Simian virus 40 early mRNA's contain multiple 5' termini upstream and downstream from a Hogness-Goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large T antigen.

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0022-538X/81/100224-17$02.00/0

Simian Virus

40 Early

mRNA's Contain

Multiple

5' Termini

Upstream and Downstream from

a

Hogness-Goldberg

Sequence;

a

Shift in

5' Termini

During the Lytic Cycle Is

Mediated by Large T Antigen

PRABHAT K. GHOSH AND PAUL LEBOWITZ*

Department of InternalMedicine, Yale University School of Medicine, New Haven,Connecticut 06510

Received 16 April 1981/Accepted 15 June 1981

We have usedprimer-directed synthesis, separation, andsequencing ofcDNA's

toidentify andlocalize the 5' termini of simian virus40earlymRNA's. We have examinedpolyadenylated RNAs obtained from whole cytoplasm and polysomes oftwotransformed linesandfrom the cytoplasmofinfected cells early and late inthelytic cycle, andwehave attempted tocorrelate the resultsof our cDNA

analyses withrecentanalyses of early cap structures. We have found that early mRNA's fromtransformedcells havethree principal5'termini, atresidues5,150,

5,154, and 5,155, with terminal transcribed sequences of CU, GC, and GG,

respectively. These termini lie 21 to 26nucleotides downstream from the early Hogness-Goldbergsequence.Transformedcell early mRNA's also containaseries ofless abundant 5' termini that are copied from DNA sequences as faras 80

nucleotides downstreamand a minimum of70 to 75nucleotides upstreamfrom theHogness-Goldbergsequence. Thetemplatesfortheupstream5' termini and thelate simian virus40mRNA'soverlap byaminimum of60to 65nucleotides. Early mRNA's isolated from cells early in infection contain the same three

principal5' termini and downstreamminor5'termini astransformedcell mRNA's,

butthey lack 5' terminiupstreamfrom theHogness-Goldbergsequence.With the

onsetof thelatelyticphase, there isaprogressive decrease intheutilization of

the three principal 5' termini and additional downstream 5' termini and a

progressive increase in theutilization offourmajor terminiatresidues5,190 to

5,194, which are 10 to 15 nucleotides upstream from the Hogness-Goldberg

sequence.Thisshift is evident incellsinfected withatsAmutant atthe permissive

temperature,but is abortedby growthat orshift-upto arestrictivetemperature.

Thus, this shift is mediated by thegeneA product,large T antigen. Wepresent twomodels, whicharemutuallyexclusive,toaccountfor the role ofTantigenin

the early-late shift. One involves transcription late in infectionon anew DNA

template synthesized duringDNAreplication. The second involves inhibition of initiation ofearlytranscriptionatresidues5,150 to 5,155andother downstream sitesand ashift oftranscription initiationprincipallytotheupstreamsitesas a

resultofthe binding ofTantigento twositesonsimian virus 40 DNA downstream

from theHogness-Goldberg sequence.

Whereas

eucaryotic

cellular genes appear to

giverise to mRNA's withsingle5' termini (3, 7,

37, 52), certain animal cell viruses produce

mRNA's withheterogeneous5' ends(2,6, 12, 13,

15, 17, 19-21, 23-25, 28, 30, 36, 38, 39). The

papovaviruses, simianvirus40 (SV40) and

pol-yoma virus, are especially remarkable in this

regard. For SV40, late wild-type mRNA's

ter-minate at a minimum of five sites with the

AU(U) sequenceandasmany as 10 to 15

addi-tional sites with other terminal sequences (17,

39). The latemRNA'sof mutants with deletions

in the late leaderregion are evenmore diverse

(18, 36). Theearly mRNA'sofSV40have been

more difficult to study than the late mRNA's

becauseoftheirrelativescarcity,butrecentcap

analyseshavesuggestedsevendifferent terminal

capdinucleotides(21,25), andcDNAsequencing

studies have suggested major 5' ends at two

proximate sites and anumber ofminor

down-stream 5' ends in transformed cells (15, 38).

WhereasSV40andpolyomaviruslategenesdo

not contain Hogness-Goldberg sequences

up-streamfrom the 5' termini of mRNA's (17, 39),

224

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the earlymRNA's are preceded by such a se-quence (38). In addition, a recent study has

shown5'-terminalheterogeneity ofan

adenovi-rus-SV40 hybrid mRNA taking origin froman

adenovirus late promoter in adenovirus-SV40

hybrid (44); in this system, too, a

Hogness-Gold-bergsequence liesupstreamfromtheprincipal

5' terminus. Thus, the presence of a

Hogness-Goldbergsequence does notrestrict 5'

termina-tion to asinglesite,and the 5' terminal

hetero-geneityofthepapovavirus late mRNA'scannot beattributedto anabsence of thesesequences.

Furthermore, nospecific structural features of

the papovavirus or adenovirus genes which

wouldaccountforthe 5'terminal heterogeneity

havebeenrecognized.

Recent studies involving in vitro transcrip-tional systems (32, 42; Lebowitz and Ghosh, submittedforpublication) andincorporation of B8-3P-labeled nucleosidetriphosphates into viral mRNA's made bynuclei isolated from infected

cells (19) and made in permeabilized infected

cells (7a) have suggested that most, ifnot all, earlyand lateSV40 mRNA's ariseby transcrip-tion initiatranscrip-tion. Coupledwiththemultiplicityof 5' termini, this observation suggests that there

aremechanisms in thepapovaviruses that gov-ern initiation and regulation of transcription frommultiple sites.

We have been studying early and late SV40 transcription in vivoand invitro inaneffortto

gaininsightinto the mechanismsbywhich viral

transcription is initiated andregulated. We have beenespecially interestedinearly transcription

since aregulatory mechanism involving binding

of theearly proteinlargeTantigentothree loci

overlapping the template for the 5' termini of

theearlymRNA's (49) andsuppression of

tran-scription by this antigen (26, 41, 47) has been elucidated and since theearlygeneisnecessary

for induction andmaintenance of transformation

(1, 5, 33, 35,46) and information regarding

reg-ulation ofearlygeneexpressionmayberelevant

tocellular transformation.

Inthispaper we present adetailedstudy of5'

termini of the SV40 early mRNA's. As in our

previous studies (15, 38),wehave used primer-directed cDNA synthesis and sequencing for identification andlocalization of5' termini, but

we have incorporated a number of advances

which have permitted a more detailed analysis

thanpreviouslypossible,and wehaveexamined the early lytic mRNA's both early and late in

infection. Wehave also attempted tocorrelate

ourresults with those derived from recent cap

sequence studies(21,25). We havefound

consid-erably more heterogeneity in the 5' termini of

the early mRNA's in transformed cells than

previously reported, and we have found

compa-rable heterogeneity inthe early lytic mRNA's.

