Functional domains of adenovirus type 5 E1a proteins

Cell, Vol. 50, 1091-1100,

September

25, 1987, Copyright

0 1987 by Cell Press

Functional Domains of Adenovirus Type 5 Ela Proteins James W. Lillie,’ Paul M. Loewenstein,t Michael R. Green,” and Maurice Greent l Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138 tlnstitute for Molecular Virology St. Louis University Medical Center St. Louis, Missouri 83110

Summary Adenovlrus Ela proteins function in transcriptional activation, transcriptional repression, cellular DNA synthesis induction, and cellular transformation. Here we examine the role of the previously undefined Ela region 1, the last of three conserved Ela regions to be characterized. Region 1 is required for transcriptional repression, transformation, and DNA synthesis lnduction, but not transcriptional activation. These results support our ptevlous suggestion that transcriptional repression is the basis of Ela-mediated transformation. Two conserved regions (regions 1 and 2), present in both early Ela proteins, are essential for transcriptional repression, transformation, and induction of DNA synthesis. In contrast, mutagenesis suggests that transcriptional activation requires only 49 amino acids (region 3) unique to the 289 amino acid Ela protein. This we prove by demonstrating that a 49 amino acid region 3 synthetic peptide efficiently activates an Ela-inducible promoter. This peptide is the smallest known protein fragment functioning as a transcriptional activator. Introduction Adenovirus Ela proteins play an essential role in both productive infection and cellular transformation. As an oncogene, Ela induces cellular DNA synthesis in growtharrested cells, and in cooperation with other oncogenes transforms nonpermissive primary cells (for a review, see Graham, 1984). During productive infection Ela is required to activate transcription of early viral genes (reviewed in Berk, 1986). Unlike other well-characterized transcriptional activating proteins, Ela activates transcription of a variety of viral and cellular genes with apparently unrelated promoter sequences (Berk, 1986). The mechanism of Ela-mediated activation is unknown. Furthermore, the transcription of some genes is repressed, not activated, by Ela. In these cases the targets of Elamediated transcriptional repression appear to be viral (Borrelli et al., 1984; Velcich and Ziff, 1985; Liliie et al., 1986) or cellular (Hen et al., 1985; Lillie et al., 1986) enhancers. These apparently diverse Ela activities are mediated by two early Ela proteins. A single Ela pre-mRNA is spliced into either a 13s or 12s mRNA (Berk and Sharp, 1978; see

Figure l), which encode 289 and 243 amino acid nuclearlocalized phosphoproteins, respectively (for reviews, see Graham, 1984; Berk, 1986). These proteins have identical amino and carboxyl termini and differ only by 46 amino acids unique to the 289 amino acid protein (Perricaudet et al., 1979; Figure 1). Yet the activities of these two proteins are significantly different. For example, the 289 amino acid protein functions primarily as a transcriptional activator, while the 243 amino acid protein is a transcriptional repressor (Liilie et al., 1986). Ela proteins contain three distinct regions strongly conserved among adenovirus subgroups and species (see Figure 1; van Ormondt et al., 1980; Kimelman et al., 1985). The vast majority of region 3 is unique to the 289 amino acid protein. Two additional, shorter conserved regions (regions 1 and 2) are common to both Ela proteins. We and others (for instance, see Moran and Mathews, 1987) have considered that each region might correspond to a separate functional domain. We have probed the functions of these regions by constructing and analyzing single-amino-acid substitution mutants. Our previous results (Lillie et al., 1986) suggest that region 2 is required for transcriptional repression and transformation but not transcriptional activation. Conversely, region 3 is required for transcriptional activation but not transcriptional repression or transformation. In this report we define the role of the previously uncharacterized Ela region 1 and further examine the functions of all three regions in the multiple Ela activities. Results To determine the functions of region 1, region 1 singlebase substitution mutations were constructed by random chemical mutagenesis and isolated by denaturing gradient gel electrophoresis (Myers et al., 1985; Lillie et al., 1986). Four Region 1 mutants (741 [A+E], 734 [E+K], 774 [L-S], and 777 [A-V]; Figure 1) that alter amino acids conserved among adenovirus serotypes 5,7, and 12 (van Ormondt et al., 1980) were chosen and tested for their effects on transformation, transcriptional activation, transcriptional repression, and the induction of cellular DNA synthesis. Since the two early Ela proteins behave differently in each assay (Lillie et al., 1986; and see below), the mutations were cloned into Ela plasmids or viral genomes that express only one of the two Ela products (Monte11 et al., 1982, 1984). Region 1 Single-Base Substitution Mutants Are Transformation-Defective To test the role of region 1 in transformation, we asked whether the region 1 mutants can cooperate with the activated cellular oncogene ras to transform primary cells (ras cooperation assay; Ruley, 1963). We have simplified this transformation assay by substituting primary baby rat kidney (BRK) cells with CREF cells, a cloned line of rat embryo fibroblasts that are transformed efficiently by adeno-

