Virus Research, 20 (1991) l-10

0 1991 Elsevier Science Publishers B.V. 0168-1702/91/$03.50

ADONIS 0168170291000875

VIRUS 00669

Review Article

Utilization of cellular transcription factors for adenovirus-induced transcription

Joseph R. Nevins

Howard Hughes Medical Insrituie, Section of Genetics, Department of Microbiology and Immunology, Duke University Medical Center, Durham. NC 27710, U.S.A.

(Accepted 26 February 1991)

Introduction

Viruses have been invaluable tools for the study of eukaryotic gene regulation. This is particularly true for the DNA viruses that replicate in the nuclei of infected cells. Multiple factors contribute to the utility of these viruses as model systems including the simplicity of the viral genomes, the realization that the transcription of the viral genes utilized host cell components, and the ability to introduce viral genes into cells at high efficiency via virus infections. However, it is the genetic analysis that viruses offer, an analysis not readily available in higher eukaryotic cells, that has more than any one thing provided an advantage over cellular systems. Most importantly, viral genetics has allowed the definition of regulatory genes that are involved in the control of viral gene expression. One such example is the ElA gene

of adenovirus, identified in 1979 as a regulatory gene whose product was essential to establish the early phase of the viral replicative cycle (Berk et al., 1979; Jones and

Shenk, 1979). A low level of transcription directed by each of the early viral promoters is markedly enhanced as a function of ElA (Nevins, 1981).

Various experiments have demonstrated that the stimulation of viral transcrip- tion by ElA is an indirect process. For instance, regulatory genes from unrelated viruses can replace ElA to activate early viral transcription (Feldman et al., 1982; Imperiale et al., 1983). It is also now clear that the ElA product is not itself a DNA binding protein (Ferguson et al., 1985) but rather must modify or alter the activity of cellular transcription factors that are utilized by the early viral promoters. The first such transcription factor to be identified as playing a role in ElA-dependent activation of transcription was the E2F transcription factor (Kovesdi et al., 1986). E2F binds to two sites in the early E2 promoter that have been shown to be critical

Correspondence to: J.R. Nevins. Howard Hughes Medical Institute, Section of Genetics, Department of

Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710, U.S.A.

2

ATF E2F E2F TATA r-

Fig. 1. Functional elements of the adenovirus E2 promoter. The essential functional elements of the

promoter have been defined through the analysis of a series of site-directed mutants (Murthy et al., 1985;

Zajchowski et al., 1985; Loeken and Brady, 1989). The elements are named in reference to the proteins

that recognize these sites.

for ElA-induced E2 transcription (Yee et al., 1987; Loeken and Brady, 1989) (see Fig. 1). Moreover, the E2F sites can confer ElA inducibility to an unresponsive promoter (Kovesdi et al., 1987; Yee et al., 1989). Finally, E2F DNA binding activity was shown to increase markedly upon adenovirus infection, dependent on ElA

(Kovesdi et al., 1986).

A series of recent experiments have begun to dissect the molecular mechanisms contributing to the activation of the E2F transcription factor in an adenovirus infected cell. These experiments have demonstrated a previously unrecognized

complexity to the process and have provided insight into the evolution of viral

regulatory function designed to maximize viral transcription.

The stimulation of E2F binding activity is dependent on ElA and EI4

The E2F transcription factor, as isolated from adenovirus-infected cells, binds to the E2 promoter with a high degree of cooperativity, resulting in the formation of a very stable DNA-protein complex (Hardy and Shenk, 1989; Raychaudhuri et al.,

1990). This cooperative binding requires the precise dyad arrangement of the E2F sites as found in the E2 promoter (Hardy and Shenk, 1989; Raychaudhuri et al., 1990) and requires the expression of the early E4 gene (Hardy et al., 1989; Huang

and Hearing, 1989; Neil1 et al., 1990; Raychaudhuri et al., 1990). The analysis of a series of viral E4 mutants has demonstrated that the orf 6/7 product of E4 (see Fig. 2), a 1PkDa polypeptide, is that which is required for the cooperative binding of

E2F (Huang and Hearing, 1989; Marton et al., 1990; Neil1 et al., 1990). Moreover, it is now also clear that the E4 protein is physically associated with E2F in the