Inboth lytic infection and transformed cells,5'

termini are located upstream as well as

down-stream from the Hogness-Goldberg sequence.

Whereas the principal 5' termini early in lytic

infection lie downstream from the

Hogness-Goldberg sequence, as the lytic cycle progresses

into the late phase there is a decrease (always

relative, usually absolute) in the utilization of

these 5' termini, and four new termini lying

about 10 to 15 nucleotides upstream from the

Hogness-Goldberg sequence become the major

5' termini of the earlymRNA's. When infection

is carried out with a tsA mutant, thisearly-late

shift of 5' termini is blocked at the nonpermissive

temperature. Thus, this shift is mediated by

geneA product, largeTantigen.

MATERIALS AND METHODS

TheSV40-transformed human andmousefibroblast lines SV80and SV101 were generously provided by David LivingstonandRobert Pollack. The tempera-ture-sensitive mutanttsA58was agiftfromSherman Weissman. For lytic infections with wild-type SV40, confluentVero African green monkey cells were in-fected with 10to20PFU of strain776virus per cell. For certain preparations of early RNA, cytosine ara-binoside (ara-C)wasaddedtocultures1 h postinfec-tion at afinal concentration of20,tg/ml.Infections withtsA58werecarriedout onconfluent CV1cellsat

amultiplicityof10 to 20PFU per cellateither 33 or

40.5°C,orcellswereinfected for24hat one temper-ature,followed by eithershift-uporshift-down to the othertemperature. Thegrowth conditions for infected and transformed cells were as previously described (38), except that calfserumwassubstituted for fetal calfserum.

Cellswereharvestedatthe times indicated below. Cells were lysed, and cytoplasmic RNAs were ex-tractedaspreviouslydescribed(18).Polysomeswere obtained from whole cytoplasm by adding sodium

deoxycholateto afinal concentration of 15% and

cen-trifuging the preparation in 7.5 to 45% sucrose

gra-dients in0.01 M Tris-hydrochloride (pH 7.5)-0.1 M

NaCl-0.003MMgCl2inanSW27rotor at25,000 rpm

for225 min. Polyadenylated RNAs, whichwere

ob-tainedbypassageofpreparationsthrough

oligodeox-ythymidylic acidcellulosecolumns, wereused in all

experiments.

5'-Terminal sequences ofSV40 early RNAs were obtainedby the primer extension method, involving primer-directed, reverse transcriptase-catalyzed syn-thesis of[nP]DNAscomplementarytothe 5' ends of RNAsfollowed by determination of the3'ends ofthe cDNA's.Theprimer used forallcDNAsyntheseswas therestrictionfragmentextending from residues 5,053 to5,089ontheearlyDNAstrand(Fig. 1). This frag-ment was obtainedbycleavageof the viralDNA at residues5,053 and 5,089withrestriction enzymesHinfl

and HindIII, respectively. For certain experiments,

thisprimerwaslabeledonboth DNA strands. Inmost

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experiments a primer labeled only at the 5' position of the early strand (at residue 5,053) was used. This primer was obtained by first isolating theHinfl frag-mentextending from residues 5,053 to 1,660, labeling the 5' position ofboth strands, cleaving the labeled fragment with HindIII at residue 5,089, separating the tworesulting labeledfragments by gelelectrophoresis, and isolating the small fragment spanning residues 5,053through 5,089.Determinations of the 3' ends of cDNA's were done in thefollowing two ways: (i) by separation of cDNA's on2-mm (standard thickness)

8% polyacrylamide-7 M urea slab gels, followed by

sequencing of

in#iyidual

cDNA's (34), or (ii) by co-electrophoresis ofcDNA's on thin (0.3-mm) DNA sequencing gei of thesame compositionalong with Maxam-Gilbert digests of the DNA fragment extend-ingfrom residues 5,053 to190and also labeled on the early strand only atposition 5,053. This fragment was obtained from the residue 5,053toresidue 1,660 frag-mentlabeled at the 5'ends of both strands by digestion withPvuII and isolation of the fragment extending from residue 5,053toresidue190. The methods used forlabeling primers,hybridizingprimerstoRNA, iso-lating DNA-RNAhybrids, extending primers with re-verse transcriptase, and separating and sequencing cDNA's havebeen describedpreviously (16).

Nucleotidesarenumberedaccordingtoa

modifica-tion(M.Piatak,K. N.Subramanian,andS. M.

Weiss-man,J. Mol.Biol.,inpress) of the system ofReddyet al. (40). This modification involves thefollowing: num-bering from 1 through 77 to correspond tooriginal residues18through 94;numbering from78through94 toincludea17-basepair insertion (51) detected after theoriginal description of the SV40 sequence; reten-tion of the original numbering from residues 95 through 5,226; and numbering 5,227 through 5,243 to correspond to original residues 1 through 17. The corresponding numbering in the BBB system (50) is shown inFig.1.

RESULTS

Asnotedabove,wehave introduced

improve-ments into ouranalytical methods in an effort

to definein greater detail than previously

pos-sible (15, 38)the number andgenomiclocations

of the 5' termini of the early SV40 mRNA's. These improvements have included scaling up

of cellgrowthsand RNA preparations, the use

in certain experiments of primers for cDNA

syntheses labeled on only the coding strand, electrophoresis of cDNA's on thin polyacryl-amide-ureagelsthatwereabletoresolvesingle nucleotides, and autoradiographic exposure of cDNAseparationgelsforlongertimes thanused previously.When combined with

co-electropho-resis of a digested DNA fragment having the

same 5' terminus as the fragment used as the

cDNA synthetic primer, electrophoresis of

cDNA's on thingels permitteddirect and

accu-rateone-step determinations of their3'termini.

Early mRNA'sin transformed celis.

Fig-ure 2A showsthin gel electrophoretic patterns

of DNAs complementary to the 5' termini of polyadenylated early viral RNAs extracted from

thecytoplasm of the SV40-transformed cell lines

SV80 (human) and SV101 (mouse). In this

ex-periment cDNA's were synthesized with a primer labeled on both DNA strands. The

cDNApatternsforthe twolineswerecomplex,

B >>

A

-u-.

s

_m3

_a

.c

d.