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12 s

138

,243

Frgure 1. Structures of the Adenovirus Early Ela Gene Products

AA

The structures of the 125 and 13s Ela mRNAs are shown. Horizontal lines, exons; carets, intrans. On the right are shown the lengths of the protein products. Below each mRNA the conserved regions of the encoded Ela protein are boxed, and numbered 1, 2, and 3 (Moran and Mathews, 1987). Region 1 spans Ela protern amino acids 46-77 (nucleotides 695-790). Region 2 spans amino acids 121-136 (nucleotides 920-967). Region 3, unque to the 289 amino acid protein, spans amino acids 142-188, nucleotides 983-1239. The position, nucleobde number, and amino acid change (single-letter code) of each of the four region 1 single-base substitution mutants characterized are marked below.

,289AA

734 741 774 777

Type 5

(E-K1 IA--El IL-S1 IA-V]

Table 1. Transformation of CREF Cells by Cotransfection or Mutant Ela 243 or 289 Amino Acid Protein

of ras and an Ela Plasmid That Expresses

Wild-Type

Foci Plasmids Transfected

Experiment

1

Experiment

2

Experiment

3

243 amino acid Ela protein ras alone d11500 (243 amino acid Ela only) d11500 + ras dl1500-741 + ras dl1500-734 + ras dll500-777 + ras dl1500-774 + ras

0 0 113 4 23 0 -

2 0 74 1 2 5 -

1 0 70 6

0 54 0 0 0 -

0 29 3 2 2 -

0 46 -

289 amino acid Ela protein pm975 (289 amino acid El a only) pm975 + ras pm975-741 + ras pm975-734 + ras pm975-777 + ras pm975-774 + ras

8

CREF cells (Fisher et al., 1982) were split 1:lO into 100 m m dishes in DMEM, 10% FBS (see Experimental Procedures) and maintained at 37OC. Twenty-four hours after seeding, one plate of cells was transfected with 40 ng of ras DNA and 5 ug of wild-type or mutant Ela plasmid, by calcium phosphate precipitation. After 4 to 18 hr the cells were trypsinized and replated. When the cells reached confluency (2 days), the medium was changed to DMEM, 5% FBS, and the cells were fed twice per week. The number of foci 17 to 21 days after transfection is shown.

virus infection (Fisher et al., 1982). We have found that CREF cells behave like primary BRK cells in this assay. ras or either Ela protein alone produces O-2 foci, whereas ras plus the 243 (d11500) or 289 (pm975) amino acid Ela protein produces 70-113 or 29-54 foci, respectively (Table 1). The growth properties and morphology of cells transformed by the 289 or 243 amino acid Ela protein are distinguishable (Monte11 et al., 1984). Therefore we determined the effect of the region 1 mutations on the transforming activity of both the 289 and 243 amino acid proteins. Significantly, all four region 1 mutations severely impair the transforming activity of both the 289 and 243 amino Ela proteins (Table 1). Similar results were obtained using BRK cells in the standard ras cotransfection assay (data not shown). We conclude that region 1 is essential for efficient Ela-mediated transformation.

Region 1 Transformation-Defective Mutants Efficiently Activate Transcription of Early Viral Genes To test whether region 1 is required for transcriptional activation, we asked whether the transformation-defective region 1 mutants were also activation-defective. Three region 1 mutations were rebuilt into a viral genome that expresses only the 289 amino acid Ela protein, the major transcriptional activator (pm975; Monte11 et al., 1982, 1984). Following infection of HEP-2 cells with an Ela wildtype or mutant virus, transcriptional activation of the Elainducible E2E and E4 genes was analyzed by primer extension (Figure 2). E2E and E4 mRNAs are readily detectable in the presence (pm975) but not the absence (d1312)of the Ela 289 amino acid protein. Thus, under these conditions early viral gene expression is Ela-dependent. The region 1 transformation-defective mutants produce

Domains 1093

of Adenovirus

5 Ela Proteins

‘, -E2E

* ?LCINEIA

E4 I Figure 2. Transcription of Early Viral Genes in Cells Infected with Ela Wild-Type or Mutant Adenovirus Confluent plates of HEP-2 cells were infected at a multiplicity of infection of 10 (lo7 PFU per 60 m m dish) with Ela wild-type or mutant virus in the presence of cytosine arabinoside (ara-C). Cytoplasmic RNA was prepared 12 hr after infection. Twenty micrograms of each RNA sample was analyzed by primer extension analysis (Lillie et al., 1986) using 32P-5’-end-labeled synthetic oligonucleotides complementary to Ela mRNA (positions +75 to +94 from the start site of transcription), E2E mRNA (positions +51 to +68 from the start site of transcription), and E4 mRNA (positions +28 to +45 from the start site of transcription) (see Sussenbach, 1984). The positions of the expected cDNA products are indicated on the right. The virus used in each infection is marked above each lane. Lane M, Mspl-digested pBR322 =P-DNA.

