(Received for publication, October 10, 1995; and in revised form, December 7, 1995)
From the
The IE2 gene of human cytomegalovirus has been implicated in the development of coronary restenosis, and the gene product appears to inhibit p53-dependent transactivation. Here we describe an analysis of the IE2-p53 interaction. Repression of p53 function by IE2 requires two separable domains of IE2. The N terminus of IE2 interacts with p53. IE2 has little effect on the ability of p53 to bind specific DNA sequences. Reduction of the transactivation activity of p53 is caused by a transcriptional repression function contributed by the C-terminal domain of IE2. These findings suggest that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53.
The tumor suppressor p53 protein is an important negative
regulator of cell
proliferation(1, 2, 3, 4, 5) .
Loss of p53 function results in genome instability (6, 7) and eliminates growth arrest at the G phase in response to inadequate or detrimental growth
conditions(8, 9, 10) . p53 functions as a
typical eukaryotic transcription factor; it binds to specific DNA
sequences termed p53-responsive elements (or
PRE)(
)(8, 11, 12, 13, 14, 15, 16) and
stimulates transcription of the target
genes(11, 17, 18, 19) .
Paradoxically, p53 also represses transcription of many viral and
cellular genes, which apparently do not have
PRE(20, 21, 22) . This function, probably
reflecting general negative effects on cellular growth via the
induction of WAF1/CIP1 by p53(23) , requires the
transactivation activity of p53. In structure, p53 is organized into
three functional domains: an N-terminal domain, involved in
transcriptional activation; a central domain, mediating specific DNA
binding; and a C-terminal domain, responsible for oligomerization,
transcriptional repression, and nonspecific DNA
binding(3, 24, 25) .
All of the major classes of small DNA tumor viruses that replicate in mammalian nuclei encode immediate-early gene products to overcome the negative effects of p53 on cell proliferation. For instance, the E1b protein of adenovirus, the E6 protein of papillomavirus and the large T antigen of papovaviruses each eliminates p53 function by interacting with a distinct domain of p53 ((3) ). The inactivation of p53 function by the viral immediate-early proteins results in promoting cell growth and in increasing the available pool of deoxyribonucleotides, which leads to the enhancement of viral replication.
Herpesviruses are the largest among the DNA tumor viruses. Although the replication strategy of herpesviruses must be fundamentally different from that of small DNA viruses, it seems logical that they still have to deal with the negative effect imposed by p53 on cell proliferation so that host cells can enter the S-phase of the cell cycle and thus promote viral replication. Indeed, it has been shown that the immediate-early protein BZLF1 of Epstein-Barr virus, a member of the herpes group, disrupts p53 function by binding directly to the carboxyl-terminal portion of the protein(26) .
Human cytomegalovirus (HCMV), another member of the herpesvirus family, contains a double-stranded DNA genome of 229,354 base pairs with a potential to encode for more than 200 proteins(27) . HCMV infection promotes DNA synthesis and causes proliferation of a variety of cells (28, 29, 30) . More recently, HCMV has been found to be involved in the development of coronary restenosis (31) by inducing smooth muscle cell proliferation. A number of immediate-early (IE) proteins of HCMV are expressed following entry of the virus into cells(32) . Among them, the IE2 86K protein (referred as IE2, hereinafter) is the most studied. IE2 appears to be a promiscuous transactivator of viral and cellular gene expression ( (33) and references therein). However, to maximally stimulate transcription, both IE1 and IE2 proteins are required(34) . Also, IE2 autoregulates its own expression by binding to a short nucleotide sequence, termed the cis repression signal, located immediately downstream of the TATA box(35, 36) . To date, only a limited number of studies have been performed to assess the functional domains of IE2. Recent evidence has demonstrated that the C terminus of IE2 is involved in such protein activities as activation, autoregulation, and binding to retinoblastoma protein, TBP, and TFIIB ( (33) and references therein).
The fact that immediate-early proteins of both large and small DNA viruses can associate with p53 (3, 26) naturally leads to the prediction that HCMV, by analogy, should employ a similar mechanism to inactivate p53 function. Actually, the IE2 protein of HCMV has been implicated in the disruption of the transactivation function of p53 (31) . Evidence collected so far suggests that the interaction between IE2 and p53 is of critical importance to cell growth and HCMV replication. Thus, to further understand the biological significance of the interaction between IE2 and p53, it is important to define the interaction domains of the two proteins and to elucidate the molecular mechanism underlying this interaction. This report demonstrates that two regions of IE2 are required to inactivate p53 function. The N-terminal portion of IE2 interacts with p53. The specific DNA-binding activity of p53, however, seems unaffected by IE2. Repression of the transactivation activity of p53 requires, in addition to the N-terminal end of IE2, a transcriptional repression activity conferred by the C terminus of IE2.