... ..:

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d e f

FIG. 2. (A) Patterns on 8%polyacrylamide-7 M

ureathinelectrophoretic gels ofradiolabeled DNAs

complementary to 5' ends of polyadenylated viral

earlyRNAs isolatedfromwholecytoplasm ofSV80

(lanea) andSVIO (lane b)transformedcells,infected

Vero cells treated with ara-C (20

lsg/ml)

and har-vestedat24hpostinfection(lane c), andinfectidVero cells harvested in latelyticphaseat48hpostinfection

(ltne d). The resultsofcontrol cDNA syntheses on

polyadenylated cytoplasmic RNA from uninfected Verocells(lane e) and SV40 cRNA (lane e)arealso shown. The DNAs complementary to cRNA were electrophoresedonthegelshown inFig.6and were transposedfor comparison with the cDNA's of in vivo RNAs. Numbers in left and right margins and to rightof lane d identify specific cDNA's discussed in text.(B) Lightly exposedthin 8%polyacrylamide-7M urea electrophoretic gel patterns ofsamples of the indicatedcDNA'sfrom (A), which bringoutthe band-ing patternsofcDNAs 1 and 2. cDNA 1 is adoublet, and cDNA 2appearstobe asinglet.

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but theyappeared to beidentical. They

resem-bled the cDNA patterns described previously

(15, 38)for theSV80 cell line withrespect to the

twoprincipal cDNA's (cDNA's 1and 2), minor cDNA's3and 4, andcDNA's6 through 8, which

appearinthepresentexperimentsas atriplet of

distinct cDNA's but appeared previously on

standard thickness gels as asingle broad band.

Because of their relative scarcities, cDNA's 5

and 9 through 20 were not visualized in our

previous studies. As described previously, cDNA's1 and2each accountedfor about40 to

45% of total cDNA's, whereas cDNA's6through

8 together accounted for about 5 to 8% ofthe

total. Theremaining cDNA'swereall minor and together accounted forat most5 to10% of total cDNA's. Identical cDNApatterns wereobtained by using the same primer labeled only on the coding strand (Fig. 3) and by using RNAs iso-lated from SV80 cell polysomes (Fig. 4). Thus, each of the cDNA's shown arises from

tran-scriptsof the viralearly DNA strand, and

tem-plate RNAsprobablyfunctionasmRNA's.

Inourpreviousstudies, cDNA's 1and2were

recovered in amounts adequate for sequencing by cleavages adjacent to guanine and cytosine residues only. Furthermore, on standard

thick-ness gels, there was no evidence that these cDNA'swerecomposed ofmorethanoneband.

Inthisstudywe haveanalyzed lightexposures

of thetransformed cell cDNA'sonthingelsand performed detailedsequenceanalysesof cDNAs

1 and2. Onlightly exposed thin gels (Fig. 2B), cDNA 1 uniformly had the appearance of a

doublet with components thatwereaboutequal in abundance and terminated at adjacent

ge-nomic sites, whereas cDNA 2 most frequently

appearedas asinglet(however, in certain

exper-iments, adoubletwas observed). Since certain cDNA'sreproduciblyremainedsingletsonthin

gels (Fig.2A, cDNA 9, andFig.2B,cDNA2;see

Fig.5and6) and since mRNA's with5' termini

at adjacent sites were detected previously by

methods which didnotinvolve cDNAsyntheses

(12), we believe that the cDNA 1 doublet, as

wellas othermultipletsdescribedbelow,

prob-ably reflect the presence of discrete in vivo mRNA's with 5' termini at adjacent genomic

loci rather thanpremature terminationof

tran-scription in the region justshort ofa true

ter-minus.OursequenceanalysesofcDNA's 1and

2 were performedboth by Maxam andGilbert

(34)degradations adjacenttoall four nucleotides (data not shown) and by co-electrophoresis of

cDNA's on a thin gel with a digested DNA

fragmenthaving the same5' terminus (Fig. 3).

Theformermethoddemonstrated 5'termini of

the two cDNA 1 components between residues

5,152 and 5,155 and the terminus of cDNA 2

between residues 5,148 and 5,150. The results with thelatter method appearedtobedefinitive, showingtermini of the cDNA 1components at

residues 5,154 and 5,155 and the terminus of cDNA2 atresidue5,150.

Recently, Haegeman and Fiers (21) and Ka-hanaetal. (25) have studied the5'-terminalcaps

of theSV40 early mRNA's. Both of thesegroups

have analyzed primarily mRNA's produced in cells infected with tsAmutants; however, Haege-man and Fiers have cited similar results for mRNA's obtained from SV80cells, and Kahana

et al. have reported comparable results for mRNA'sproduced byaline ofpermissive cells transforined by UV-treated SV40 and by

wild-type virus in early lytic infection. In certain

respects the results of these two groups are

similar, whereas in others they differ. Both

groupshave found relatively abundant termini

with the transcribed 5' -* 3'sequences GCand

GG. Haegeman and Fiers have also found abun-danttermini with thesequencesAUU and GA (both of which Kahanaetal. havenotfoundas

abundant components), whereas Kahanaet al. have demonstrated CU as the most abundant terminus and AG and GU as additional rela-tively abundant termini (Haegeman and Fiers have notfound these three sequencesincaps).

Thereasonsfor thedisparateresults of thetwo

groups are notclear, and the disparities hinder

attempts to assign 5' termini to certain ofour

cDNA'sonthebasis of combinedcapand primer

extension analyses. Nevertheless, the 5' -. 3'

sequencein earlySV40 mRNA's from residues

5,157 through 5,147 is UCGGCCUCUGA, and

onthe basis ofourcombinedanalyses itseems

virtually certain that two abundant early

mRNA'sstartat residues5,155 and5,154with

the sequences GG and GC, whereas another

abundantmRNA starts atresidue5,150withthe

sequenceCU (Fig.1). These termini lie21to26

nucleotides downstream from the early Hog-ness-Goldbergsequence.

Of theminorcDNA's shorter than cDNA's 1

and 2, cDNA 3 is adoublet, cDNA's 4 and 5 are

singletsor have single majorcomponents, and

cDNA's6through8 aresinglets. We also

local-ized the 3' termini of these cDNA's both by

Maxam-Gilbert sequencing (data not shown)

andbyco-electrophoresis withadigestedDNA

fragment havingthe same 5'terminus (Fig. 3).

These twoanalysesgave consistentresults

sug-gesting that the 3' terminilie at or within one

nucleotide of residues 5,145 and 5,146(cDNA 3),

5,140(cDNA 4),5,134 and 5,135 (cDNA 5),and

5,125through5,123(cDNAs 6through8,

respec-tively) (Fig. 1).

