO-1-modified isoform accumulated throughout the time course of HCMV infection (Fig. 2A). The same set of lysates were also subjected to immunoblot analysis with MAb CH810, which is directed against epitopes shared by IE1 and IE2. The same 90-kDa band was also detected in increasing amounts during the time course of HCMV infection (Fig. 2B). In addition, MAb CH810 also detected the unmodified (86-kDa) and SUMO-1-modified (105-kDa) IE2 isoforms that we and others have already described (7, 26). A similar fraction of 90-kDa IE1 was also detected in HCMV-infected HF cells (not shown).

To obtain direct evidence that the 90-kDa IE1 isoform is indeed covalently conjugated by SUMO-1, we infected U373-MG cells with HCMV(Towne) and immunoprecipitated the whole-cell lysates with anti-SUMO-1 PAb FL101. Immunoblot analysis of the immunoprecipitates with MAb 6E1 detected the 90-kDa IE1 isoform (Fig. 2C, lane 4), while MAb CH810 was able to detect sumoylated isoforms of both IE1 and IE2 on the same immunoblot (Fig. 2C, lane 2). These results establish that IE1 is covalently modified by endogenous SUMO-1 during HCMV infection.

Mapping of SUMO-1 conjugation sites in the IE1 protein.

The consensus KXE motif (34) is a well-conserved pattern in almost all SUMO-1 substrates except when the target lysine residues are located within the RING finger domains of PML and MDM2 (10, 36). There are four KXE motifs at positions 163, 197, 331, and 450 in IE1 that represent potential SUMO-1 attachment sites. Each of these four candidate lysine residues was mutated to arginine, and the resulting mutant IE1 proteins were tested for SUMO-1 modification by transfecting them with or without Flag-SUMO-1 into 293T cells. Immunoblot analysis of the cell lysates showed that K163R, K197R, and K331R single point mutants were all still modified by SUMO-1, whereas no 90-kDa isoform was detected in cells transfected with the K450R mutant (Fig. 3A).

We confirmed these mapping data with an in vitro sumoylation assay system. In contrast to the high in vitro sumoylation efficiency of IE2 (7), wild-type IE1 was only modestly sumoylated in our assay system (Fig. 3B, lane 2). When we incubated in vitro-translated K450R mutant IE1 protein with the same reaction mixture, there was clearly no detectable SUMO-1 modification (Fig. 3B, lane 5). Taken together, these results mapped lysine residue 450 as the only major SUMO-1 conjugation site in the HCMV IE1 protein. Therefore, the known SUMO-1 conjugation target motifs in IE1 and IE2 are DTVSVKSEPVSEI and MLPLIKQEPIKPE, respectively (conjugation sites are in boldface).

IE1 does not interact with SUMO-1 or Ubc9 in yeast two-hybrid assays.

Because most SUMO-1 substrates, including HCMV IE2 and PML, interact with SUMO-1 and/or Ubc9 in yeast two-hybrid assays (7, 26, 53), we were interested to test whether IE1 also interacts with SUMO-1 or Ubc9. Surprisingly, we detected no IE1–SUMO-1 or IE1-Ubc9 interactions after transforming GAL4-DB/IE1 together with GAL4-A/SUMO-1 or GAL4-A/Ubc9 into Y190 yeast cells, whereas IE2 and PML both showed a high level of interaction with both SUMO-1 and Ubc9 (Fig. 4). In a parallel control experiment, this same GAL4-DB/IE1 expression plasmid was able to interact with GAL4-A/PML1 at a level similar to that described previously (2), confirming that the protein was stable and available for interaction in the yeast cells. In agreement with the results in yeast two-hybrid assays, GST-SUMO-1 and GST-Ubc9 both failed to bind to in vitro-translated IE1, whereas both bind to GST-IE2 with high affinity (7). These results suggest that the mechanism of IE1 sumoylation might be very different from that of most previously described SUMO-1 substrates, whose direct binding to Ubc9 and SUMO-1 presumably facilitates the transfer of the SUMO-1 moiety from the Ubc9 thioester intermediate.