wild-type levels of Ela, E2E and E4 mRNAs (Figure 2; pm975734, pm975741, and pm975774). Thus these region 1 mutations severely decrease El& transformation activity but not its ability to activate transcription, supporting the previous conclusion that Ela’s transformation and activation functions are unrelated (Lillie et al., 1988; Moran et al., 1988). Region 1 Transformation-Defective Mutants Cannot Efficiently Repress Transcription from an Enhancer-Linked Target Gene Our previous Ela mutagenesis study suggested that Elamediated transformation and transcriptional repression are linked (Lillie et al., 1988). To test this correlation further and to determine the role of region 1 in transcriptional repression, we asked whether transformation-defective region 1 mutants were also repression-defective. Since the 243 amino acid protein is the major transcriptional repressor (Lillie et al., 1988) region 1 mutations were rebuilt into viral genomes that express only the 243 amino acid protein (d11500; Monte11 et al., 1984). HEP-2 cells were coinfected with an Ela wild-type or mutant virus and

Figure 3. Transcription of a Human 8-Globinenhr+) Gene in Cells Infected with Ela Wild-Type or Mutant Adenovirus In the presence of ara-C, confluent plates of HEP-2 cells were coinfected at a multiplicity of infection of 20 (2 x 10’ PFU per 60 m m dish) with Ela wild-type or mutant virus and at a multiplicity of infection of 100 with the in340-2@-globinenhf+) virus, which contains a human 8-globin gene (linked to the SV40 enhancer) in place of the Ela gene (Lillie et al., 1986). Forty hours after infection, cytoplasmic RNA was prepared. Twenty micrograms of each RNA sample was analyzed by primer extension analysis using 32P-5’-end-labeled synthetic oligonucleotides complementary to 8-globin mRNA (positions +127 to +I42 from the start site of transcription; Lawn et al., 1980) and to Ela mRNA (see Figure 2). The positions of the expected cDNA products are indicated on the right. The virus used in each infection is marked above each lane. Lane M, Mspl-digested pBR322 SP-DNA. Below the autoradiogram the structure of the in340-2/f3-globinenh(+r recombinant virus is shown. Adenovirus sequences, thick, solid lines; 8-globin exons, open, numbered boxes; 8globin 5’and 3’flanking and intervening sequences, thin lines; SV40 72 bp repeats, hatched boxes. The human 8-globin transcriptional start site and direction of transcription are indicated by an arrow. The numbers refer to nucleotide positions on the adenovirus 5 genome.

a recombinant adenovirus containing the human f3-globin gene, linked to the SV40 enhancer, cloned in place of the Ela gene (Lillie et al., 1988). In the absence of Ela protein (coinfection with dl312), P-globin mRNA is readily detectable (Figure 3). In contrast, in the presence of the wild-type 243 amino acid Ela protein (coinfection with dl1500), P-globin mRNA levels are reduced at least 20-fold. Thus, as previously shown (Lillie et al., 1988) the 243 amino acid Ela protein efficiently represses transcription of the 5-globin gene linked to the SV40 enhancer. In the presence of region 1 mutant proteins, however (coinfection with dl1506741, dl1500-734, dl1500-774, or dl1500-777), Pglobin mRNA levels are either unaffected or only slightly reduced. Thus transformationdefective region 1 mutants are also repression-defective. We conclude that region 1 is essential for both Ela’s ability to transform and its ability to repress transcription.

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Table 2. DNA Synthesis Ela Plasmid Microinjected

in Growth-Arrested

Cells Microinjected

with Wild-Type

Number of Experiments

or Ela Mutant Plasmids Cells Incorporating [3H]Thymidine (o/o)

Wild-type Buffer pm975 Ela d11500 El a

alone (289 amino acid protein) (243 amino acid protein)

6 6 10

3+2 13 + 4 32 f

6

Region 1 mutants dll500-A 1 dl1500-741 dll500-734 dl1500-774 dl1500-777

6 7 7 7 7

5*2 16 -t 4 8+3 18 r 3 18 k 4

Region 2 mutants dll500-936 dll500-953

6 6

6&2 723

Region 3 mutant pm975-1098 10% Fetal bovine serum added to medium

2

12

29 k 2

81 t

9

Serum-starved Cl27 mouse fibroblasts were microinjected with plasmid DNA as indicated, labeled with 13H]thymidine for 40-48 hr, and autoradiographed (see Experimental Procedures). The numbers of radioactive nuclei in microinjetted areas were counted by microscopic examination. In control experiments 3%-10% of uninjected quiescent cells were labeled with radioactivity. This background level was subtracted from the values shown. Data are presented as the mean f standard deviation.

