Multiple, distinct trans-activation functions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain.

0022-538X/93/127684-06$02.00/0

Copyright © 1993, American SocietyforMicrobiology

Multiple,

Distinct trans-Activation Functions Are Encoded by

the Simian Virus 40 Large

T

and Small t Antigens, Only

Some of Which

Require the 82-Residue

Amino-Terminal Common Domain

MARY R. LOEKEN

Joslin DiabetesCenterand Harvard Medical School, OneJoslin Place, Boston, Massachusetts02215 Received24May1993/Accepted 15 September 1993

Simian virus 40 (SV40) small t and large Tantigens can each transactivate the adenovirus (Ad) E2A and

the Ad VA-I promoters. The first 82 amino acids of large T and small t are identical. However, this large

T-small t commondomain between residues 1 and 82 does not transactivate, suggesting that large T and small

teach encode separate trans-activation functions. To determine whether the large Torsmall tuniquedomains,

which are required for trans activation of the E2A promoter, are sufficientfor thisactivity, we have employed

expression plasmids separately encoding the common and unique domains of large T and small t.

Cotransfection ofa large Tunique domainexpression plasmid efficiently transactivated the E2A promoter.

Optimaltransactivationby large Trequiredthemotif thatbinds cellularproteinssuchastheretinoblastoma

gene product, which is located in the large Tunique domain, andadditional largeT structures outside this

motif.In contrast,the small tunique domain did not trans activate the E2A promoter.Experimentsutilizing

E2A promoter mutantscontaining only the ATF- or EIIF-binding sites demonstrated that transactivation by

small t involvesonly the EIIF transcription factor and that this function requiresboth thecommon (residues

1to82) and the small t uniquedomains expressed as a colinear protein. transactivation by large T, in contrast,

involves at least three mechanisms. There appear to be at least two mechanisms that involve the EIIF

transcription factor, at least oneof which does not require the common domain (residues 1 to 82) and one

mechanism thatinvolves the ATF factor and does require both the common and the large Tunique domains.

The simian virus 40 (SV40) genome encodes two early protein products, large T and small t antigens (large T and small t, respectively), that are generated from the same

primary transcript (32). As a result of differential splicing, these proteins contain identical 82-residue amino-terminal domains (which are referredto here asthe 1-82 or common

domain),whereas thecarboxy-terminal domainsareuniqueto

eitherlargeT orsmall t (theseare referred to here asunique domains) (Fig. 1). We have previously reported that during transient transfection ofplasmid DNA, SV40 largeTandsmall tantigenscaneach transactivatetheadenovirus(Ad)E2Aand

VA-I promoters (17). Structures that are unique to large T

(residues83 to 708

[T/u])

or tosmallt(residues83 to 192[t/u])

are required for trans activation of the E2A promoter, since insertion ofastopcodon inplace of aminoacid83producesa

stable common-domain protein product that does not

stimu-late chloramphenicol acetyltransferase (CAT) activity

con-trolled by the E2A promoter

(17).

This suggests that trans

activation by large Tand small t does not occur bya shared

mechanism activatedbytheircommonamino-terminal domain

but occurs by distinct mechanisms activated by structures

includingsequencesfrom withineach of their unique domains. We wished to determine the biochemical mechanism by

which small t activates transcription and to establish whether this activity can be distinguished from those of other trans

activators. We have previously found that the effects of smallt

can be distinguished from those oflargeT onthe SV40 late

promoter (5, 17) and ofthe Ad ElA 289- or 243-amino-acid

products on the Ad E2A promoter (16). Therefore, study of

transcription regulation bysmall t may elucidatenovel

regula-toryprocesses that would not be revealed withother systems.

Inaddition, itis not clear whether the amino-terminal domain

shared by both large T and small t is required for the trans-activation activities of either protein. Mutations within

thecommondomain have beengenerated whichdemonstrate

thatthisregionisimportant for largeTstabilityanditscellular

transformation activity (20). Forthe present study, we asked whether small t or large T structures that are necessary for trans activation are sufficient when expressed as separate

molecules. Furthermore,we employedreporterplasmids con-taining mutations ineither the ATFortheEIIF

transcription

factorbinding sitestodetermine howmanylargeT-and small

t-specific

trans-activation processes

might

be involved in

regu-lationof the E2A promoter and whether anyofthese processes

mightbe sharedby large Tand small t.