DNA-protein complex (Huang and Hearing, 1989; Marton et al., 1990; Neil1 et al., 1990). Although it is tempting to speculate that the cooperative binding of the E2F molecules is brought about by a dimerization of the E4 proteins, there is as yet no

evidence for such. That this E4-induced cooperative binding of E2F is important for E2 transcrip-

tion is indicated by the finding that E2 transcription is reduced in the absence of E4

(Babiss, 1989; Reichel et al., 1989) and that the E4 gene is a trans-activator of the E2 promoter (Goding et al., 1985; Reichel et al., 1989). Moreover, it is the E4 orf 6/7 product, that which associates with E2F to allow cooperative binding, that functions as a trans-activator of E2 transcription (Neil1 et al., 1990). Finally, E4 orf 6/7 mutant proteins that still interact with E2F but that do not allow cooperative binding are impaired for truns-activation (Neil1 and Nevins, in preparation).

9.0 9.5 IO,0

AA e -0RF2

AA- - ORF3

AA- -cl Et - ORF 3/4

AA- - CftF4

AA- - @RF6

AA -iI - ORF 6/7

Fig. 2. The E4 transcription unit. The various IS4 mRNAs that derive by alternative splicing of the EA primary transcript are depicted. The open reading frames (ORF) are indicated by the open boxes.

Of course, many experiments have shown that ElA alone can truns-activate the E2 promoter (Imperiale et al., 1983; Leff et al., 1984; Zajchowski et al., 1985) and that this trans-activation is dependent on E2F sites (Loeken and Brady, 1989). In fact, isolated E2F sites and even a single E2F site can confer ElA inducibility to a normally unresponsive promoter (Kovesdi et al., 1987; Yee et al., 1989). Taken together, these results indicate that ElA trans-activates the E2 promoter through the E2F factor. Although some assays have suggested that the role of ElA in the

activation of E2F DNA binding during a virus infection is indirect, to merely allow

E4 transcription activation (Hardy et al., 1989), a direct role for ElA was evident from experiments in which E4 expression was forced in the absence of ElA. Under conditions where E4 expression was equal in a wild type infection (ElA + ) or in an

infection with the ElA mutant d1312 (ElA - ), E2F activation was markedly reduced in the d1312 infection (Kovesdi et al., 1986; Reichel et al., 1989). Thus, rather than to merely allow E4 transcription, ElA must directly contribute to the

activation of E2F. Although ElA alone or E4 alone can trans-activate the E2 promoter, the

combination of ElA and E4 is much more effective (Reichel et al., 1989). This result suggests that the two truns-activators are likely working via different mechanisms. Through the analysis of E2F in viral infections in the absence of E4, it appears that one function of ElA is to increase the level of active E2F that is capable of DNA

binding (Raychaudhuri et al., 1990). That is, there is an increase in E2F levels,

independent of the influence of E4-induced cooperative binding. Thus, as schemati- cally pictured in Fig. 3, ElA action results in an increase in the pool of E2F that is capable of binding to DNA, thus leading to a stimulation of E2 transcription by driving more factor onto the promoter. The production of the E4 protein would convert any E2F that was active (with respect to DNA binding) to a cooperatively

ElA * l E4

**a -

1 E4

n

Fig. 3. Role of ElA and E4 in the activation of the E2F transcription factor. An inactive form of the E2F

factor, with respect to capacity to bind to DNA, is depicted as an open figure and a form that is active

for DNA binding is depicted as a solid figure. The E4 1PkDa protein (hatched figure) interacts with E2F

to allow cooperative binding, dependent on the arrangement of the E2F sites found in the E2 promoter. Although the E4 protein clearly forms a complex with E2F, and can be found in the DNA-protein

complex, the depiction to suggest an interaction between two E4 molecules to allow the stable complex is

only speculation.

binding form that could stably interact with the promoter. Thus, E4 alone would stimulate transcription due to an increase in factor bound to the promoter. ElA and E4 together would lead to an even larger increase in transcription by increasing the pool of active E2F and converting this pool to a stably binding form.