Regions 1 and 2 Are Required for Efficient Induction of Cellular DNA Synthesis The results with the region 1 mutants described above complete the map of Ela protein domains required for transformation and regulation of transcription. To define the regions required to induce DNA synthesis, we analyzed region 1, 2, and 3 mutants in a microinjection assay. The Ela 243 amino acid protein induces synthesis of DNA in serum-starved cells (Spindler et al., 1985; Stabel et al., 1985; Table 2). Microinjection of buffer alone into serumstarved cells induces only 3% of injected cells to incorporate [3H]thymidine, whereas injection of an Ela gene that expresses the wild-type 243 amino acid protein (dl1500; Table 2) stimulates 32% of cells to synthesize DNA. Consistent with previous results (Spindler et al., 1985) region 3 (which is specific to the 289 amino acid Eta protein) is not only unnecessary for this Ela activity but is even inhibitory. In contrast to the wild-type 243 amino acid Ela protein, the wild-type 289 amino acid protein (pm975; Table 2) induces DNA synthesis in only 10% of injected cells. A region 3 mutation that destroys the 289 amino acid protein’s potential to activate transcription (pm975-1098; Lillie et al., 1986) improves its ability to induce synthesis of DNA 2-fold (Table 2; compare mutant pm9751098 with wild-type pm975). Region 1 is required for efficient induction of DNA syn-

thesis. All four region 1 single-base substitution mutants induce DNA synthesis less efficiently than does the wildtype 243 amino acid protein (Table 2). In addition, an inframe deletion mutant within region 1 (dl1500-Al; deletion of nucleotides 662-757) that synthesizes normal levels of a smaller Ela protein and is severely transformationdefective (data not shown) does not induce cellular DNA synthesis in serum-starved cells (Table 2). Region 2, like region 1, is also required to induce synthesis of cellular DNA. Two region 2 mutants that are repression-defective but have normal transcriptional activation potential (dl1500-936 and dl1500-953; Lillie et al., 1986) are severely defective in induction of cellular DNA synthesis (Table 2). In a previous study, however, another region 2 transformation-defective mutant was reported to induce wild-type levels of DNA synthesis in non-serumstarved BRK cells (Zerler et al., 1987). We therefore present additional data that confirm that region 2 is required for efficient induction of DNA synthesis in serum-starved cells. Quiescent CREF cells were infected with a wild-type or a region 2 Ela mutant virus and then labeled with [3H]thymidine. At 48 hr after infection the wild-type 243 amino acid Ela protein (d11500; Figure 4) induces cellular DNA synthesis 5-fold over background (dl312; Figure 4). In contrast, the transformation-defective region 2 mutant protein (dl1500-953; Figure 4) does not induce DNA synthesis above background until 72 hr after infection. The

Domains of Adenovirus5 Ela Proteins 1095

!

dllSOO--953

10

5

hours

post-

infection

Figure 4. DNA Synthesisin Serum-StarvedCREF Cells Infectedwith Ela Wild-Typeor Mutant Adenovirus Confluent plates of CREF cells were serum-starvedas described by Spindler et al. (1985)and infectedat a multiplicityof infectionof 5 (5 x iti PFU per 60 m m dish) with Ela wild-typeor mutant virus, as indicated. At the indicatedtimes after infection,the cells were labeled for 12 hr with PH]thymidine.13H]Thymidineincorporationwas determined and is plotted for all time points. dl15W encodes the wild-type 243 amino acid Ela; dll500-953 encodes the region 2 mutant 243 amino acid Ela; pm975 encodes the wild-type 289 amino acid Ela; dl312 encodes an Ela deletion mutant.

previous conclusion that region 2 is not required for induction of DNA synthesis (Zerler et al., 1987; Moran and Mathews, 1987) may be due to the cell type, the use of serum-supplemented media, or the particular region 2 mutant used in this study (Zerler et al., 1987). Based on these combined results we conclude that efficient Ela-mediated induction of cellular DNAsynthesis requires both regions required for transformation and transcriptional repression, region 1 and region 2. Disruption (pm9751098) or deletion (d11500) of the region required for activation of transcription, region 3, increases Ela’s potential to induce synthesis of DNA. Region 3 Is an Autonomous Transcriptional Activator Extensive mutagenesis of Ela has identified only one region (region 3) required for transcriptional activation (Glenn and Ricciardi, 1985; Lillie et al., 1988; Moran et al., 1988; reviewed in Moran and Mathews, 1987; Figure 2). To test whether region 3 is sufficient to activate transcription, we synthesized a 49 amino acid peptide that contains region 3 and asked whether it could activate transcription of an Ela-inducible early viral gene. This 49 amino acid peptide contains the 48 amino acids unique to the 289 amino