IE1 sumoylation is not required for its transient targeting to and subsequent disruption of PODs.

Our previous studies have shown that both the HCMV IE1 and IE2 proteins are targeted to PODs within the first a few hours after virus infection (5). However, IE1 only transiently colocalizes with PML in the PODs, and subsequently both IE1 and PML become redistributed in a nuclear diffuse pattern, whereas IE2 alone has no effect on the distribution of PML. It has been suggested that modification by SUMO-1 is required for the normal localization of PML and some other associated proteins such as hDaxx in the PODs (59, 83). Therefore, after generating the K450R IE1 mutant, which is not modified by SUMO-1, we asked whether the properties of transient targeting to and disruption of PODs might be affected in DNA-transfected HF cells. Double-label IFA analysis was carried out with the PML(C) PAb to detect endogenous punctate POD structures and with MAb 6E1 to detect the IE1 proteins. However, both wild-type IE1 and the mutant IE1(K450R) proteins were found to efficiently colocalize with PML in either nuclear punctate forms (Fig. 5A to D, insets) or as nuclear diffuse patterns (Fig. 5A to D). We confirmed these results by infecting HF cells with either Ad-IE1 or Ad-IE1(K450R) for 12 h in a similar double-label IFA experiment. Again both wild-type and K450R mutant IE1 displayed either nuclear punctate or nuclear diffuse colocalization with PML in the infected cells (data not shown). Therefore, the lack of SUMO-1 modification had no obvious effect on either the targeting to or the disruption of PODs by IE1.

Stable expression of exogenous PML enhances the level of IE1 sumoylation during HCMV infection.

We previously generated a stable U373 cell line overexpressing PML1 (referred to as U373-PML cells) and showed that both the disruption of PODs and the formation of viral DNA replication compartments were delayed during HCMV infection of this cell line (4). Given that IE1 is a substrate for SUMO-1 modification and that the IE1-PML interaction is important for the disruption of PODs by IE1, we were interested to examine the extent of IE1 sumoylation in U373-PML cells. Both U373-PML and the control U373-Neo cells were infected with HCMV(Towne), and cell lysates harvested at various time points after infection were examined by immunoblot analysis with MAb CH810. Although the 90-kDa IE1 isoform accumulated in both cell lines during HCMV infection, the relative abundance of the sumoylated IE1 band was significantly higher in U373-PML cells especially at 24 h after infection (Fig. 6A). Interestingly, the fraction of sumoylated IE1 remained at close to 30% from 24 to 144 h after HCMV infection in U373-PML cells, whereas the sumoylated fraction only gradually increased from 5 to 18% in the control HCMV-infected U373-Neo cells over this same time course (Fig. 6A). These results suggest the existence of a dynamic equilibrium between IE1 sumoylation and desumoylation in HCMV-infected cells. The stable expression of exogenous PML evidently enhanced the steady-state level of IE1 sumoylation, either directly or indirectly, revealing a novel aspect of IE1-PML and IE1-POD interactions that may contribute to the slower virus growth in this cell line.

Because PML is a RING finger protein and because a growing number of RING finger domains have been shown to have E3 ubiquitin ligase activities (33, 82), we wondered whether PML might have a direct enzymatic effect on IE1 sumoylation given the fact that PML physically interacts with IE1, SUMO-1, and Ubc9. Therefore, to test whether the PML protein has any direct effect on IE1 sumoylation, we introduced purified GST-PML-L protein into the in vitro SUMO-1 conjugation assay. This form of GST-PML was able to interact with the in vitro-translated IE1 protein in GST binding assays (data not shown). However, no significant increase or decrease of the 90-kDa sumoylated IE1 was observed when the eluted GST-PML-L protein was added to the reaction (Fig. 3B; compare lanes 3 and 2). Since the PML protein is efficiently sumoylated in vitro, GST-PML might compete with IE1 for SUMO-1, thereby masking its potential catalytic effect on IE1 sumoylation. To exclude this possibility, we also fused an N-terminal RING finger fragment of PML with GST and introduced the resulting GST-PML(1-96) protein into the in vitro IE1 sumoylation assay. There is only one SUMO-1 target lysine residue (K65) present in this PML fragment, and Kamitani et al. have shown that sumoylation of K65 is dependent on the sumoylation of K160 and K490 in vivo (36). Therefore, GST-PML(1-96) is not expected to greatly reduce the amount of available SUMO-1 in the in vitro IE1 sumoylation assay. Again, no significant increase or decrease of IE1 sumoylation was observed upon addition of GST-PML(1-96) (data not shown).