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5' SV40 EARLY mRNA's 229 With the exception of cDNA 10, which

ap-g,nm pearedtobeadoublet in theelectrophoresisof

a

OCz° SV101 cell cDNA's, it was

difficult

to determine

s_ro > ufrom Fig.> 2Aand3whether minortransformed

3 D C T G A u cellcDNA's 9 through 20 were singlets or

mul-_*NK&

1W

^qtiplets.

Each of these cDNA'swasalsosubjected

to Maxam-Gilbert sequenceanalysis (data not

shown). cDNA's 9 through 16 contained only SV40early strandsequences up totheir 3'

ter-mini. cDNA's 17 through 20 contained only SV40earlysequences up toresidues 15through

20,but sequencescould notbereadaccurately

* beyond this point; thus it isnotknown whether

these cDNA's terminate with viral or host

se-quences,and if theformer, how farupstream on

theviralgenometheir terminilie. Thesequence

studiesfurther revealed terminiatorwithin one ortwonucleotides of residues 5,160, 5,184 and

-16 5,185, 5,193 and 5,194, 5,206, 5,213, 5,222, 5,233

-15

through

5,237,

and 9

through

13 for cDNA's 9

through 16,respectively(Fig. 1).

-14 We have attempted to determine the

signifi--13 cance of minor cDNA's 3 through 20 in the

following threeways. (i) We triedtodetermine

-12 whether these cDNA's appeared constantly in

-t1 thereversetranscripts ofallin vivoearly RNAs

or specifically intranscripts of RNAs obtained

5182-89 A8--. - from only certain sources. The presence of a

l-10 cDNA which appeared amongthe transcripts of

certain RNAs butnotamongthetranscripts of othersorwhichappeared invarying quantities

amongthe transcripts ofearlyRNAs obtained

5l164,65 C2- -- 9 from various sources wouldhavesuggested

de-5160 C rivation froma discrete in vivo mRNA. On the

5154,55 C2 other hand, the presence of a cDNA which was

-2

uniformly

present in the reverse

transcripts

of

5150 G- .

al

earlyRNAs would have been consistent with

14P;

-- 3 either derivationfromadiscrete mRNApresent

*k.

5139,40 G

-_-5135

TK

-...

5134 Cl

5130 A

--C2

---:5125T

C2-_

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FIG. 3. Determinationofthe3'endsofDNAs

com-plementaryto5'endsof earlymRNA's by co-electro-phoresis ofcDNA's witha digestedDNAfragment havingthesame5'endasthecDNA's.cDNA'swere

synthesizedoncytoplasmic polyadenylatedRNAs

ex-tractedfrom Vero cells infected for48h with wild-type(WT) SV40(lane 1)andmutantdl 892(lane 2) andfrom SV80 cells (lane 7); forcontrolpurposes,

cDNA'swerealsosynthesizedonRNApolymeraseII

invitrotranscripts of BglI-cleaved SV40DNA (lane

8). Theprimer forcDNAsynthesesand thedigested DNAfragmentwerebothlabeled with32Ponlyatthe 5' endoftheearlystrand,atposition5,053. The DNA fragmentwassubjectedtonucleotide-specific diges-tionsby the methodofMaxamandGilbert(34) (lanes 3through 6). Electrophoresiswas on athin 8% poly-acrylamide-7 Mureagel. cDNA's arenumberedat

therightin accord with thenumberinginFig. 2A.

The sequenceofthedigestedDNAfragmentshould be readfrombottomtotop, correspondingto a5'

-3' direction ontheearlyDNA strand. The relevant SV40 DNA sequence is shown inFig.1.BglIcleaves the SV40early strand between residues 5,159 and 5,160. Comigration ofacDNA synthesizedon tran-scripts of the BglI-cleaved DNA (arrow) with the

guanine-terminal band corresponding to residue

5,159ofthedigested DNAfragmentindicates initia-tion ofin vitro transcription from the end ofthe

fragmentand allowed directreading ofthe3'

termi-nus of each cDNAfrom the residue number ofthe comigratingbandofthedigestedDNAfragment. VOL. 40, 1981

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FIG. 4. Patternson8%opolyacrylamide-7 Murea

electrophoretic gels of radiolabeled DNAs comple-mentarytothe 5' ends ofpolyadenylated RNAs ob-tainedfrom the polysomes (P) and whole cytoplasm (C) Of SV80cells.

in vivo under a wide variety of conditions or

derivation by a constant artifact in our

proce-dure (e.g., premature termination of reverse

transcriptionordegradationof invivo RNAs at

specific sites). (ii) We compared the terminal

sequences of these cDNA's with the earlycap

sequencesmentioned above. (iii) Weperformed

control reverse transcriptions by using SV40

cRNA asatemplateand compared the gel

elec-trophoretic patterns of the cDNA's transcribed

oninvivo early RNAs and cRNA. SV40 cRNA

is transcribed solely from the early strand of

form I DNA with preferred initiation sites

dis-tant from the template for the 5' ends of the

early mRNA's and transcription through this

region (29, 53). Since procedural artifacts were

likely to affect reverse transcription of in

vivo-and invitro-synthesized mRNA's similarly, the

presence on electrophoresis gels of cDNA's

de-rivedfrom in vivo RNAs which had no

counter-partsamong thecDNA'stranscribed on cRNA

wouldhavesuggestedderivationfromintact in

vivomRNA's. On the other hand, cDNA's

tran-scribed on in vivo RNAs which comigrated with

cDNA's synthesized on cRNA had to be

sus-pected of arising artifactually. However, since

initiation oftranscription is not wholly specific

and can occur with low efficiency at a great

number of sites on the viral early strand (P.

Lebowitz, P. K. Ghosh, and S. M. Weissman,

unpublished data), comigration ofcDNA's

de-rived from in vivo RNAs and cRNA could also reflect initiation of transcription from identical

sitesunder in vivo and invitro conditions.

We didnotobserve cDNA'sthatcomigrated

with in vivocDNA's 5, 9, and 11 through20in

any of our cRNA experiments. In addition,

cDNA's 5, 9, and 11 through 16 terminate at

sites with sequencesidentifiedinearlycaps(21,

25) andarealso present inreversetranscriptsof

RNA polymerase II transcripts of SV40 DNA

(Lebowitz and Ghosh, submitted for

publica-tion). Thus, it is likely that these cDNA's are

derived from intactearlymRNA's.Further

sup-portfor the significance ofRNAsgiving riseto

cDNA's5and11 comesfrom thefact that cDNA

11isusuallythemostabundant cDNA inreverse

transcripts ofearly RNAsisolated late inlytic

infection and cDNA 5 isnotpresent inreverse

transcripts of early mRNA's produced by the

origin-defective mutants 6-1, 6-17, and 8-4,

al-though these mRNA's do have termini at up-streamsites (14) (Fig. 1).