acid Ela protein and the first 3 amino acids of exon 2, which are highly conserved among different adenovirus serotypes and species (Kimelman et al., 1985, and references therein; Figure 1). To test the transcriptional activation potential of this and other synthetic peptides, we have used a microinjection assay similar to that described by Guilfoyle et al. (1985). The Ela-inducible E2 gene (pE2) is microinjected into HeLa cells, and E2 expression is monitored by indirect immunofluorescence using an EPspecific antibody. When the E2 gene alone is microinjected, 15% (8%-22% in 12 experiments) of the cells fluoresce (Table 3). Similar background E2 expression was found in microinjection assays by Guilfoyle et al. (1985) and in transfection experiments (for example, see Leff and Chambon, 1988). When the E2 gene is coinjected with the wild-type Ela gene (pLAl), 55% (40%-75% in seven experiments) of the cells express E2 protein (Table 3). As expected, an Ela gene encoding only the 289 amino acid protein (pm975) activates E2 expression with wild-type efficiency (Table 3; Figure 5A). A region 3 mutant defective in transcriptional activation (pm975-1098), however, fails to activate the E2 gene. Thus, both in plasmid microinjection (Table 3) and virus infection (Lillie et al., 1988), Ela’s transcriptional activation function requires an intact Region 3. Remarkably, 55% (42%-720/b in 28 experiments) of cells express the E2 gene following injection of the 49 amino acid region 3 peptide (Iable 3; Figure 5B). Thus, the region 3 peptide induces E2 expression in the same percentage of cells as does the Ela gene. Several control experiments indicate that activation of E2 transcription by the region 3 peptide is specific to its amino acid sequence. First, coinjection of several other peptides, including a 40 amino acid region 1 peptide and an 18 amino acid region 2 peptide, fail to activate E2 expression (Table 3; Figure 5C). Moreover, a partial region 3 peptide containing only the C-terminal 31 amino acids of the intact 49 amino acid region 3 peptide does not activate E2 transcription. Second, preincubation of the region 3 peptide with antiserum raised against the N-terminal 15 amino acids of region 3 prevents transcriptional activation of E2 (Table 3; Figure 5D), while incubation with preimmune serum has no effect on the peptide’s transcriptional activation function. The antibody is not toxic nor does it inhibit transcription nonspecifically since preincubation and coinjection of this antibody with an SV40 early gene does not affect SV40 early transcription (data not shown). The results presented above demonstrate that the region 3 peptide can activate transcription of an early viral gene introduced into cells as naked DNA. We next asked whether the region 3 peptide can also activate transcription of an early viral gene introduced into cells in viral chromatin, the natural template of Ela action. HeLa cells were microinjected with peptide and then infected with an Ela mutant virus (dl312; Table 3). In the absence of Ela protein (d1312 alone), E2 expression from the viral genome is undetectable. The substantially lower level of Ela-independent expression with viral infection versus plasmid microinjection is almost certainly due to the packaging of the E2 gene in viral chromatin. Significantly, injection of

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Table 3. Transcriptional Microinjected

Activation

of the Ela-Inducible

E2 Promoter

Materials

by Region 3 Peptide Immunofluorescent-Posrtive

A. pE2 + Ela plasmid pE2 pE2 pE2 pE2

alone + pLAl (Ela 243 + 289 amino acid) + pm975 (Ela 289 amino acid) + pm9751098 (Region 3 289 amino acid mutant)

15 55 58 21

+ f f +

5 9 3 3

55 19 14 20

+ f f f

8 2 2 5

6. pE2 + Ela peptide pE2 pE2 pE2 pE2

+ + + +

region region region partial

3 peptide 2 peptide 1 peptide region 3

C. pE2 + Ela peptide pE2 pE2 pE2 pE2 pE2 pE2

31 amino acids)

+ antiserum

alone + region 3specific serum + preimmune serum + region 3 peptide + region 3 peptide + region 3-specific serum + region 3 peptide + preimmune serum

D. Ela peptide dl312 dl312 dl312 dl312

(49 amino acids) (18 amino acids) (40 amino acids) peptide (C-terminal

15 17 13 58 8 53

+ dl312 virus infection

alone + region 3 peptide + region 2 peptide + region 1 peptide

0 38 0 0

HeLa cells were microinjected with the materials indicated. At 18-24 hr after injection, cells were fixed, incubated with EP-specific antibody (anti-peptide 67 rabbit serum; see Experimental Procedures), and then stained with fluorescein-conjugated goat anti-rabbit immunoglobulin. The percentages of microinjected cells that showed fluorescence are listed. In (C), pE2 or peptide + pE2 was incubated with region 3-specific antibody (anti-peptide 34 antisera; Experimental Procedures) or preimmune serum before microinjection. In (D), cells were microinjected with peptide alone and then infected 1 hr later with Ela deletion mutant dl312 (Jones and Shenk, 1979). Data in (A) and (8) represent the average of 3-26 experiments and are presented as the mean * standard deviation. Data in (C) and (D) are representative experiments repeated at least twice.