Stable expression of exogenous SUMO-1 also enhances the level of IE1 sumoylation during HCMV infection.

The above observations imply that the effect of overexpressed PML on IE1 sumoylation is indirect. One possibility is that overexpressed PML provides a larger intranuclear pool of conjugated forms of SUMO-1. Upon HCMV infection and subsequent IE1-dependent PML desumoylation (58), higher levels of free SUMO-1 may become available for IE1 sumoylation in U373-PML cells. To test this hypothesis, we established another stable U373 cell line overexpressing exogenous Flag-SUMO-1 (U373-SUMO-1 cells) and examined the level of IE1 sumoylation in HCMV-infected U373-SUMO-1 cells with MAb CH810. Similar to the situation in U373-PML cells, the fraction of sumoylated IE1 remained constant at between 28 and 33% from 6 to 72 h after HCMV infection in U373-SUMO-1 cells, whereas the sumoylated fraction again increased gradually from being undetectable at 6 h to up to 12% at 72 h after HCMV infection in the control U373-Neo cells (Fig. 6B). Therefore, stable expression of exogenous SUMO-1 enhances IE1 sumoylation to a level similar to that in U373-PML cells as early as 6 h after HCMV infection.

The displacement of PML from the PODs by IE1 is independent of proteasome activity.

The disruption of PODs by the HSV-1 IE110 (ICP0) protein has been shown to correlate with the rapid proteasome-dependent degradation of PML, and Sp100 and proteasome inhibitors such as MG132 proved to stabilize PODs in HSV-1-infected cells (12, 18, 61). However, the PML signal is just displaced, not lost, in HCMV-infected cells (5), and therefore we were interested to test if proteasome inhibitors have a similar effect on the disruption of PODs during HCMV infection. HF cells were infected with HCMV(Towne) in the presence or absence of proteasome inhibitor MG132 and stained for IE1 and PML at 6 h after infection. Surprisingly, the infected cells showed similar efficiencies of POD disruption in both situations (Fig. 7A to D). In contrast, as a positive control for MG132 activity, Vero cells were infected with HSV-1(KOS) in the presence or absence of MG132 and stained for IE110 and PML at 4 h after infection. Without MG132, almost all of the infected cells had a barely detectable PML signal (Fig. 7E and F). However, in the presence of MG132, a majority of the infected cells retained punctate POD structures, with only a few cells still losing the PML signal (Fig. 7G and H). Therefore, we conclude that the displacement of PML from the PODs by IE1 during HCMV infection does not require proteasome activity and that IE1 utilizes a different mechanism for POD disruption than does HSV-1 IE110.

PML1 represses basal transcription.

Several studies have documented that the 641-aa PML-L isoform can function as a transcriptional repressor when fused to the GAL4 DNA-binding domain (GAL4-DB) (75, 80). We were interested to test whether the 560-aa PML1 isoform used in all of our studies also has repressor activity in the mammalian GAL4 targeting assay. Therefore, we cotransfected GAL4-DB or GAL4-DB/PML1 with our standard target (GAL4)₅-TK-CAT reporter used for repressor assays (27, 28). GAL4-DB/CBF1 (27) and GAL4-DB/Daxx (42) were also used as positive controls for transrepression. The basal activity of the (GAL4)₅-TK-CAT target alone in HeLa cells gave 85% acetylation (Fig. 8, lane 1). Cotransfection with both PML1 and Daxx showed a 50-fold inhibition of CAT activity from the (GAL4)₅-TK promoter, whereas CBF1 gave a 10-fold inhibition in the same experiment (Fig. 8, lanes 2, 4, and 6). Therefore, targeting PML1 to a promoter can also repress basal transcription despite the lack of the unique C-terminal domain of PML-L that is known to interact with HDAC. Control experiments confirmed that the GAL4-PML1 fusion protein still localizes normally in PODs and is displaced by IE1 cotransfection (not shown).