Toaddressthesequestions,weemployed plasmidsencoding

large T cDNA (5), the 82-residue large T-small t common domain (5), or the large Tunique domain

(22)

all under the control of theRoussarcomavirus

(RSV)

long terminalrepeat. Ithaspreviously been demonstrated that thecommondomain isexpressedas astableprotein product (17)and that thelarge

Tunique domain(T/u)appearstobeless stable thanwild-type large T (22). We also constructed anexpression plasmid that

would encode the small t unique domain by using the poly-merase chain reactionto

amplify

the small t

unique

domain coding sequence. As shown in Fig. 2, by using amonoclonal

antibodywhichrecognizesstructureswithin the smalltunique domain (21), a protein product with the molecular weight expected of the small t

unique

domain

(t/u)

was detected by

Western blot analysis following transient transfection of

ex-pression plasmids into CV-1 cells. Expression of full-length

small tbythe plasmid containingthe smalltcDNA sequence

under the control of the

promoter-enhancer employed

for

expressionof the smallt

unique

domainwascomparabletothe 7684

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T-unique (T/u)

.gm

174

u)

FIG. 1. Diagrams ofprotein products ofSV40large T andsmallt. As aresult ofdifferentialsplicing ofaprimary transcript, largeTand small thave anidentical 82-residue commonamino-terminal domain (solid area) andunique carboxy-terminal domains (largeTresidues83

to708 [diagonal striped area] and small tresidues 83 to 174

[cross-hatchedarea]).Theplasmids used in this study expressed only large T, the 1-82common domain, the largeTunique domain, smallt, orthe smalltunique domain.

Y v

-S

E

0 :

4-z

0*

14.3

level of small t expressed by the control plasmid, pRSV-T/t

(which contains the entireSV40 early coding region controlled

by the RSVlong terminal repeat [19]). That the small tunique

product does not appear tobe as abundant as thefull-length

small t protein may mean that the 1-82 common domain is

important for the stability of the small t unique domain just as

it isfor the largeTunique domain (20, 22).

trans activation of the E2A promoter by large T does not

require the commondomain, whereas trans activation by small

tdoes. The abilities of thelarge T or small t unique domains to

trans activatethe wild-typeAd E2A promoter linked to a CAT

reporter gene(18) were tested (Fig. 3A). As expected,

cotrans-fection ofcontrol plasmids encoding Ad EIA or SV40 large T

stimulated pE2A-w.t. CAT activity. Cotransfection of a

plas-mid encodingthe large Tuniquedomain(T/u)also stimulated pE2A-w.t. CAT activity. Since the large T unique domain, even

when expressed without the common domain, binds cellular

proteins such as the retinoblastoma gene product (RB) and

p107(22) thatarefound incomplexes with theEIIF

transcrip-tion factor (7, 10, 29),trans activation by the large T unique

domain could occur upon binding to any of these

EIIF-associated proteins. However, RB or p107 binding does not

appear to be the only mechanism by which large T trans

activates the E2A promoter, sinceaplasmid encodingalarge

T cDNAwith apoint mutation that destroys the binding site

for cellularproteins such as RB and p107 (T/Kl) can also trans

activate the E2A promoter. However,there is aslight

reduc-tion in the trans activation of the E2A promoter by the KI

mutant.Since this mutation doesnotaffect the stability oflarge

T (15), this suggests that the large T structure that binds

cellularproteins such asRB andp107 isnecessary forpartof

thetrans-activation function oflargeT, atleast withregard to

theE2A promoter. Thus, thelargeTunique domain, which is

necessaryfortransactivation, issufficient. Furthermore, there

appear to be structures both within the RB/p 107-binding

pocketandoutside it thatparticipatein transactivation of the

E2A promoter. Aspreviously reported (17),the 1-82common

domain could not trans activate the E2A promoter, while

6.2

B

_ 0)

0 _

_ m

z w

'E

[image:2.612.58.297.77.182.2]

C

FIG. 2. (A) Western blot analysis of products of plasmids encoding

small torthe small tunique domain. The plasmid encoding small t

(pSRaB-t/cDNA)wasconstructedbyLaurenSompayrac byinsertinga

small tcDNAsequence (kindly provided by Kathleen Rundell) into the plasmid pSRotB, which is a modified pSRo vector (31). The plasmid encoding the small t unique domain (pSRaB-t/u)was

con-structed by polymerase chain reaction amplification of the small t

unique domain with primersdesigned togeneratea translation

initi-ation site andto allow insertion into the pSRoaB expression vector.