the region 3 peptide activates E2 expression in 38% of the cells, whereas E2 expression is still undetectable following injection of region 1 or region 2 peptides. Taken together, these results indicate that only 49 of the 289 amino acids in the larger Ela protein are necessary and sufficient for Ela-mediated transcriptional activation. We conclude that Ela region 3 is an autonomously functioning transcriptional activation domain. Discussion The Ela gene encodes three highly conserved regions essential for Ela activities. A variety of mutagenesis studies have failed to reveal any additional region required for any known Ela function (see Moran and Mathews, 1987). In this report we demonstrate that region 1 is required for transformation, induction of DNA synthesis, and transcriptional repression, but not transcriptional activation. We further show that Ela region 3 is sufficient for transcriptional activation. Based on these results and previous mutagenesis studies (Glenn and Ricciardi, 1985; Lillie et al., 1986; Moran et al., 1986) a model for Ela functional domains is presented (Figure 6) and discussed below.

Transformation and Transcriptional Repression It seems likely that Ela transforms cells by directly or indirectly modulating the expression of cellular genes involved in regulated growth (see Kingston et al., 1985). We have attempted to define the relationship between transformation and transcriptional regulation by mutational analysis. We find that transformation, induction of cellular DNA synthesis, and transcriptional repression are thus far inseparable, which suggests that repression is the basis of Ela-mediated transformation. These activities require two regions (regions 1 and 2) that act in concert. Whether regions 1 and 2 must be on the same molecule to function is unknown. Previous studies (Lillie et al., 1986; Moran et al., 1986) have shown that transformation can be uncoupled from transcriptional activation of viral gene expression. In light of our present study, we conclude that these activities are completely separable; no region is required for both activities (see Figure 6). The 243 amino acid protein and the 49 amino acid peptide efficiently mediate transformation and transcriptional activation, respectively, yet these proteins overlap by only three amino acids. In fact, point mutations in region 3 that interfere with transcriptional activation ac-

y;gyins

of Adenovirus

5 Ela Proteins

forming) activity of the 289 amino acid protein. We suggest that the transforming and reported repression (Borrelli et al., 1984; Velcich and Ziff, 1985) activities of the 289 amino acid protein exist in spite (and not as a result) of the region 3 activation domain. Region 3 can, however, alter the transformed cell phenotype (for example, see Monte11 et al., 1984) and does so most likely by activating the expression of cellular genes.

Figure 5. Transcriptional

Activation

of the E2 Gene by Ela Region 3

HeLa cells were microinjected with the E2 gene (pE2) and Ela peptide or plasmid, as specified below, and then analyzed by immunofluorescence 16 to 24 hr after injection with an EP-specific antibody (antipeptide 67 antiserum; see Experimental Procedures). The photo graphs show typical immunofluorescence patterns but represent only a small patch of the total quadrant and therefore are not accurate quantitative representations. See Table 3 for complete quantitative analysis of these experiments. (A) pE2 + pm975 (encodes 289 amino acid Ela protein). (6) pE2 plus region 3 peptide. (C) pE2 plus region 1 peptide. (D) pE2 plus region 3 peptide preincubated with antibody specific to the N terminus of region 3 (anti-peptide 34 antiserum; see Experimental Procedures).

tually increase the repression (unpublished observations) and transforming activities (Lillie et al., 1988) associated with the 289 amino acid protein. The region 3 activation domain may impair the repression (and thus the trans-

ltanscriptional Activation Extensive mutagenesis of Ela has identified only one region (region 3) required for transcriptional activation (Glenn and Ricciardi, 1985; Lillie et al., 1988; Moran et al., 1986; reviewed in Moran and Mathews, 1987; Figure 2). Here we have proved that region 3 is sufficient for transcriptional activation by demonstrating that a 49 amino acid region 3 peptide transcriptionally activates the Elainducible E2 promoter. The mechanism by which Ela activates Ela-inducible promoters is unknown. Ela activates a variety of viral and cellular genes with extremely divergent promoter sequences and does not appear to be a sequence-specific DNA binding protein (Ferguson et al., 1985; Ko et al., 1986; reviewed in Berk, 1986). In contrast, other well characterized transcriptional activating proteins, such as the prokaryotic h. repressor, yeast GAL4 and GCN4, and the mammalian glucocorticoid receptor, bind to and activate only those genes that contain the cognate binding site. Furthermore, these activating proteins contain, in addition to a sequence-specific DNA-binding domain, a separate domain required for activation (Ptashne, 1986; Keegan et al., 1988; Ma and Ptashne, 1987; Hope and Struhl, 1986; Miesfeld et al., 1987; Hollenberg et al., 1987). We have demonstrated that the Ela region that is necessary and sufficient for activation is very small and may contain only a single functional domain. This activation domain does share sequence homology with a DNA-binding motif, the “zinc finger:’ first identified in the transcriptional activator TFIIIA (Miller et al., 1985; Berg, 1986). But we note that the central component of the zinc finger is a metalbinding site that is also present in many metalloenzymes that do not interact with DNA (Fersht, 1977; Berg, 1986). Thus the presence of a zinc finger motif does not imply that Ela directly interacts with DNA. Although lacking region 3, the 243 amino acid protein has a limited transcriptional activation function according