As shown in Fig. 1A, IE2 repressed p53 function efficiently (compare lanes 2 and 3). Repression was specific for IE2 because another HCMV IE protein, IE1, had no effect on the transactivation function of p53 (data not shown). Removal of IE2 residues 136-289 consistently resulted in a small increase in the protein's repression activity toward p53 (compare lane 4 to lane 3). However, a further deletion of IE2 residues 45-135 largely reduced the protein's repression activity (compare lane 5 to lane 2). We thus concluded that a region encompassing IE2 residues 45-135 was required for the inhibition of p53 function. Besides, Fig. 1A also indicates that the C terminus of IE2 was also involved in the repression, because both C-terminal truncation mutants, IE2CD80 and IE2CD189, weakly repressed p53 activity (Fig. 1A, compare lanes 6 and 7 to lane 2), suggesting that the intactness of IE2's C terminus was required for full repression activity. (Further supports for this point were provided in the experiment of Fig. 4A.) Note that the inability of IE2 derivatives in repressing p53 function was not due to a low level of protein being made. In fact, all three inactive IE2 mutants were more abundant than the other two active derivatives (Fig. 1B, left panel, compare lanes 5, 6, and 7 to lanes 3 and 4). Neither could the low CAT activity be attributed to a fluctuation in the level of p53 (Fig. 1B, right panel), indicating that IE2 derivatives had little effect on the expression of p53.
Figure 1: Two IE2 regions are required for repression of p53 function. A, repression of the transactivation activity of p53 by IE2 derivatives. 5 µg of reporter, 1 µg of p53V143A expression plasmid, and 10 µg of each plasmid expressing IE2 derivatives were transfected per sample. Experiments were repeated 3 times. 1 µg of plasmid pCH110 (Pharmacia Biotech Inc.) containing a functional LacZ gene was used as an internal control to monitor transfection efficiency. After transfection, cells were incubated at 37 °C for 36 h, and the temperature was then shifted to 30 °C for another 24-h incubation. An autoradiogram of a typical experiment is shown. Diagrams of the structure of the effectors and reporter are shown below the autoradiogram. The presence (+) or absence(-) of p53V143A, the IE2 derivative and relative CAT activity (or RCA) are indicated above each track of the autoradiogram. RCA is the mean -fold stimulation of transcription compared with basal activity. The standard deviations were 0, ±8.0, ±1.1, ±0.8, ±7.1, ±5.2, ±5.6 for lanes 1-7, respectively. B, protein levels of IE2 derivatives (left panel) and p53V143A (right panel). Transient transfection was performed as in Fig. 1A. Proteins of Saos-2 cells transfected with the vector alone (lane 1), with p53V143A (lane 2), or with p53V143A and IE2 derivatives (lanes 3-7) were fractionated on a 10% SDS-polyacrylamide gel. IE2 derivatives (left panel) and p53V143A (right panel) were detected by immunoblotting. The position of p53V143A is indicated by arrowhead (right panel). The presence (+) or absence(-) of p53V143A as well as the IE2 derivative are indicated above each track of the immunoblot. The positions of molecular mass markers in kilodaltons are shown on the left. The effectors are the same as in Fig. 1A; therefore, their diagrams are omitted.
Figure 4: GAL4-IE2 functions as a direct transcriptional repressor. A, GAL4-IE2 represses CAT activity driven by a thymidine kinase promoter with five upstream GAL4-binding sites. 5 µg of reporter and 5 µg of each GAL4 derivative were transfected per sample. The effector and RCA are indicated above each track of the autoradiogram. The CAT activity of the reporter alone was set at 100. Diagrams of the structure of the effectors and the reporter are shown below the autoradiogram. Otherwise as in Fig. 3. The standard deviations were 0, ±18.2, ±0.5, ±0.03, ±6.4, ±9.4, ±21.1, 0, ±0.8, ±12.8 for lanes 1-10, respectively. B, protein levels of GAL4-IE2 derivatives. Transfection was performed as in Fig. 4A. The GAL4-IE2 derivatives are the same as in Fig. 4A; thus, their diagrams are omitted. GAL4 derivatives were detected with an anti-GAL4 antibody. Each of the positions of GAL4-IE2 derivatives was indicated by a dot. Otherwise as in Fig. 1B.
