V irus Research

EISEVIER Virus Research 33 (1994) 89-97

Multiple pathways for activation of E2A expression in human KB cells by the 243R ElA protein

of adenovirus type 5

Joe S. Mymryk a,1, Stanley T. Bayley b9*

’ Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada, LX? 4Kl b Department of Biology, McMaster University, Hamilton, Ontario, Canada, LBS 4KI

(Received 2 November 1993; revision received and accepted 16 February 1994)

Abstract

Adenovirus type 5 (Ad5) mutant d1520, which produces only the smaller 243 residue (243R) ElA protein, induced efficient production of the viral E2A 72-kDa DNA binding protein (DBP) in human KB cells, but not in human W138, 143, or HeLa cells. In transient expression assays, the 243R ElA protein induced transcription from the E2 early promoter in KB but not in HeLa cells; there was no transcription from the E3 promoter in either cell line. In KB cells, truncation of the E2 promoter from - 285 to - 97 basepairs dramatically reduced transactivation by the 243R ElA product but not by wt ElA, suggesting that the 243R protein acts through factors binding in this region. Multiple deletions in both exon 1 and exon 2 of the 24313 ElA protein failed to disrupt its ability to induce DBP expression. The possible redundant pathways for this induction are discussed. The multiplicity of these pathways and the fact that they are all inactivated in the WI38 and 143 lines are surprising.

Key words: Transcription; Adenovirus; ElA, E2A, Mutant

The ElA region of human adenovirus 5 (Ads) produces two major proteins of 289 and 243 residues (289R and 243R). These proteins are identical except that the larger contains a unique internal sequence of 46 residues (Fig. IA). It has been known for some time that, by virtue of this unique sequence, the 289R protein is a strong activator of transcription from a variety of viral and cellular genes (Shenk and Flint, 1991). However, even though the smaller ElA product does not contain

* Corresponding author. Fax: + 1 (905) 522-6066 or 521-2955. 1 Present address: London Regional Cancer Centre, London, Ontario, Canada, N6A 4M.

0168-1702/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1702(94)00024-7

J.S. Mymtyk. S. T. Rayley / Virus Research 33 (1994) 89-97 90

A

B

C

D

243R Unique

dZO1/07/520/15 n - D I-

d~~~/520/16 i - D _-

diOl/O7/520/31 I - m

d102/06/520/31 m m -

d102/07/520/31 m-m

d~~lOS/520/31 - - -

Cellular p300 - - Binding Proteins W D

Induction of Gene Expression

- m

Fig. 1. Map of Ad5 ElA proteins, deletion mutants, binding sites for cellular proteins and regions required for induction of gene expression by the 243 residue ElA protein. (A) Map of the 289R and 243R ElA proteins and the deletions incorporated into complex mutants used in this study. The deletions are indicated as open rectangles with the first and last residues deleted. (B) The ElA proteins produced by mutants containing deletions in exon 1 and exon 2. In each case, the portions of the 289R ElA protein produced are shown by black bars. These mutants were constructed by ~mbining appropriate restriction fragments from plasmids containing the required deletions, and rescuing the recombinant mutant ElA sequences into d1309 by the method of McGrory et al. (1988). (CD) Regions of the protein required for: (C) binding to the human cellular proteins ~300 and pRb (Egan et al., 1988: Howe et al., 1990). Filled rectangles represent regions essential for binding, the hatched rectangle. a region of secondary importance. (D) induction of gene expression by the 243313 protein (Mymryk and Bayley, 1993a; 1993b). Rectangles with different shadings represent redundant domains.

this sequence, this protein is also able to induce gene expression. The most studied pathway by which it does this involves the transcription factor E2F. E2F exists i- cells in an inactive form bound to cellular proteins pRb and ~107, and by competing for binding to these proteins, ElA releases E2F to activate transcription (Nevins, 1992). This mechanism depends on sequences in exon 1 by which ElA binds to pRb and ~107. Recently, we reported two other pathways for induction of gene expression by the 243R protein, one requiring sequences in exon 1 for binding to a ceIlular protein ~300 (Gedrich et al., 1992; Mymryk and Bayley, 1993a), and the other involving excm 2 (Mymryk and Bayley, 1993b). It was evident from our results that for the induction of some genes, the three pathways, involving

J.S. Mymryk, S.T. Bayley / virus Research 33 (1994) 89-97 91

exon 2, binding to ~300, and binding to pRb, are redundant (Mymryk and Bayley, 1993a).