,243AA

Figure 6. Summary mains

of Ela

Functional

Do-

Symbols are as in the legend to Figure 1. Regions required (+) or dispensible for activity (-) are indicated.

Cell 1098

to some studies (Leff et al., 1984; Krippl et al., 1985). We suggest that in these instances the 243 amino acid protein activates transcription by a different (perhaps more indirect) mechanism than does the 289 amino acid protein. It will be interesting to test whether the region 1 and 2 mutations, which do not affect activation by the 289 amino acid protein (Lillie et al., 1986; Moran et al., 1986; this study), affect activation by the 243 amino acid protein. In conclusion, single-base substitution mutants have been used to define the functional domains of the adenovirus Ela protein. These results show how a single gene can express two proteins with antagonistic functions: transcriptional activation and transcriptional repression. Differential splicing of a common pre-mRNA retains or excises an RNA segment that encodes a small, autonomous transcriptional activation domain.

Microinjection-Induction of Cellular DNA Synthesis Cl27 cells (a gift of R. Baserga) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and were growth-arrested as follows. Approximately 1 x 105 cells were seeded in DMEM, 10% FBS onto 22 mm2 coverslips in 35 m m plastic petri dishes. The coverslips were transferred to DMEM, 1.0% FBS when the monolayer was about 60% confluent, and cells were injected 4 to 5 days later. Only 3%-10% of nuclei in quiescent cultures were labeled during a 40-48 hr exposure to [3H]thymidine. whereas 70%90% of nuclei incorporated radioactivity upon addition of 10% FBS Plasmid preparations for injection were dissolved in 10 m M Tris (pH 7.6) 1 m M EDTA at a concentration of 400 wglml. The nuclei of 100 to 200 cells in etched quadrants were microinjected with glass capillaries (Graessmann et al., 1980) using the constant pressure of a glass syringe (Capecchi, 1980). After injection, cells were exposed to 1.0 &i/ml of [3H]thymidine for 40-48 hr and then fixed in methanol. Coverslips were coated with Kodak NTB2 photographic emulsion and autoradiographed for 2 days at 4“C. The numbers of radioactive nuclei in microinjected quadrants were counted at 200x magnification under phase microscopy.

Experimental

Microinjection-Transcriptional Activation of E2 HeLa cells were grown on 22 mm2 coverslips in DMEM, 5% FBS, and when the cells were 50%-70% confluent the nuclei of 100-200 cells were coinjected with peptide (120 Ng/ml) or Ela plasmid (100 uglml) and with the E2 plasmid pE2 (250 ug/ml). For antibody-inhibition experiments 50 pl of peptide (150 &ml) was incubated for 1 hr at 37oC with 1 PI of anti-peptide 34 antibody (see below) or preimmune serum, mixed with pE2, and then microinjected. In another assay cells were microinjected with peptide alone and then infected with the adenovirus type 5 Ela deletion mutant dl312 (Jones and Shenk, 1979). Timecourse experiments showed that in all cases the number of EP-positive cells was constant 18-36 hr after injection (data not shown). In all assays cells were fixed 18-24 hr after microinjection with ethanol-acetone (i:l), and E2 protein synthesis was analyzed by immunofluorescence. Cells (on coverslips) were rehydrated for 5 min in phosphatebuffered saline (PBS) and incubated for 1 hr at 37oC with a 1:20 dilution of anti-peptide 67 rabbit serum (see below) in PBS containing 5% FBS. After being rinsed with PBS, the cells were incubated for 1 hr at 37°C with a I:20 dilution of fluorescein-conjugated F(ab’k fragment of goat anti-rabbit immunoglobulin (Cooper Biomedical, Malvern, PA), washed with PBS, and mounted. Fluorescent cells were scored with a Zeiss fluorescence-phase microscope at 250x magnification and photographed with Kodak TriX film.