Plasmid sequences were verified by dideoxy sequence analysis of double-stranded DNA (27). To examine steady-state levels of the products of these plasmidsupontransienttransfection, monkey CV-1 cells in 60-mmculturedishesweretransfectedwith 10 ,ug of plasmid

DNA and30,ug of calf thymus carrier DNA by the calcium phosphate coprecipitation method.Forty-eight hours after transfection, the cells

were harvested andanalyzedasdescribed elsewhere (6) with 2 ,ug of

smallt-specific Pab 280 (Oncogene Science) and horseradish peroxi-dase-coupled sheep anti-mouseimmunoglobulin G (Amersham). Asa

control for small t expression, a plasmid encoding the entire SV40 early region controlled by the RSV long terminal repeat(pRSV-T/t)

wasemployed. The migration positions of molecular weight markers areindicated.(B) Western blot analysis of the products of the plasmids

encodingthe smalltuniquedomainorwild-type smallt.The

concen-tration of the plasmid encoding the small t unique domain was

transfectedatahigher concentration than that of the plasmid encoding

wild-type small t to see whether increased levels of the t/u product

could be achieved. Cells were transfected and cell extracts were

assayed as described forpanel A,except that 40 ,ug of the plasmid encodingthe smalltunique domainor10,ugof theplasmid encoding wild-type small tplus 30 p.gof calfthymus carrier DNAwasused for

transfection.

1 82

1-82 LargeT

smallt

1 82

1-82 Vunique (Vt

A

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A

4

*a

w-.- - X

-e

B

...

*----*-

040 1404

_ _ Tt~+

0

FIG. 3. CATactivities of pE2A-w.t. CAT.(A) CV-1 culture disheswere transfectedasdescribed elsewhere

of pE2A-w.t. CATalone (-) orwith2 p.gof plasmids

(13), large T cDNA(T), large Tuniquedomain (T/u), withapointmutationwithintheregionbetween residue (15), the large T-smalltcommondomain (1-82), small tunique domain (t/u), and carrier DNAto maintain a

DNA concentration of 15 jLg per plate. Forty-eigh

transfection, one-half of the cell extractwasassayed fo for I h with ['4C]chloramphenicol and acetyl coenzym

weretransfectedasdescribed for panel A,exceptthat 2(

plasmidencoding the small t uniquedomain wasempi

cells cotransfected with small t plus t/u plasmids (t transfected with 2jig of the small t and 1(1 ,ugof the ,

domainexpressionplasmids. In allcases,the total DNA

waskeptconstant at 15 jLgperplate with carrierDNA

full-length small tcould. However, unlike the lar

domain, the small tunique domain (t/u) did nott

the pE2A-w.t. CAT plasmid.

It was not totally unexpected that trans activ

small tuniquedomain might be reduced, since ita

less stable than wild-type small t.Wetestedwheth

theamountoftheplasmid encoding the smallt un

transfccted into cells might raise the cellular con(

the small tunique domaintoaconcentration suffic

in trans activation. As shown in Fig. 2B, transfecti

expression plasmid at a concentration four times that ofthe

wild-type small t plasmid generated a steady-state concentra-tion of the small t unique domain that was similar to that of

wild-type

small t.

However,

even a

fivefold-higher

concentra-tion of the small t

unique

domain

plasmid

(10

compared

with

2

pg)

wascompletely inactive with regard to transactivation of

the

pE2A-w.t.

CAT

plasmid (Fig. 3B).