To investigate these pathways further, we have studied induction of gene expression by the 243R protein of the Ad5 E2A gene for the 72-kDa DNA binding protein (DBP), first in a number of established human cell lines, second in terms of the E2A promoter sequences required, and third with ElA deletion mutants to define ElA sequences that are involved. The DBP gene was chosen as it can be induced to high levels by the 243R ElA protein (Mymryk and Bayley, 1993a), and it is induced in KB cells by both exon 1 and exon 2 of this protein (Mymryk and Bayley, 1993b).

Cells from human WI38 diploid embryonic lung, 143, HeLa, and KB lines were infected at a multiplicity of infection (moi) of 30 plaque-forming units (pfu) per cell with Ad5 d/520, producing the 243R but not the 289R ElA protein (Haley et al., 1984); d/309, which is wild-type for ElA (Jones and Shenk, 1979); or d1312, which lacks ElA entirely (Jones and Shenk, 1979). Cells were labelled with [ 35S]methionine 12-14 h post-infection, harvested, lysed and immunoprecipitated for the viral 72-kDa DBP, as previously described (Mymryk and Bayley, 1993a). The immunoprecipitates were analyzed on SDS-polyacrylamide gels and autora- diographed (Fig. 2). In all four cell lines dZ309 induced a substantial increase in DBP expression as compared to d1312. However, induction by d1520 varied with the line: it was high in KB, low in HeLa, and negligible in WI38 and 143 cells.

We also tested the ability of the ElA 243R protein to induce gene expression in KB and HeLa cells using transient expression assays. Cells were transfected with a reporter plasmid containing the E2 early promoter directing expression of the CAT gene (Imperiale et al., 19851, together with plasmid pLE2, containing a wt ElA region producing both ElA proteins (Jelsma et al., 1988); pLE2 d1520, producing only the 243R ElA protein (Jelsma et al., 1989); or a control plasmid. In both KB and HeLa cells, pLE2 strongly induced transcription from the E2 early promoter (Fig. 3). pLE2 d/520 induced a large increase in transcription from the E2 promoter in KB cells, but had little or no effect in HeLa cells. In other similar experiments, a reporter construct containing the E3 promoter (Weeks and Jones, 1983) was strongly induced by pLE2, but not by pLE2 d/520 in either cell line (Fig. 3). In KB cells, therefore, although the smaller ElA protein induced transcription from the E2 early promoter, it was unable to do so from the E3 promoter, where it consistently repressed transcription (Fig. 3).

Thus results from both infected and transfected cells showed that the 243R ElA protein was able to induce DBP expression in only one of the four human cell lines we examined. This probably explains the failure of many earlier studies, including our own, to observe induction of gene expression by the 243R ElA protein (Shenk and Flint, 1991; Jelsma et al., 1988), as they were performed mainly in HeLa cells, which supported little of this activity. The cell type specificity suggests that a component of the cellular transcription apparatus necessary for induction by the 243R ElA protein must frequently be lost when human cells are immortalized or transformed to establish a line. This is in contrast to rodent cells, where the smaller ElA protein induced DBP expression in primary baby rat kidney cells and

92 J.S. Mymryk, S. T. Bayley / Eus Research 33 (1994) 89-97

143

HeLa

Fig. 2. Autoradiographs of SDS-polyacrylamide gels of E2A 72-kDa DNA binding protein immunopre- cipitated from extracts of [35S]methionine labelled human cells infected with Ad5 mutant viruses. The cell line is indicated for each row, and the infecting virus for each lane.

in all five rat and mouse lines that we tested previously (Mymryk and Bayley, 1993a).