Inthree cRNAexperimentswedetectedonly

an extremely weak band that comigratedwith

cDNA 10, whereas intwoanalysesweobserved

weak bands thatcomigratedwith cDNA's8and

10andbarelydetectable bands thatcomigrated

with cDNA's1through4and,possibly,cDNA's

6and7(Fig. 2A). However,whereas the cDNA

patternsof transformed cell RNAs shown inFig. 2A wereobtainedbyhybridizingtranscripts

con-tainingapproximately0.05jgof viral RNA with

primer and exposing the cDNA separation gel

for 2 days, the cDNA pattern of cRNA was

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obtained by hybridizingreversetranscripts

con-taining 10 ytgof>16S cRNA with primer

con-tainingapproximately thesame amountof total

radiolabel and the same specific activity and

exposing thegel for2 weeks. Given the

approx-imately 1,000-fold excess of template cRNA and

exposuretime used in this andourotherstudies

with cRNA, the presence of weak cDNA's of

cRNA comigrating with certain cDNA's of in

vivo RNAs has minimal significance.

Further-more, the presence of weak cDNA's of cRNA

comigrating with cDNA's 1and 2 copied on in vivo early RNAs, taken together with the

evi-dencethat these cDNA's are derived from intact

early mRNA's (together, they constitute 80 to

90% of the total cDNA, their termini lie the expected distance downstream from the early Hogness-Goldberg sequence, their termini

cor-relatewell with the available sequences ofthe

SV40earlycaps,and directsequenceanalysis of theearly SV40 RNAs has revealedone ormore

termini in the region of the termini of cDNA's1

and2[9]),stronglysuggeststhatearly transcrip-tion with Escherichia coli RNA polymerase

maybe initiated extremely inefficientlyat

cer-tain sitesof in vivo initiation. Thus, webelieve that cDNA's 1 through 4, 6 through8, and 10 arealso all derived from in vivo RNAs andnot

from a systematic methodological artifact.

In-deed, thefollowingobservationsprovidefurther evidence that the 3' termini of these cDNA's mark the 5' termini ofspecific intact mRNA's. (i) Exceptfor cDNA's4and10,all terminate in RNA's withsequencesfound inearlycapsfrom transformed cells (21, 25). However, under

cer-tain conditions cDNA 10 becomes aprominent cDNAlate in lytic infections (see below), and

capscorrespondingtothe termini of cDNA's4

and10mayhaveescaped detection duetotheir uniqueness and scarcity. (ii) With regard to

cDNA3, twoorigin-defective mutants(mutants

6-1and 6-17) utilize residues5,145and5,146(the termini of cDNA3components)asthe 5' termini

of their principal early mRNA's; on the other hand, theearlymRNA's of anothermutant

(mu-tant 8-4) hasno detectable 5' termini atthese sites, although principal termini lie just

up-stream (14) (Fig. 1). (iii)WithrespecttocDNA

4, mutant 6-17 synthesizes an mRNA with a

terminusatresidue 5,140, whereasmutants8-4

and 6-1 withmajorupstream 5' termini do not

(14) (Fig. 1). (iv) The relative quantities of

cDNA's6 through8varyconsiderably

depend-ing upon the source of in vivo RNA; whereas

cDNA's derived from transformed cell RNAs

demonstrate a relative abundance of cDNA 6

comparedwith cDNA's 7 and 8, cDNA's ofearly

RNAsproducedlate in thelytic cycle

reproduc-ibly demonstrateaprominentcDNA 8,but very

weakor nocDNA's6and7(Fig. 2A).Inaddition,

RNAs extracted from cells transformed by cer-tain ofthe origin-defective mutants yield rela-tively prominent cDNA's 7 and 8, but barely detectable cDNA6 (14) (Fig. 1).

In summary,the evidencecited above suggests

amultiplicity of 5' termini of early mRNA'sin

SV40-transformed cells. It now appears that there arethree principal5' termnini (at residues

5,150, 5,154,and5,155),several minor 5' termini

at downstream sites, and a multiplicity of 5' terminiatupstreamsites. Most ofthelatterlie

upstream from the early Hogness-Goldberg

se-quence atresidues 5,176through5,181 (Fig. 1),

within the genomic region coding for the late mRNA leaders. One 5' terminus lies 70 to 75

nucleotides upstream from the

Hogness-Gold-bergsequenceand within10to15nucleotidesof

two 72-base pair tandemly repeated sequences

(Fig. 1), and it is likely that the termini of cDNA's17through20liewithin these repetitive

sequences.

Early mRNA's inlytic infection. Most of

our experiments on the 5' termini of early

mRNA'sproduced

early

in the

lytic cycle

were

performed with RNAs isolated from infected cellsgrownin thepresence ofara-C. The elec-trophoreticpattern of cDNA's copiedonthe 5' termini ofearlyRNAsfrom ara-C-treated cells (Fig. 2A) was similartothatof cDNA's copied

on transformed cell RNAs with respect to the twomajor cDNA's(cDNA's1and2) and minor downstream cDNA's3 through8. Asfor

trans-forned cell cDNA's, in mostexperiments early

lytic cDNA 1 was composed of two adjacent

bands, whereas cDNA 2 appeared as a singlet (Fig. 2B).Althoughbands thatcomigrated with transformed cell cDNA's9through11 were vis-ualized among the cDNA's of the early lytic

RNAs shown in Fig. 2A, the appearance of

cDNA 9 was variable, and cDNA's 10 and 11

were notvisualized whenaprimerlabeled only

on the early strand was used forreverse

tran-scription(Fig.5and6).(Indeed,the band inFig.

2A that comigrated with cDNA 11 is of host

originsince RNA isolatedfrom uninfected Vero

cellsuponreversetranscriptionwithprimer la-beled on both strands yielded a similar band

[Fig. 2A].) Thus,theearlymRNA'ssynthesized

in infectedcellsin thepresence of ara-C appear

to have the same principal and minor

down-stream5' endsastransformedcellmRNA's,but

theylackessentially all minor upstream 5'

ter-*mm.