In contrast, trans

I

activation

by

the

large

T

unique

domain,

whose

stability

is also

reduced compared with full-length large T, did not appear to

be dramatically compromised. Thus, the common domain is

P _* required fortrans activation by small t but not by large T. We

considered that the common domain might be necessary for trans activation

by

small t either because it influences the * * conformation of the small t unique domain (but not of the

large T unique domain) required for this activity or because

the common and unique domains together form a structure

that complexes with a cellular factor(s) involved in trans

activation of the E2A promoter. If a cellular factor(s) does

complex with both ofthe small t domains, it must do so only

when they are expressed as one contiguous molecule, since

cotransfection of plasmids encoding the common and unique

domains on separate plasmids did not stimulate pE2A-w.t.

CATactivity (Fig. 3B). Finally, cotransfection ofan excessof

plasmid encoding the small t unique domain did not inhibit

trans activation bysmall t, at least when theconcentrationsof

plasmids which we were able to transfect together were used

(Fig. 3B), suggesting that the small t unique domain without

the common domain does not titrate a limitingcellular factor

involved in trans activation.

Involvement of large T and small t domains inactivation of

specific E2A promoter-binding transcription factors. Full

ac-tivity of the Ad E2A promoter appears to beregulated within

a 55-nucleotide enhancerelement containing a single binding

site for the transcription factor ATF and two invertedbinding

sites for thetranscription factor EIIF(18). We havepreviously

determined that transactivation of the E2A promoterby small

t involves the binding sites only for EIIF (16). The results of

the experiment shown in Fig. 3A demonstrate that both the

cells in 35-mm common and the unique domains are required for trans

(18)with 2 jig activationbysmall t. Fromthis,weconclude thatwe can detect

encodingEIA one trans activation function of small t that requires an

large T cDNA interaction of the common and unique domains and affects the

es 105and 114 EIIF transcription factor. However, we have previously

deter-t,orthe small mined that trans activation bycoexpressed largeTand small t

constant total involves both the ATF and the EIIF transcription factors (18). it hours after This suggests that trans activation of the E2A promoter by

e

A.

(B) Cells

large

T involves ATF and could involve EIIF. To determine

or 10jlgof the which of the transcription factors that

regulate

the E2A

loyed, and the promoter are targeted by large T and which domains of

large

t+ t/u) were Tare responsible for activating these transcription factors,we

small t unique employed CAT plasmids containing mutations in the ATF or

concentration the EIIF site of the E2A promoter (18). As shown in Fig. 4,

CAT activity was stimulated upon cotransfection ofa plasmid

encoding only the large T unique domain (T/u) with the

plasmid containing mutations in the ATF site. Thus, the

rge T unique common domain is notrequired for activation of EIIF bylarge

trans activate T, whereas the common domain is required for activation of

EIIF by small t. As discussed above, trans activation of an

,ation by the EIIF-dependent promoter by the large T unique domain is not

appears to be unexpected, because the binding site forEIIF-associated

pro-erincreasing teins maps within the large Tunique domain. However, trans

iiquedomain activation of the EIIF-dependent promoter by the large T

centration of mutant (T/K1) that does not bind RB, p107, and related

,ientto result proteins is only slightly reduced when compared with that of

ion ofthe t/u wild-type large T, suggesting that additionaldomainsof large T

Adlabb,

IP

4

lie

*,

4.- 0 Z)

4.- co_I Z)

v- -A-

1-

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pE2dIATF pE2dIEIIF

FIG. 4. CAT activity of E2 promoter mutants. Cells were

trans-fected as described in the legend to Fig. 3, except that the CAT plasmids employed contained mutationsatthe ATF site(and hence containedwild-typeEIIFsites)orthe EIIF sites(andhence contained

awild-type ATF site). Extracts ofcells transfected withpE2 dlATF

wereassayed for 2 h, and extracts of cells transfected with pE2dlEIIF

were assayed for4 h.

outside theRB/plO7-binding pocket participateinactivation of EIIF-dependenttranscription.

Study oftrans activation of the E2A promoter containing only the ATF site (pE2

dlEIIF)

isdifficult because of theweak

stimulation of the ATF site. Whereas activation of the E2A

promoter containing only the EIIF sites by EIA, largeT, or

smallt is

consistently

10-to20-fold

(Fig.