We have examined the role of upstream sequences of the E2 early promoter for induction by ElA. The promoter proximal region of the E2 early promoter consists of a TATA-like sequence, a pair of E2F sites, and an ATF site within the first 80 basepairs (bp) upstream of the cap site (Murthy et al., 1985; Zajchowski et al., 1987; Loeken and Brady, 1989). Binding sites for other, uncharacterized factors are present further upstream between -110 and -150 bp (Boeuf et al., 19871, but these are not required for induction by wt ElA (Imperiale et al., 1985; Loeken and Brady, 1989). We transfected KB cells with a plasmid containing the CAT gene driven by either the full length E2 promoter, containing 285 bp of upstream sequence, or truncated E2 promoters containing 97 bp or 36 bp of the promoter. Fig. 4 shows the results of co-transfecting these reporter plasmids with pLE2 or pLE2 d1520. The full length E2 promoter was strongly induced by both pLE2 and pLE2 d1520. Ninety-seven bp of the promoter were sufficient for strong induction

J.S. Mymryk, XT. Bayley /Eus Research 33 (1994) 89-97 93

E2 CAT E3 CAT

HeLa Fig. 3. Response of the E2 early and E3 promoter to ElA. HeLa and KR cells were transfected with 5 pg of reporter plasmid containing the E2 early or E3 promoters driving CAT expression and 10 pg of either pLE2, containing wt EM, or pLE2 d1520 containing ElA from d1520. Extracts were prepared 36-48 hours after transfection and analyzed for CAT activity as described previously (Jelsma et al., 1988, 1989).

15

1 I wt ElA

m 243R ElA

0

-265/+40 -97/+40 -36/+40 13) (4) (2) (4) (3) (2)

E2 Reporter Plasmid

Fig. 4. Effects of Ad5 ElA proteins on E2 early promoter deletions. The deletions were constructed by standard methods using convenient Nar 1 and Ssp 1 restriction sites within the E2 promoter. KJ3 cells were transfected with plasmids containing the indicated fragments of the E2 early promoter and tested for response to ElA as in Fig. 3. The numbers of replicates are indicated in brackets.

94 J.S. Mymryk, ST. Buyley / Virus Research 33 (1994) 89-97

Fig. 5. Autoradiographs of SDS-polyacrylamide gels of E2A DBP immunoprecipitated from extracts of [3SSjmethionine labelled and infected KB cells. The infecting virus is indicated for each lane. A) Induction of DBP expression by mutants of Ad5 dl520 ~ntaining two deletions in ElA exon 1. The construction of these mutants has been described previously (Howe et al., 1990). Results are shown for mutants that bind both ~300 and pRb (dlO2/06/520), bind one or the other (d/01/06/520, d/02/08/ 520, dlO5/07/520), or bind neither (d101/07/520, dl01/08/520, d104/08/520, d143/08/520) (see Fig. IA). B) Induction of DBP expression by mutants of Ad5 d1520 containing two deletions in ElA exon 1 and one in exon 2 of ElA.

by wt ElA, in agreement with previous studies (ImperiaIe et al., 1985). In contrast, this truncated promoter responded poorly to pLE2 dl520, indicating that for full induction, the 243R protein differed from the larger ElA protein in requiring sequences further upstream, possibly including the binding sites between - 110 and - I50 bp (Boeuf et al., 1987).

To map the regions of the 243R ElA protein required to induce DBP expres- sion in KB cells, we employed a variety of deletion mutants. Exon 1 and exon 2 of the 243R protein can each, on its own, induce DPB in KB cells (Mymryk and Bayley, 1993b). To determine whether the pathways requiring these two exons were additive or redundant for this induction, we used mutants in which induction pathways in exon 1, requiring binding to ~300 and pRb, had been inactivated by deleting the regions required for binding to these cellular proteins (Mymryk and Bayley, 1993a). With mutant ElA proteins containing an intact exon 2, single deletions that abolished binding to ~300 or to pRb, and double deletions that abolished binding to both, had no adverse effect on induction of DBP. This is illustrated in Fig. 5A for mutants containing pairs of the exon 1 deIetions shown in Fig. 1A. Thus the induction pathway operating through exon 2 was able to compensate fully for the loss of induction by exon 1 pathways. To learn more about