Theelectrophoreticpatternof DNAs

comple-mentarytothe 5' ends ofearlyRNAsmade late

in thelytic cycle (Fig. 2A) differed greatlyfrom

http://jvi.asm.org/

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8

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FIG. 5. Patternson8%polyacrylamide-7Murea

thinelectrophoretic gelsof radiolabeled DNAs

com-plementary to the 5' termini of early lytic RNAs isolatedfrom wild-type(W.T.) SV40- andmutantdl 892-infectedVero cellsattheindicatedtimes postin-fection.Inbothwild-typeandmutantinfections,

ara-C(A.C.)wasaddedtoonecell cultureat1h

postin-fection, and the cells were harvested at 30 h. The numbersonthe right refertothe cDNA's shown in Fig.2A. UnnumberedcDNA's between cDNA's 9 and 10wereobserved in certainexperimentslate inlytic

infectionandmayreflectadditionalearly5' termini.

thepatternsof cDNA'scopiedonthe5'endsof

RNAs isolated from either transformedcellsor

[image:9.496.84.242.62.513.2]

infected cells treated with ara-C. Essentially

FIG. 6. Patternson8%polyacrylamide-7Murea

thinelectrophoretic gels of radiolabeled DNAs

com-plementarytothe 5'endsof early lyticRNAs isolated from CVI cellsinfected withmutanttsA58. Lanes 1

and3,Infectionsat33°C for48and24h, respectively; lanes 4 and6, infectionsat40.50Cfor48and 24h,

respectively; lane 2,infectionat33°C for24hfollowed by shift-up to 40.5°C for24 h; lane 5, infection at

40.5°C for24 hfollowed by shift-downto33°C for24 h; lane 7, control infectionat33°C for48 h in the

presence of ara-C. cDNA's synthesized on SV40

cRNA wereco-electrophoresedinlane 8. The

num-bersontheleft refertothe cDNA'sshown inFig.2A. Theunnumbered cDNA's betweencDNA's 9 and 10

were observed in certain experiments late in lytic infectionsandmayreflectadditionalearly5' termini.

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identical patterns were obtained with primers

labeled on both DNA strands or on the early strand alone.Comparedwith the pattern for

ara-C-treated cells, the pattern of DNAs

comple-mentary tothe5' endsofearlyRNAs madelate

ininfectionwas mostnoteworthy for its marked reductions in the relative quantitiesof cDNAs

1, 2, 6,and7,its increase in therelativeamount

of cDNA8,and theappearanceofanumberof cDNA's withterminiupstreamfromthe termini of cDNA's1and2.The latter cDNA'sincluded themajorspeciesobservedatthistime,cDNA's

10.5and11,moderatelyabundantcDNA's9and

10, faint cDNA's 12 through 16, and several cDNA's longer than cDNA 16 which did not

appear tocomigratewithanyof the transformed

cell cDNA's.In contrast to cDNA 1from

trans-formed cells and early in infection, late lytic

cDNA 1 was atriplet, whereas cDNA 2 was a

doublet. In addition, cDNA's 10.5 and 11 were

bothdoublets (Fig. 7A). Weperformed

nucleo-tidesequenceanalysesoncDNA's 1through16

and confirmed that all of these cDNA's were

composed of viral early sequences. We also found that the principal cDNA's, cDNA's 10.5

and11,hadterminiat orwithinonenucleotide of residues 5,190 and 5,191 and residues 5,193

and 5,194, respectively (Fig. 7B), whereas the

terminiof the remaining cDNA'swerethesame as the termini for the comigrating cDNAs of transformed cell mRNA's. The specificity of

cDNA 10.5for lateinfection, the prominence of

cDNA11atthistime, the absence of thesetwo

cDNA's in reverse transcripts of cRNA (Fig. 2A), and the prominence of these twocDNA's

in reverse transcripts of RNA polymerase II

transcripts of SV40 DNA (Lebowitz andGhosh,

submitted forpublication) suggestthat the ter-miniof cDNA's10.5and11reflect the 5' termini of specific early mRNA's. Furthermore, caps

with the sequences AU, CU, and AC,

corre-spondingtoterminiatresidues5,190, 5,193, and

5,194,respectively,arethemostabundantearly capslateinlytic infections (Y. Groner,personal communication), arguing that these upstream

termini donotarisefromprematuretermination

of reverse transcription (due to annealing of

earlymRNA's with late mRNA'sterminatingat

residue 5,189) but ratherrepresenttheprincipal

early 5' termini late in the lytic cycle. Thus,

there is striking heterogeneity of the early

mRNA's lateinthe lytic cycle, with four

prin-cipal 5' termini at residues 5,190, 5,191, 5,193,

and5,194 upstream fromtheHogness-Goldberg

sequenceand less abundantterminiconforming

tothe terminiof transformed cell mRNA'sboth

upstream and downstream from the

Hogness-Goldbergsequence (Fig. 1).

The very different cDNA patterns obtained

for mRNA's from ara-C-treatedcells and from cells in the latelytic phase suggested the

exist-enceofatleastapartial switch in the 5' termini used by the early mRNA's early and late in infection. To study this switchfurther,weasked the following three questions. Could the ara-C pattern be duplicated early ininfection in the absence of this agent? Does this switch occur

abruptlyat somespecific pointinthelytic cycle,

or does it occur gradually as the cycle

pro-gressed? And do viable mutants with lesions close tothe origin ofreplication also manifest thisswitch? We answeredthefirsttwoquestions

by examining DNAs complementary to the 5'

ends of RNAs thatwere isolated from infected cells harvestedatdifferent timesduring the lytic cycle (Fig. 5).At 16hpostinfection, the cDNA

patternwasidenticaltothepattern obtained for RNAs isolated fromcellsgrownin thepresence

ofara-C; cDNA's1and2constituted thebulk of the cDNA's, cDNA's 3 through 8 were minor constituents, and cDNA'swith termini at more

upstream locations were completely absent.

Thus, the cDNApattern obtained with RNAs from cells treated withara-Cwas not anartifact, butcharacteristic of theearly phaseof infection.

Atlatertimes cDNA's1through8first increased and thenleveled off in absolutequantity.