4) (18), activation of

thepromotercontaining only the ATF site is usuallynot more

than 5-fold

(18).

In the

experiment

shown in Fig. 4,

cotrans-fection ofplasmids encoding EIAorlargeT stimulated CAT

activity

from pE2 dlEIIF

eight-

and sevenfold, respectively.

Thecommon domain appears toberequired for activation of

the ATF

site,

since cotransfection of the

plasmid

encoding the

large

T

unique

domain did not stimulate CAT activity from pE2 dlEIIEF. Interestingly, cotransfection with the plasmid encoding largeTwithamutationintheRB-binding site (T/K1) stimulated CAT

activity only

3.8-fold

(about

halfaseffectively

as wild-type large

T),

suggesting that the same largeT struc-ture that is involved in activation of EIIF is also involved in

activation ofATF. Aspreviously reported (16),small tdidnot transactivateapromotercontaininganATFsite(only twofold activationwas observed in the experiment shown, which is in

the range of

experimental

variation normally observed).

The results of these experiments suggest that there are at

leastfour trans-activation functions encoded in the SV40 early

genes that can be detected by assaying gene expression

di-rected by the Ad E2A promoter. There is one function

encodedby smalltthat

requires

both thecommonandunique

domains and involves the EIIF transcription factor. Unlike large T, Ad EIA, or human papillomavirus E7, which are

thought

toactivate

EIIF-dependent transcription

by bindingto RB or a

p107-cyclin

A complex and thereby releasing free,

activeEIIF

(2,

3, 7, 8,

23),

small tdoesnotbindtoanyknown

EIIF-associated factors. Thus, small t must employ a

mecha-nism distinct from that oflargeT toactivate EIIF-dependent

transcription.

It is conceivable that trans activation bysmall t occursupon

binding

toand

inhibiting

the cellularenzyme, type 2Aphosphatase (PP2A) (24, 28, 33, 35), perhaps by

suppress-ing the dephosphorylation of cellular factors that associate

withEIIFonlyin theirunderphosphorylated state. Ithas been

suggested

that both the common and the small t unique domains

participate

in PP2A

binding

(25). Our results,which

indicate that these domainsarealsorequired fortrans

activa-tion,

areconsistent with amodel inwhich both of the small t domains are

required

to bind to and inhibit PP2A activity,

which results instimulation ofEIIF-dependent transcription.

Although

it has been difficult to

study

the

linkage

between

PP2A binding and trans activation because many small t

mutants are unstable (4, 14), study of stable small t mutants

will benecessary to test thismodel.

There appear to be at least three separate functions encoded

inlargeTthatwe candetectduringtransactivation of theE2A

promoter. One of these functions involves theATF

transcrip-tionfactor andrequiresthe common domain in addition to the

large T unique domain. This suggests either that a factor(s)

involved in activation of ATF binds to large T within a

structureformedby boththe commonand theuniquedomains

orthat the commondomaindirects a conformation of the large

T unique domain which activates ATF. Since activation of ATF-dependent transcription by large T is reduced upon

mutation of the RB/plO7-binding site within the unique

do-main, it is possible that large Tforms a structure inwhich a

factor(s) that regulates ATF contacts both the common

do-main and the RB-binding pocket. Very little is known about

regulation of ATF-dependent transcription to explain how it might be activated in response to large T. Since ATF is a

member of aleucinezipper family of proteins including cyclic

AMPresponseelement-binding (CREB) proteins whose

activ-ities are regulated by phosphorylation, it is possible that the

phosphorylated state of ATF affects its ability to direct

tran-scription. Phosphorylation of bacterially expressed ATF by

microtubule-associated kinase stimulates DNA binding by

ATF (1). It is possible that large T directly or indirectly regulatesakinase activity that phosphorylates ATF.