Figure 3: IE2 does not inhibit the specific DNA-binding activity of p53. 1 µg of reporter pL6EP1C (lanes 1-5 and 10-14) or pL6EC (lanes 6-9) was cotransfected with each expression plasmid for activator (1 µg), blocker (2 µg), and IE2 (6 µg). The amount of expression plasmid for SV40 large T antigen (TAg) is 3 µg (lane 13) and 6 µg (lane 14). The presence (+) or absence (-) of activator, blocker, IE2, and large T antigen as well as RCA are indicated above each track of the autoradiogram. After transfection, cells were incubated at 37 °C for 48 h, and CAT activity was measured. Diagrams of the structure of the activator, blocker, and reporters are shown below the autoradiogram. Otherwise as in Fig. 1A. The standard deviations were 0, ±16.5, ±0.3, ±0.6, ±16.1, 0, ±43.7, ± 5.2, ±40.6, 0, ±10.7, ±0.8, ±4.5, ±12.6 for lanes 1-14, respectively.
Figure 2: The C terminus of p53 is required for the interaction with IE2. Transfection was performed as described in Fig. 1A, except that effectors were p53V143A, p53V143ACD30, p53V143ACD55, and IE2. Otherwise as in Fig. 1A. The standard deviations were 0, ±3.5, ±0.6, ±14.5, ±6.9, ±3.7, ±5.5 for lanes 1-7, respectively.
As shown in Fig. 4A, GAL4-IE2 repressed transcription of the reporter pG5TKCAT (compare lane 3 with lane 1). The repression was dependent upon both the GAL4 and IE2 modules of the chimeric protein, since neither the GAL4 DNA binding domain nor the IE2 alone was able to significantly repress transcription from the promoter of G5TK (compare lanes 2 and 10 with lanes 1 and 8). The repression domain was mapped to the C-terminal half of IE2, whereas the N-terminal half of IE2 possessed no detectable repression activity (compare lanes 4 and 7 with lane 1). The entire C-terminal half of IE2 seemed to be required for full repression activity, because a further reduction in length of the IE2 module from GAL4-IE2(290-579) resulted in a large decrease in the repression activity (compare lanes 5 and 6 with lane 4). A Western blot shown in Fig. 4B demonstrated that most of the chimeric proteins were expressed to a similar level. Proteins GAL4-IE2(290-579) and GAL4-IE2(1-289) were two exceptions. The former's high level of expression, in conjunction with the removal of an activation domain located in the N terminus of IE2 (33) might be responsible for its strong repression activity toward reporter pG5TKCAT (Fig. 4A, lane 4 and Fig. 4B, lane 3), whereas the latter's high level of expression demonstrated that the N terminus of IE2 indeed had little repression activity (Fig. 4A, lane 7, and Fig. 4B, lane 6).
Figure 5:
GST-p53 fusion protein retains S-labeled IE2 protein in pull-down assays. A,
retention of IE2 to wild-type p53 fusion protein (GST-p53) but not
mutant p53 fusion protein (GST-p53CD55). Lane 1, input IE2
protein; lanes 2, 3, and 4, retention of IE2
protein by GST, GST-p53, and GST-p53CD55, respectively. The position of
IE2 protein is indicated by an arrowhead. The GST protein
ligand, whose structure is shown below the autoradiogram, is indicated
above each track of the autoradiogram. The positions of molecular mass
markers in kilodaltons are indicated on the left. B, retention
of IE2 derivatives by GST-p53 protein. The left panel shows
one-hundredth each of the input IE2 derivatives directly loaded onto
the gel. The right panel shows retention of IE2 derivatives by
GST-p53 protein. The IE2 derivative whose structure is shown below the
autoradiogram is indicated above each track of the autoradiogram. The
positions of molecular mass markers in kilodaltons are indicated on the
left.
Previous studies have demonstrated a functional as well as a physical interaction between HCMV IE2 and p53 proteins(31) . In this report, we defined the domains required for the IE2-p53 interaction and analyzed the molecular mechanism underlying this interaction. IE2 can be divided into two domains, regarding the repression of p53-mediated transcriptional activation. Data obtained from in vitro protein-protein interaction studies demonstrate that the N terminus of IE2 contains a p53-interacting domain (Fig. 5B). Evidence supporting that the C terminus of IE2 functions as a transcriptional repression domain comes from experiments with derivatives of the GAL4-IE2 fusion protein (Fig. 4A). Importantly, the IE2 domain required for transcriptional repression is mapped to the C terminus containing residues 290-579 of the protein, no matter how the IE2 is brought to the promoter, either by fusion to the GAL4 DNA-binding domain (Fig. 4A) or by interacting with p53 (Fig. 1A). Furthermore, IE2 does not affect the ability of p53 to bind PRE in vivo (Fig. 3). Thus, we conclude that IE2 inhibits p53-mediated transcription by tethering a repression domain to p53. We note, however, that IE2ID(136-289) interacts with p53 less strongly in vitro (Fig. 5B, lanes 1 and 2) but represses p53 better in vivo than IE2 (Fig. 1A, lanes 3 and 4). We currently do not have an explanation for it.