Procedures

Plasmids pm975 (Monte11 et al., 1962) and dl1500 (Montell et al., 1984) contain the left-terminal 6.1 map units of adenovirus type 2, including the entire Ela region and a small part of the Elb region. pm975 contains a singlebase substitution at adenovirus 2 nucleotide 975, a T-G transversion that alters the Ssplice site consensus sequence used to construct the 12s mRNA (Monte11 et al., 1982). Thus this plasmid expresses only the 13s mRNA and the 269 amino acid Ela protein. dl1500 contains a deletion of nucleotides 1110-I 118. which removes the 5’splice site used to construct the 13s mRNA (Monte11 et al., 1964). Thus this plasmid expresses only the 12s mRNA and the243 amino acid protein. Mutations in region 1 were inserted into these plasmids by replacing the wild-type 322 bp BstXl fragment (adenovirus type 2 nucleotides 616-937) with the corresponding mutant adenovirus type 5 fragment. prasl contains the Ha-rasl gene isolated from T24 human bladder carcinoma cells (Goldfarb et al., 1962). pLA1 contains the Bglll fragment of adenovirus 5 DNA, O-9.4 map units (Tamanoi and Stillman, 1982). pE2 contains a BamHI-EcoRI fragment of adenovirus 5 DNA, 59.5-75.9 map units. Mutagenesis Single-base substitutions throughout the Ela BstXl fragment (adenovirus type 5 nucleotides 617-936) were generated by random chemical mutagenesis and then selected by denaturing gradient gel electrophoresis (Myers et al., 1985). All mutations were identified by DNA sequencing. Four of the mutations isolated by this method (at adenovirus type 5 nucleotides 734 [G-A], 741 [C-A], 774 [T+C], and 777 [C-T]) are characterized in this report. Viruses and Cells Growth of cells and virus stocks was by standard procedures (see Green and Wold, 1979). All viruses were propagated and titered on 293 cells, which express wild-type Ela proteins (Graham et al., 1977). The single-base substitution mutations were inserted into the viral chromosomes of pm975 and d11500 by the method of Stow (1981). in340-2/ 6-globinenhr+) (Lillie et al., 1986) contains the human f3-globin gene cloned in piace of the Ela gene at the left terminus of the in340 genome (Hearing and Shenk, 1983). The 293 cells were a gift of H. Young. HEP-2 cells were obtained from M.A. Bioproducts. HEP-2 cells, a human epidermoid carcinoma line, were used for the transcription assays because they show high levels of Ela-dependent expression of early viral genes and Elaindependent expression of the enhancer-linked f3-globin gene after viral infection. CREF cells were a gift of l? Fisher and were chosen for the ws cotransfection assays because this cell line is transformed efficiently by adenovirus infection (Fisher et al., 1982). In fact, CREF cells proved to be a very convenient, inexpensive, and useful source of cells for these assays. We were able to obtain reproducibly high transformation frequencies with ras and Ela gene cotransfections.

Peptide Synthesis and Anti-Peptide Antisera Preparation Peptides representing region 1 (40 amino acids [PTLHELYDLDVTAPEDPNEEAVSQIFPDSVMLAVQEGIDL]), region 2 (18 amino acids [DLTCHEAGFPPSDDEDEE]), and region 3 (49 amino acids [EEFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSPVS]) were synthesized by the solid-phase method of Merrifield (1963) using a manual adaptation of the automated procedure outlined in ClarkLewis et al. (1986). A portion of the resin was removed after addition of 31 residues of the region 3 peptide to create a 31 amino acid C-terminal region 3 peptide. Composition analysis showed that the peptide products contained all amino acids in the predicted ratios. Details of peptide synthesis and characterization will be published separately. The region 3 peptide was purified by reverse-phase chromatography. One major peak with a shoulder was isolated and shown to be as active as the crude peptide in the transcription assay. Peptide 34 (GEEFVLDYVEHPGHGC), representing the N terminus of region 3, and peptide 67 (EYRNVSLPVAHSDA[C]), representing a C-terminal region of the adenovirus 2 E2 73 kd DNA-binding protein, were synthesized as above and used to generate anti-peptide antibodies as previously decribed (Green et al., 1983; Symington et al.. 1986). Acknowledgments We gratefully acknowledge M. McCaffrey for superb technical assistance. We thank Dr. Janey S. Symington for her enthusiastic participation in some of these studies and for her expertise in immunofluores-

Domains of Adenovirus 1099

5 Ela Proteins

cence. We also thank K. Lee, A. Brand, E. Giniger, A. Ephrussi, and J. Fisher for comments on the manuscript. J. W. L. was supported by a National Institutes of Health predoctoral training grant. This work was supported by grants from the NIH and The Chicago Community TrusVSearle Scholars Program to M. R. G. and NIH grants CA21624 and CA29561 to M. G. M. G. holds a Research Career Award from the National Institute of Allergy and Infectious Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisefnent” in accordance with 16 USC. Section 1734 solely to indicate this fact.

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