J.S. Mymryk, S. T. Bayley / Krus Research 33 (1994) 89-97 9.5

the exon 1 pathways therefore required studies with mutants lacking exon 2. We attempted this by combining double mutants in exon 1 with d11151, which truncates the ElA protein just after exon 1 (Fig. lA, Mymryk et al., 1992). Unfortunately, this approach was unsuccessful as all the resulting mutants were uniformly defective. Although d11151 on its own is sufficient to induce DBP expression to a level comparable to d/520 (Mymryk and Bayley, 1993b), this mutant produces a low level of ElA protein, indicating that the protein may be unstable. Possibly when additional deletions were made in it, this protein was destabilized even more, decreasing the amount of it in the cell to a level where it ceased to produce an observable effect.

To investigate whether particular regions of exon 2 were essential for induction, we combined double deletions in exon 1 that inactivate the exon 1 pathways, such as d101/07/520 and d104/08/520, with mutations d/1115, d11116 and pm1131, that collectively delete the whole of exon 2 (Fig. 1A); for clarity, proteins from these mutants are shown in detail in Fig. 1B. Surprisingly, all of these mutants efficiently induced expression of the DBP at levels comparable to that of d1520 (Fig. 5B). From this it appeared that no single region of exon 2 was essential for induction, and that full induction was possible with as little as the first 34 residues.

However, this conclusion must be qualified for the following reason. While the deletion in pm1131 did not reduce induction here (Fig. 5B), we found earlier that in an ElA protein consisting of exon 2 alone, small deletions in the region of the pm1131 deletion did reduce this induction, although they did not abolish it (Mymryk and Bayley, 1993b). Various explanations for this difference are possible. First, if we assume, as seems probable, that exon 2 exerts its effect by interacting with a cellular protein, a small deletion within exon 2 may disrupt this interaction more than a large deletion at the C-terminal end of the polypeptide chain. A second possibility is that the combinations of deletions we tested did not inactivate all exon 1 pathways. It seems unlikely that the weak binding of d104/08/520 to ~300 or the binding of ~107 by d101/07/520 caused the induction as neither of these mutants induced DBP expression in any of the rodent cells we tested previously (Mymyrk and Bayley, 1993a). It is also unlikely that residues 90-105 were responsible, as no effects of deletions in this region have been reported. However, if say residues 3 to 25 and 36 to 69 were involved with separate pathways, none of the mutants we used here would have inactivated both at the same time. A third possibility is that induction is independent of the coding regions of ElA altogether and depends instead on the 5’ or 3’ untranslated regions of the mRNA. This seems unlikely because if it were the case, we should not have observed reduced inductions with small deletions in exon 2 (Mymryk and Bayley, 1993b). Furthermore, virus producing only the 9s ElA mRNA, which differs from the larger ElA mRNAs only within the coding regions, does not induce DBP expression in KB cells (Mymryk and Bayley, 1993b).

We are left at present, therefore, with two possibilities for induction pathways in addition to the two in exon 1 described earlier (Mymryk and Bayley, 1993a). One is that the exon 2 pathway (Mymryk and Bayley, 1993b) involves the whole of exon 2, but no single part of it is essential. Alternatively, the exon 2 pathway

96 J.S. Mymtyk, S. T. Bayley /virus Research 33 (1994) 89-97

involves roughly the C-terminal 70 residues only, and there is yet another unidenti- fied pathway in exon 1.

Even though we have failed to map precisely all the regions in the 243R ElA protein responsible for inducing DBP expression, this work demonstrates two remarkable features of this induction. One is the ability of this ElA protein to induce expression despite extensive deletions throughout its polypeptide chain. The other is that despite the multiplicity of pathways through which this protein induces DBP expression, they are all inactivated in the WI38 and 143 lines, suggesting that these pathways must have some key feature in common.

We thank Susan Shepherd and Anar Lakhani for excellent technical assistance, Drs. Phil Branton, Joe Nevins, Lud Prevec, and Peter Whyte for generous gifts of antiserum, plasmid, and cell lines and Don Van Meyel for assistance with the photography. This work was supported by grants from the National Cancer Institute of Canada and the Natural Sciences and Engineering Research Council of Canada. J.S.M. was a research student of the National Cancer Institute of Canada.

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