The basis for this El A-dependent increase in E2F appears to be an induced phosphorylation of E2F resulting in an activation of DNA binding capacity (Bag&i et al., 1989). E2F DNA binding activity is inactivated by treatment with phos- phatase. This phosphatase-inactivated E2F can be re-activated with an extract from adenovirus-infected cells, as well as with a purified kinase, but not with an extract from uninfected cells. Since there is no evidence to suggest that ElA itself is a kinase, a cellular kinase is most likely involved in the activation of E2F. Perhaps ElA stimulates the activity of such a kinase or alternatively, ElA might inhibit the activity of a phosphatase, thus achieving the same net result, an increase in the phospho~lation of E2F.

The induction of cooperative E2F binding by the interaction of the E4 protein with E2F is striking in view of the specificity. This cooperative binding requires the exact arrangement of E2F sites as found in the E2 promoter (Hardy et al., 1989; Raychaudhuri et al., 1990). Although E2F binding sites have been identified in a variety of cellular promoters (Blake and A&khan, 1989; Hiebert et al., 1989; Thalmeier et al., 1989; Mudryj et al., 1990), no other instances have been found where E2F sites are arranged in the manner found in the E2 promoter. It thus appears that through the action of the E4 protein, the cellular E2F factor is converted into an E2-promoter-specific factor. In short, the virus has evolved a mechanism to redirect a cellular transcription factor for viral purposes not unlike the re-utilization of the cellular Octl factor by the herpesvirus VP16 protein

5

(McKnight et al., 1987; Gerster and Roeder, 1988; O’Hare and Goding, 1988;

Preston et al., 1988).

Trum-activation by the 12s ElA product

The finding that the viral E4 protein could form a specific complex with the E2F

transcription factor suggested the possibility that E2F might normally interact with cellular proteins. Although there was no evidence of such interactions in HeLa cells,

the cell line widely used for studies of adenovirus gene control, E2F was indeed found to be complexed to cellular factors in extracts of a variety of other cell lines

(Bag&i et al., 1990). Although the normal cellular function of these complexes is not known, they are significant with respect to a viral infection since the E4 protein

cannot interact with E2F that is already complexed to a cellular factor, thus preventing the formation of a stable complex on the E2 promoter (Bag&i et al., 1990). Strikingly, however, ElA proteins can dissociate these E2F-containing com- plexes, releasing free E2F that can associate with the E4 protein. It would therefore appear that the virus has evolved a two-step mechanism, as depicted in Fig. 4, to efficiently utilize the cellular E2F factor and redirect it for viral specific purposes.

It is now clear that this function of ElA is independent of the sequences found within conserved region 3 (CR3) of the protein, those sequences normally associated with ElA truns-activation function (Ricciardi et al., 1981; Monte11 et al., 1982; Lillie et al., 1986; Schneider et al., 1987) (Fig. 5). Although there have been previous reports of truns-activation by the 12s ElA product, the relevance of this to viral

1 4 - - - - -

Fig. 4. Role of the ElA 12s gene product to dissociate E2F complexes and allow E4 interaction. E2F is depicted as the solid figure. In most cell types, E2F is complexed to a cellular factor (open figure). This

complex can bind to the E2F sites in the E2 promoter (shown below), but without cooperativity and apparently non-functionally. The ElA protein dissociates this complex, releasing free E2F that can bind

to the promoter and stimulate transcription. Free E2F does not bind to the E2 promoter cooperatively

and thus usually only occupies one site. The release of R2F allows the E4 protein to bind to E2F,

resulting in a complex that can bind cooperatively to the adjacent sites in the E2 promoter, form a stable complex, and a large stimulation of transcription.

CRI CR2 CR3 I 40 80 120 140 188 289

, .‘.‘.‘_‘.‘.‘.‘.‘. ,,,

w-1 Transformation

Fig. 5. Domains of the ElA proteins required for E2F complex dissociation. Depicted at the top is a schematic of the 289 aa product of the 13s ElA mRNA, containing the CR1 and CR2 domains as well as

CR3 that is unique to the 13s product. The segments of the ElA protein that are required for E2F

complex dissociation are depicted. These regions extend from residues 38-73 and residues 124-135,

based on assays of various ElA mutants (Raychaudhuri et al., submitted). Shown below is a schematic

depicting regions of the protein required for ElA transformation, as assayed in conjunction with the activated ras oncogene (Lillie et al., 1986; Moran et al., 1986; Zerler et al., 1986; Whyte et al., 1988;