With regard to the EIIF sites, the common domain is not

required for trans activation by large T. Mutation of the

RB-p107-binding site reduced trans activation by large T but

didnoteliminate it. Since theKI mutantoflargeTappears to

be completely defective for binding ofRB, pIO7, and related proteins (9, 11, 15a), thissuggeststhat there are at least two waysforlargeT toactivate EIIF-dependenttranscription,one

of which involves the RB/p107 pocket and one of which involveslarge T structuresoutside the

RB/p1O7

pocket. EIA has been found to induce the phosphorylation of the RB

protein independently of its association with RB (34). Coex-pression of SV40 large T and small t also stimulated phos-phorylation of RB. Since EIIF appears to complex with

underphosphorylated formsof RB (7), large Tcould conceiv-ably activate EIIF-dependent transcription without bindingto RBby activating an RBkinase.

Itisinterestingto notethat when Zhu et al.tested the effects

ofseveral mutations in largeT on transactivationofthe RSV

andSV40 latepromoters,they also observedthat structures in

the commondomain and structureswithin the large Tunique

domain involved in DNAbindingorzinc bindingwere

neces-sary for optimal trans activation (36). However, the

RB-binding site was not necessary for trans activation of these

promoters. Thus, trans activation of the RSV and SV40 late

promotersby largeTmayinvolvesome common mechanisms

and some mechanisms distinct from those involved in trans

activation of the E2A promoter. Consistent with our results,

Srinivasanetal. foundthat atruncatedlarge Tcontainingthe

amino-terminal 121 residues, which retained the binding site

for RB and

p107,

could transactivate the E2A promoter (30).

Nevertheless, this mutant did not trans activate the E2A

promoter aseffectivelyasdidwild-type largeT.However,since

thismutant also lost its nuclear translocation signal, it is not

clear from their experiments whether the decrease in trans

activation indicates that structures carboxy terminal to the

RB/plO7-binding site are involved in trans activation of the

E2A promoter or whether translocation of large T to the

nucleusis requiredforoptimaltrans activation.

Significance of multiple activation processes encoded by

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large T and small t. It is not clear why there are multiple

pathways by which

SV40

early gene productsactivate the Ad

E2A

promoter.

Several viruses, including pseudorabies virus

(13),

SV40

(19), polyomavirus (16), and papillomavirus (26)

encode proteins that trans activate the E2A promoter. Thus,

even though there are no ATF- or EIIF-binding sites within

the

SV40

genome, productive SV40 infection may require

activationof a

cellular

process, perhaps transcription of

cellu-lar genes containingATFandEIIFsites. These events may be

central to the subversion of host cell processes that lead to

cellular DNA synthesis during productive viral infection (see

reference 12 for a review). It is possiblethat several

indepen-dent pathways leading to activation of ATF- or

EIIF-depen-dent transcription must occur. By encoding large T and small

tproteinsthat each can interact with distinct pathways leading

to activation of EIIF- and ATF-dependent

transcription,

the

virus ensures that all essential pathways are active. Alterna-tively, there may be different pools of large T and small t that form complexes with various cellular proteins, each of which performs a specific function during regulation of viral

tran-scription,

DNA synthesis, and activation ofcellular processes

needed for viral assembly. Not allof these complexes may be

present simultaneously, but they each may form at different

times within the cell or lytic cycle. Further study of the

mechanisms of transcription regulation by small t and by large

T with structures outside the RB/p107pocket will be necessary

and will advance our understanding of the diverse biochemical

processes by which the

EIIF

and ATF transcription factors can

be regulated.

I am grateful to Sunanda Babu and Jeannine Richard for expert

technical assistance; to James DeCaprio, William Kaelin, Robin

Bachelder,

and Maria Bennet for helpful comments; and to Alan Smith for critical reading of the manuscript. Lauren Sompayrac kindly

provided the pSRa.B and pSRetB-t/cDNA plasmids, and Ilan Bikel

provided the pRSV-T/u andpRSV-T/Kl plasmids.

This work was supported in part by grant MV-477 from the American Cancer Society and grant CA50599 from the National Cancer Institute.

REFERENCES

1. Abdel-Hafiz, H. A.-M., L. E. Heasley, J. M. Kyriakis, J. Avruch, D. J. Kroll,G. L. Johnson, and J. P. Hoeffler. 1992. Activating transcription factor-2 DNA-binding activity is stimulated by phos-phorylation catalyzed by p42 and p54 microtubule-associated protein kinases. Mol. Endocrinol. 6:20)79-2089.