HCMV IE1 specifically inhibits PML-mediated transcriptional repression.

PML1 has been reported recently to inhibit Daxx-mediated transcriptional repression via a physical interaction with Daxx and the consequent sequestration of Daxx to the PODs (42). Given that the proteasome-independent displacement of PML from PODs by IE1 may be mediated through a specific IE1-PML physical interaction (2), we also asked whether IE1 could regulate PML1-mediated transcriptional repression in a GAL4 targeting assay similar to that described above. GAL4-DB/PML1 was transfected with or without the wild-type IE1 expression plasmid into HeLa cells, and the activity of the target (GAL4)₅-TK-CAT reporter gene was measured. Interestingly, IE1 coexpression reduced the 50-fold PML repression to less than 5-fold (Fig. 8, lanes 2 and 3). In contrast, IE1 coexpression had little effect on CBF1- or Daxx-mediated repression of the (GAL4)₅-TK promoter (Fig. 8, lanes 4 to 7). These data suggest that expression of IE1 specifically overcomes PML-mediated transcriptional repression, presumably either through direct physical interaction with PML or through some associated effect connected to POD disruption.

Disruption of PODs by IE1 but not IE1 sumoylation is required for efficient inhibition of PML-mediated repression.

Our previous studies showed that a truncated IE1(1-346) mutant lacking the acidic C terminus was still targeted to and colocalized with PODs but that it nevertheless failed to displace PML from the PODs, thus uncoupling the requirements for targeting from subsequent disruption (2). Muller and Dejean found an IE1(L174P) point mutant that did not interact with PML in yeast and showed that this same mutant protein also failed to target or disrupt PODs (58). Therefore, we generated an L174P mutant IE1 expression plasmid by site-directed mutagenesis and confirmed that the mutant protein was localized exclusively in a nuclear diffuse pattern and failed to displace PML from PODs (data not shown). Given the observation that wild-type IE1 could inhibit PML-mediated transcriptional repression, we asked whether any of the three IE1 expression vector mutants, IE1(K450R), IE1(1-346), or IE1(L174P) (Table 1) retained this derepression activity. In this experiment, HeLa cells were cotransfected with either the (GAL4)₅-TK-CAT or the (GAL4)₅-TK-LUC reporter gene (67) together with GAL4-DB or GAL4-DB/PML1 in the presence or absence of wild-type or mutant IE1. The control samples (Fig. 9, lanes 3) showed that PML repression occurred equally in both the CAT and luciferase reporter assays, confirming that the effect is likely to be transcriptional and not a reporter gene-specific posttranscriptional-level event such as that mediated by the Epstein-Barr virus MTA protein. Furthermore, the sumoylation-negative IE1(K450R) mutant still overcame PML-mediated repression with an efficiency of greater than 60% of the level obtained with wild-type IE1 (Fig. 9, lanes 5), whereas both the IE1(1-346) and IE1(L174P) mutants failed to inhibit the PML-mediated repression to any measurable extent (Fig. 9, lanes 4 and 6). Importantly, although we cannot exclude the possibility that IE1 alone might have upregulated the (GAL4)₅-TK-CAT target, the different kinetics of the luciferase assay show that IE1 alone has no significant effect on the (GAL4)₅-TK-LUC target. Therefore, these data indicate that the inhibitory effect of IE1 on PML-mediated transcriptional repression may require its ability to target to and displace PML from the PODs but does not depend on its ability to be sumoylated.