The IE2-p53 interaction shows many parallels with the well established interaction between E1b and p53. First, like E1b(49) , IE2 can repress p53-dependent transcriptional activation (Fig. 1A and Fig. 2). Second, like E1b(43) , IE2 does not affect the ability of p53 to bind specific DNA sequences (Fig. 3); rather, it tethers a transcriptional repression domain to p53 (Fig. 4A and 5B). Despite these similarities, different regions of p53 are involved in the E1b- and IE2-p53 interactions; E1b binds to the N terminus of p53(3) , whereas IE2 targets the C terminus ( Fig. 2and 5A).
p53-dependent transactivation was not completely repressed by IE2 (Fig. 1A and Fig. 2). The strength of IE2's repression activity may not be a viable explanation for the incomplete inhibition, because IE2 appears able to efficiently repress transcription when brought to a promoter by fusion to the GAL4 DNA binding domain (Fig. 4A). Thus, some other mechanism has to account for the incomplete repression of p53 activity. We speculate that in the IE2-p53 complex, unlike the E1b-p53 one, the N-terminal activation domain of p53 is not involved in the interaction and is, therefore, probably free to contact the transcription machinery, explaining why IE2 fails to efficiently repress p53 function (Fig. 1A and Fig. 2). Alternatively, the incomplete repression of p53 function by IE2 could be caused by the existence of activation domains in the latter(33) . Perhaps, a putative interaction between the activation domains of p53 and IE2 could partially overcome the negative effect of IE2 and thus result in incomplete repression. It should be noted, however, that Speir et al.(31) observed a good repression of the transactivation activity of p53 by IE2 in primary human coronary smooth muscle cells(31) . The reason for the observed discrepancy is not known. However, since the current studies were carried out in a p53-negative tumor cell line, the discrepancy may reflect the physiological differences of the recipient cells used for transient transfection assays.
It is noteworthy that the C terminus of p53 is indispensable for the interaction with IE2 of HCMV and BZLF1 of Epstein-Barr virus, both immediate-early proteins of herpesviruses ( (26) and the present work). Although the C terminus of p53 is rarely involved in tumor mutations(3) , this region has been shown to interact with TBP and the hsc-70 protein(3) . In addition, this region, which contains two phosphorylation sites, also mediates the oligomerization, transformation, and transcriptional repression activities of p53(3, 24, 25) . By targeting this region, IE2 can potentially affect any of these p53 functions. In contrast, very few functions have been assigned to the N terminus of IE2(33) . However, an examination of the primary sequence of this IE2 domain reveals several interesting features characteristic of eukaryotic transcription factors: a repeated motif of proline-N-proline, a helix-loop-helix-turn-helix structure and stretches of polyglutamic acid and polyserine(50) . This observation implies that p53 can potentially interact with nuclear proteins containing such structural motifs.
Both IE2 and p53 interact with a number of cellular proteins, such as TBP and TFIIB ( (33) and (51) and references therein). The possibility that interaction between IE2 and p53 is mediated through some cellular protein(s) has not been totally excluded, since each of the assays used to examine the IE2-p53 interaction either included the reticulocyte lysate or was performed within cells. Nonetheless, it is very unlikely that interaction between IE2 and p53 is mediated through TBP or TFIIB, since TBP and TFIIB interact with the C terminus of IE2 ( (33) and references therein), whereas this region is dispensable for the IE2-p53 interaction (Fig. 5B).
IE2 has been shown to transactivate homologous and heterologous gene expression as well as to negatively autoregulate. The present studies identify a third activity of IE2; it functions as a direct transcriptional repressor when brought to a promoter either by fusing to the GAL4 DNA-binding domain (Fig. 4A) or by interacting with p53 (Fig. 1A and Fig. 2). Since p53 possesses a transcriptional repression domain (24, 25) and p53 itself mediates transcriptional repression of heat shock 70 promoter by interacting with the CCAAT-binding factor(52) , these raise the question of whether p53 is a cofactor required for IE2 repression. However, the finding that GAL4-IE2 repressed transcription of a target promoter in cells lacking p53 does not favor this idea (Fig. 4A). In light of this and of the fact that IE2 possesses specific DNA binding activity (36) , it is important to determine whether IE2 can repress transcription of genes bearing IE2 binding sites in the promoter/enhancer region. Moreover, transcriptional repression domains have been shown to be alanine-rich(53) , highly basic (54, 55) and rich in proline and hydrophobic amino acids(56, 57) . As a transcriptional repressor, IE2 appears unique in that it bears no similarity to the aforementioned repression domains. Further studies of the repression activity of IE2 should elucidate the molecular mechanisms concerning how direct transcriptional repressors work.