Jelsma et al., 1989; Whyte et al., 1989; Stein et al., 1990). These regions extend from residues l-26, 35-77. and 121-139.

infection and to the action of the CR3-dependent process has been unclear. In particular, various reports have suggested no trans-activation function at all for the 12s product (Lillie et al., 1986; Moran et al., 1986; Zerler et al., 1986), or that activity was limited to a few cellular promoters (Zerler et al., 1987). In contrast, other experiments have demonstrated that the 12s product could trans-activate viral promoters, including the E2 promoter, but that the efficiency of this event was variable (Leff et al., 1984; Winberg and Shenk, 1984; Ferguson et al., 1985; Simon et al., 1987). A resolution of these discrepancies was suggested by an analysis of the E2F dissociating activity in comparison to the ability to truns-activate (Bagchi et al., 1990). Specifically, it would appear that there is a correlation between the presence of E2F in a complexed state and the ability of the ElA 12s product to truns-activate the E2 promoter. That is, in HeLa cells where E2F is largely free, the 12s product was inactive in tram-activation whereas the 13s product could efficiently stimulate E2 transcription. In contrast, in mouse L cells where the majority of E2F is complexed, the 12s product could truns-activate.

Role of 12s ElA tram-activation in oncogenesis

Through the analysis of a large series of ElA mutants it has become clear that the E2F dissociating activity is dependent on sequences within both CR1 and CR2 (Raychaudhuri et al., submitted). Moreover, rruns-activation assays have demon- strated that mutations within CR1 or CR2 that disrupt E2F dissociation also disrupt nuns-activation function. Of considerable additional interest is the realiza- tion that the sequences required for dissociation of E2F complexes are also those sequences in ElA that are required for transforming activity (Raychaudhuri et al., submitted), suggesting the possibility that this biochemical activity of ElA contrib- utes in part to the ability of ElA to act as an oncogene. Interestingly, these are also

the amino acid sequences that are shared with two other viral oncogene products, SV40 T antigen and human papillomavirus E7. Moreover, previous experiments

have shown that both T antigen (Loeken et al., 1986; Loeken and Brady, 1989) and E7 (Phelps et al., 1988) possess truns-activation function and this was determined

using the adenovirus E2 promoter. In fact, analysis of promoter mutants has shown

that it is the E2F sites in the E2 promoter that are the targets for both T antigen and

E7 (Loeken and Brady, 1989; Phelps et al., submitted). Indeed, recent experiments have shown that the E7 protein is capable of altering the E2F complexes in a

manner similar to ElA (Phelps et al., submitted). It therefore appears that these viral regulatory proteins have evolved a common function, directed at a cellular transcription factor, that may be a critical part of the ability of these proteins to induce oncogenic transformation.

Conclusions and perspectives

The process of transcription initiation is clearly a complex event, involving the

interaction of a large array of factors with the promoter and enhancer of a gene. Controlling the frequency of transcription could therefore involve the alteration of one or more of these factors. The experiments summarized in this review, that

address the mechanisms controlling the activity of one such factor, have highlighted an additional complexity. These experiments demonstrate that multiple separate events contribute to enhancing the interaction of this factor with the promoter.

These results are of perhaps most interest in revealing the evolution of viral

regulatory strategies. The interaction of the E4 protein with E2F allows cooperative binding but dependent on the arrangement of binding sites found in the E2

promoter. Since this arrangement appears to be unique to the adenovirus E2 promoter, this action of E4 generates an ‘EZspecific’ transcription factor. More- over, since the E4 protein cannot accomplish this task in the presence of cellular

proteins that compete for the E2F factor, the role of the 12s ElA product can be seen as a facilitator of this event. Thus, the virus has evolved a mechanism to re-direct a cellular transcription factor for viral purposes and do so in an efficient manner.

Although future experiments must address the mechanism by which the E4 protein induces cooperative binding, perhaps the question of most importance relates to the consequence of ElA-mediated dissociation of E2F complexes in the absence of E4. Since this event appears to correlate with the oncogenic capacity of ElA, a detailed understanding of the mechanism, the normal promoter targets of

E2F, and the identity of the E2F-binding factors may well contribute to our understanding of oncogenesis.

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(Received 12 February 1991)