How-ever,themostsignificant changelate in infection

was the appearance and gradual increase of

cDNA's 9 through 16 (especially cDNA's 10, 10.5, and 11). In certain experiments the

amounts of cDNA's 1 through 8 actually

de-creasedinabsolutetermsduring lateinfections,

whereas the amounts of cDNA's9 through 11

increased. Indeed, in certain experiments, the ratios of cDNA's9through11 tocDNA's 1and

2reached5:1 to10:1late in infection(Fig.2and 7A). Thus, the late phase is characterized bya

relativeand, undersomeconditions,anabsolute decrease in earlymRNA's with termini atand downstream from residues 5,150through 5,155 and theappearance and thenabsolute increase

inmRNA's whichutilize terminiupstreamfrom residue 5,155(especiallyanincrease in mRNA's

withterminiatresidues5,190through 5,194).

dl 892 isaviablemutantofSV40which lacks

19DNAbasepairs,extendingfromresidue5,196

to residue 5,215 (45). Wechose this mutant to

studytheearly-lateshift in the5' terminiof the

early mRNA's for the following reasons: the

deletionabutsonthetemplatefor the principal

5'terminilateininfection and italsoremoves a

large part of the third (or lowest affinity) T-Thearrowsindicate two cDNA'sof cRNA, the

upper-mostof which appearedtocomigratewith cDNA 10. A darkerprint of the cDNA's of cRNA appears in Fig.2A.

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[image:11.496.148.390.64.459.2]

. 0

FIG. 7. (A) Patterns on 8%1vpolyacrylamide-7 M urea thin electrophoretic gels ofradiolabeled DNAs complementarytothe5'endsof early RNAs isolated fromwild-type(W.T.) SV40-infected Vero cells early and late in thelyticcycle. Cellswereinfectedasdescribed in thelegend to Fig.2.Theprimer for cDNA syntheses waslabeledonlyontheearly strand. The numbersontheright refer to cDNA's shown in Fig.2A. (B) Maxam-Gilbert (34) sequenceanalyses of cDNA's10.5and11complementary to 5'termini of early RNAs isolated from infected Vero cells late ininfection (see Fig. 2A). The residues are numbered as indicated in the legend to Fig.

1.Sequenceswerereadasdescribed inthelegendtoFig. 3.Asterisks markposition 5,127,where acytosine

residue appears in thecDNA.In cDNA's derivedfrom transformed cellRNAs,athymineresidue appearsin thisposition.

antigen binding site (49) (Fig. 1). Figure5shows

that the early mRNA's of dl 892 undergo the

sameshift in their 5' terminilate in infectionas

wild-typeSV40. Thus, deletion of sequences

im-mediately adjacenttothetemplate for the

prin-cipal 5' termini used late in infection does not

block the utilization of these termini at that

time. The normal early-late shift in dl892 also

indicates that the thirdT-antigen binding site is

notinvolved in this shift.

Mechanism of early-late shift of 5'

ter-mini of early lytic mRNA's. Since large T

antigen suppressesearlySV40transcriptionand

istheonly knownregulatorofearlytranscription

(26, 41, 47), it seemed likely that this antigen

might also mediate theearly-lateshift in the 5'

terminiof the early lyticmRNA's. Totestthis

hypothesis, we infected permissivecells with a

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mutantofSV40that synthesizeda

temperature-sensitive geneA orearlygeneproduct (tsA58),

andby using cDNA analyses weexamined the

5'termini of theearly mRNA's thatweremade by the mutant at the permissive temperature

(330C),

at the nonpermissive temperature

(40.5°C), andafter shifts fromonetemperature to the other (Fig. 6). Since the Vero line of Africangreenmonkeykidney cells used inmost

ofourstudies didnotgrowwellat40.5°C, these experiments were performed with CV1 cells,

which grewsatisfactorilyatthistemperature.

Upon reverse transcription, RNAs obtained

frommutant-infected cellsgrown at330Cfor24

and 48 hyielded cDNA's with the gel electro-phoreticpattern characteristic of the latephase oflytic infection. Growthat40.5°C followedby ashiftto330Cfor24halsoyieldedRNAs which

gavethetypical latelyticcDNApattern.On the otherhand, RNAs extracted from cellsgrownat or shifted up to 40.50C yielded cDNA's with a

pattern characteristic of the earlyphase of the lytic cycle. Thus, growth of the virus at the nonpermissive temperature freezes the 5' ter-miniof theearly mRNA'satgenomic sites char-acteristicof theearlyphase oflyticinfection and

does notpermitthemtotake onthe additional

5' terminiatupstreamsites characteristic ofthe latephase of thelytic cycle. Therefore,we

con-clude that thegeneAproduct, largeTantigen,

is involved in the shift of the 5' termini of the

earlylyticmRNA's late in the infectiouscycle.

DISCUSSION

We have reached the following four conclu-sions from the present study: there is striking

heterogeneityofthe 5' termini of theSV40early

mRNA's in both transformed and lytically in-fectedcells; 5' termini arelocatedupstream as

well as downstream from the early

Hogness-Goldberg sequence; there is a shift in the 5'

terminiof theearly lyticmRNA'sfromaseries of downstream sitesearlyinthelytic cycleto a

series ofupstreamsites late ininfection;and the

upstreamshift is mediatedbythegene A

prod-uct,largeTantigen.

5'-Terminalheterogeneity: correlation of

cap and cDNA terminal sequences. It has become clear over the past several years that

one of the characteristics ofpapovavirus early andlate transcriptionisheterogeneityof the5'

terminiof therespectivemRNA's (6, 12, 13, 15,

17, 19-21, 25, 28, 30, 38,39). For theSV40early

mRNA's,capstructureanalyseshavesuggested

that asmanyas sevenseparate di-or

trinucleo-tides lieadjacent toterminalcaps (21, 25),and

ourcDNAanalyses suggestedthreetofour

ma-jor termini and amultiplicityof minor termini

for both transformed cell and early lytic mRNA's. 5'-Terminal heterogeneity has also beenreportedrecently foranadenovirustype2

DNAbinding protein mRNA (2),other

adeno-virustype 2early mRNA's(23, 24), and a hybrid

adenovirus-SV40 mRNAoriginating froma

ma-jor adenovirus latepromoter in an

adenovirus-SV40hybrid (44).

Figure 1 summarizes our findings on the

lo-cations(plusorminusone or twonucleotides in certain cases) of the 5' termini of the early mRNA's of wild-type SV40 and a number of

origin-defectivemutants(14). Thesefindingsare

basednotonlyonlocationsof thetermini of the

cDNA'swe havestudied, but alsoonattempts to correlate these locations with the available information onearly caps (21, 25). Indeed, the

locations of the ternini of both majorandminor mRNA'swehave deduced from cDNA analyses

agreewell with the availablecapdataexceptfor

RNAs in transforned and infected cells

repre-sentedby cDNAs4and10.Thepossibility that

these RNAsareuncapped orthatthecaps are

too scarce to detect must be considered. One

discrepancy between our results and those of Kahana et al. (25) on the one hand and the results ofHaegeman and Fiers (21)onthe other

concernsthefrequency ofmRNA's with an AUU

terminus.Despite thepresenceofthis sequence atthe 5' termini ofanumber of early mRNA's, accordingtoouranalyses and those of Kahana

etal. these mRNA's are minor and donot

ac-countformorethanafewpercentoftotalearly

mRNA's. However, by sizing cDNA's reverse

transcribedon RNAsfromcells infected for 12

h with SV40 strain 776, Thompson et al. (47)

identifiedamajorearly 5' endatresidue 5,143,

where the RNA sequence is AUU. We do not

findanyterminusatthissite, and the basisfor

thisdiscrepancyhasnotbeendetermined.