2. Bagehi, S., R. Weinmann, and P. Raychaudhuri. 1991. The retinoblastoma protein copurifies with E2F-I, an EIA-regulated inhibitor of the transcription factor E2F. Cell 65:1063-1t)72.

3. Bandara, L. R., and N. B. La Thangue. 1991. Adenovirus E1A prevents the retinoblastomagene product from complexing with a cellular transcription factor. Nature (London) 351:494-497. 4. Bikel, I., and M. R. Loeken. Unpublished results.

5. Bikel, I., and M. R. Loeken. 1992. Involvement of simian virus40

(SV40) small t antigen in trans activation of SV40 early and late

promoters. J. Virol. 66:1489-1494.

6. Brady, J., J. B. Bolen, M. Radonovich, N. Salzman, and G. Khoury. 1984. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc. Natl. Acad. Sci. USA 81:2040-2044.

7. Chellappan, S. P., S. Hiebert, M. Mudryj, J. M. Horowitz, and J. R. Nevins. 1991. TheE2F transcription factor isacellulartarget for the RBprotein. Cell65:1053-10)61.

8. Chittenden, T., D. M. Livingston, and W.G.Kaelin, Jr. 1991. The

T/EIA-binding domain of theretinoblastomaproductcaninteract selectively with a sequence-specific DNA-binding protein. Cell 65:1073-1082.

9. DeCaprio, J. A., J. W. Ludlow, J. Figge, J.-Y. Shew, C. M.Huang, W.-H. Lee, E.Marsilio, E.Paucha,and D. M. Livingston. 1988. SV40 large tumor antigen forms a specific complex with the product oftheretinoblastoma susceptibilitygene.Cell 54:275-283. 10. Devoto, S. H., M. Mudryj,J. Pines, T.Hunter,andJ.R.Nevins. 1992. A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex.Cell68:167-176.

11. Ewen, M. E.,J. W. Ludlow, E. Marsilio, J. A. DeCaprio, K. C. Millikan,S. H.Cheng,E.Paucha,and D. M.Livingston. 1989.An N-terminal transformation-governing sequence of SV40 large T antigen contributesto thebindingofbothpllORb andasecond cellular protein, p12(). Cell58:257-267.

12. Fanning,E. 1992. Simian virus 40largeTantigen: thepuzzle,the pieces, and theemerging picture.J. Virol. 66:1289-1293. 13. Imperiale,M. J.,L.T.Feldman,and J. R.Nevins.1983. Activation

of gene expressionbyadenovirus andherpesvirusregulatorygenes acting in trans and by acis-acting adenovirus enhancer element. Cell 35:127-136.

14. Jog, P., B. Joshik,V.Dhamankar,M. J.Imperiale, J.Rutila,and K. Rundell. 1990. Mutational analysis of simian virus 40 small-t antigen. J. Virol.64:2895-2900.

15. Kalderon, D., and A. E. Smith. 1984. in vitro mutagenesis ofa

putativeDNAbindingdomain of SV40large-T.Virology 139:109-137.

15a.Livingston, D.Personal communication.

16. Loeken, M. R. 1992. Simianvirus 40 smalltantigentransactivates the adenovirusE2A promoterbyusing mechanisms distinct from those used byadenovirus EIA. J. Virol. 66:2551-2555.

17. Loeken, M. R., I. Bikel, D. M. Livingston, and J. Brady. 1988. trans-activation ofRNApolymerase II andIII promotersby SV40

small t antigen. Cell 55:1171-1177.

18. Loeken, M. R., and J. Brady. 1989. The adenovirus EIIA

en-hancer: analysis of regulatory sequences and changes in binding activity ofATFand EIIFfollowing adenovirus infection. J. Biol. Chem. 264:6572-6579.

19. Loeken, M. R., G.Khoury, and J.Brady. 1986. Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or EIA occursbydifferent mechanisms. Mol. Cell. Biol. 6:2020-2026. 20. Marsilio, E., S. H.Cheng,B.Schaffbausen,E. Paucha,and D. M.

Livingston. 1991. TheT/t commonregion of simian virus40large T antigen containsa distinct transformation-governing sequence. J. Virol. 65:5647-5652.

21. Montano, X., and D. P. Lane. 1984.Monoclonal antibodytosimian virus 40small t. J.Virol. 51:760-767.

22. Montano, X., R.Millikan, J. M.Milhaven, D. A.Newsome, J.W. Ludlow,A. K.Arthur,E.Fanning, I.Bikel,and D. M.Livingston. 1990. Simianvirus40smalltumorantigen andanamino-terminal domain of large tumor antigen share a common transforming function. Proc. Natl. Acad. Sci. USA 87:7448-7452.

23. Mudryj, M., S. H. Devoto, S. W.Hiebert,T.Hunter,J.Pines,and J. R. Nevins. 1991. Cell cycle regulation of the E2Ftranscription factor involvesan interaction with cyclinA. Cell 65:1243-1253. 24. Pallas, D. C., L. K. Shahrik, B. L.Martin,S.Jaspers,T. B.Miller,

D. L. Brautigan, and T. M. Roberts. 1990. Polyoma small and middleTantigens andSV40 smalltantigenform stablecomplexes with proteinphosphatase 2A.Cell 60:167-176.

25. Pallas,D. C.,W.Weller,S.Jaspers, T. B.Miller,W. S.Lane,and T. M.Roberts. 1992. Thethird subunit ofprotein phosphatase2A (PP2A),a55-kilodaltonproteinwhich isapparentlysubstituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A

subunits,bears little resemblance to Tantigens. J. Virol.

66:886-893.

26. Phelps,W. C., C. L. Yee, K.Munger,and P. M.Howley. 1988.The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similartothose ofadenovirus EIA. Cell 53:539-547.

27. Sanger,F., S.Nicklen,and A. R.Coulson. 1977. DNA sequencing

with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

28. Scheidtmann, K. H., M. C. Mumby, K. Rundell, and G. Walter. 1991. Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t

on November 9, 2019 by guest

http://jvi.asm.org/

antigen. Mol.Cell. Biol. 11:1996-2003.

29. Shirodkar, S., M. Ewen, J. A. DeCaprio, J. Morgan, D. M. Livingston,andT. Chittenden.1992.The transcriptionfactorE2F interacts with the retinoblastoma product and a p107-cyclin A

complexinacellcycle-regulatedmanner.Cell 68:157-166. 30. Srinivasan, A., K. W.C.Peden,andJ. M. Pipas. 1989.The large

tumor antigen of simian virus 40 encodes at least two distinct transformingfunctions. J. Virol.63:5459-5463.

31. Takebe, Y., M.Seiki,J.-I.Fujisawa, P.Hoy, K. Yokota, K.-I. Aria, M.Yoshida,and N.Arai. 1988. SRcx promoter: an efficient and

versatile mammalian cDNAexpression system composed of the simian virus40early promoterand the R-U5 segmentof human T-cellleukemia virustype1long terminalrepeat.Mol.Cell. Biol.

8:466-472.

32. Tooze, J. (ed.). 1981. DNAtumorviruses: molecular biologyof

tumor viruses. Cold Spring Harbor Laboratory, Cold Spring

Harbor, N.Y.

33. Walter, G., R. Ruediger, C. Slaughter, and M. Mumby. 1990.

Association of protein phosphatase 2Awith polyoma virus

me-dium tumorantigen. Proc. Natl. Acad. Sci. USA 87:2521-2525. 34. Wang, H.-G. H., G. Draetta, and E.Moran. 1991. EIA induces

phosphorylation of the retinoblastoma protein independently of direct physical association between the ElA and retinoblastoma products. Mol. Cell. Biol. 11:4253-4265.

35. Yang, S.-I.,R.L. Lickteig, R.Estes, K. Rundell,G. Walter, and M.C.Mumby. 1991. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol. Cell. Biol. 11:1988-1995.

36. Zhu, J., P. W. Rice, M. Chamberlain, and C. N. Cole. 1991. Mapping the transcriptional transactivation function of simian virus 40 large T antigen. J. Virol. 65:2778-2790.

on November 9, 2019 by guest

http://jvi.asm.org/