Asnotedabove, theearlymRNA's from

trans-formed cells and from the early phase oflytic infectionappear to have threeprincipal5'

ter-minilocatedatresidues5,150, 5,154,and 5,155,

whereas theearly mRNA's from late lytic

infec-tionappear tohave four main terminilocatedat

residues 5,190, 5,191, 5,193, and 5,194. The

clus-tering of early 5' termini at adjacent sites is

reminiscent of the close clusteringof the 5'

ter-miniof certainlate polyoma virus(12) andSV40

(39) mRNA's.Recently,wehavefound that the

5' ends ofessentially all of the early mRNA's

described here arise by transcription initiation

(Lebowitz and Ghosh, submitted for publica-tion). Thus, clusteringof 5' ends suggests that

initiation of transcription may be directed

to-wardspecific genomic regions but that there is

flexibilityinthe number andselectionofspecific

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initiation sites within such regions.

One of the most interesting findings of our

study and that of Kahana et al. (25) is that

pyrimidines are present in the 5'-penultimate

position of certain SV40 early mRNA's. As noted

above, Kahanaet al. have found that the

prin-cipal earlycapcontains the transcribedsequence

CU, andwehavefound 5'termniniwith

pyrimi-dines in the penultimate position among the

early mRNA's of wild-type virus and certain

origin-defectivemutants(14) andamongthelate

mRNA's of several late leadermutantsofSV40

(Ghosh, Piatak, Barkan, Mertz, Weissman, and Lebowitz, manuscript in preparation).

Further-more,auridine-terminalcaphasbeenidentified

recently in an adenovirus type 2 mRNA (22).

Thus, 5' termini containing pyrimidines may

now be extended fromprocaryotic mRNA'sto

include certain animalvirus mRNA's. Whether

the relative abundance ofpyrimidine termini in

the early SV40 mRNA's and their absence or

virtual absence in the wild-type late mRNA's

have anysignificance withregard tothe

mech-anism by which the respective 5' termini are

formed(i.e., initiationoftranscription by

differ-entmechanisms) is unknown.

Span of the template for 5' termini and

generation of 5' terminii.We havelocated the

5' termini ofwild-type SV40 early mRNA's as

far downstream as residues 5,095to 5,097 (14)

andasfar upstreamasresidues 9to 13(Fig. 1).

In addition, we have identified cDNA's with

viral sequences extending further upstream,

probably into the pair of tandemrepeats

span-ning residues 25 through 96 and residues 97

through 168onthe viralgenome.However,we

cannot be certain how far upstream viral

se-quencesextend and whether they terminate in

virusorcovalently linked hostsequences.Thus,

thetemplatefor the 5' termini of thewild-type

early mRNA's spans at least 160 nucleotides

and,probably, considerablymore.Furthermore,

in certain origin-defective mutants, we have

identified 5' termini asfardownstreamas

resi-dues5,066to5,070 (14) (Fig. 1), indicating that

aregion ofatleast 190nucleotides iscapable of

servingasthetemplatefor the 5'termini of the

early mRNA's. In comparison, the SV40 late

mRNA'sarederived fromatemplate almost300

nucleotideslong (17). With the template for the

viral latemRNA'sextendingonthe late strand

to residue 5,189 or evenbeyond (17), many of

theupstreamearly 5' termini fall within the late

genomic region. Ourdatashowthat there isan

overlap ofat least 65 to 70 nucleotides in the

templates for the early and late mRNAs.

All SV40 early mRNA's with 5' termini

up-stream from the Hogness-Goldberg sequence

contain AUG initiationcodons neartheir5'

ter-mini.Although theyareinphasewithT-antigen

coding sequences, these AUGsare followed by

downstreamtermination signalsand thus cannot give rise to minor T antigens containing

lengthened amino termini. Whether the

up-stream AUG triplets interfere with the use of

the AUG atresidues 5,081 through 5,079

nor-mallyused for initiationof T antigen is presently

unknown. Of interest is the fact that the AUG triplet from residues5,178 through 5,176 is

fol-lowed by23aminoacid-codingtriplets andthen

a UAG terminator. A protein containing 62

amino acids encoded by the SV40 late leader region, designated the agnogene product, has

recently been identified (G. Khoury, personal

communication;J. Mertz,personal communica-tion), and itmaybe questioned whether these

23 triplets code for an anologous small early

protein. Furthermore, since the late protein

bindstonucleic acids(G. Khoury,personal

com-munication), it appearsreasonable tospeculate

that this protein and a possible early analog

functioninregulation of late and early transcrip-tions,respectively.

As Fig. 1 shows, the Hogness-Goldberg

se-quenceforearly transcription is located21 to 26

nucleotidesupstreamfrom the major 5' endsat

residues 5,150, 5,154, and 5,155. Although the Hogness-Goldberg sequence has been

consid-ered important in initiation oftranscription at

downstream sites, recent experiments, both in vivo (4) andinvitro (Lebowitz and Ghosh, sub-mitted for publication), have shown that this

sequence isnot essential for initiation of

tran-scription from these sites. Other sequences,

which arealmostcertainlyupstream, must

con-stitute theearly transcriptionalpromoter. Still, the Hogness-Goldberg sequence and possibly

certain adjacent sequences appear to have the

following twoimportant functions: theyplay a

crucial role infixing initiation oftranscriptiona

specificdistance(i.e.,21to26nucleotides)

down-stream from the Hogness-Goldberg sequence

(14), andtheyappear to beimportantin maxi-mizing theefficiency of transcription from

down-streamsites(8).

The factorsresponsibleforgeneratingthe

up-stream 5' termini of the early mRNA'sare not

known. Primerextension studiesof RNA

polym-eraseII in vitrotranscriptional productsofSV40

DNAhavesuggestedthat theterminiatresidues

5,184, 5,185, and 5,190 through 5,194 and sites

further upstream arisebytranscriptioninitiation

(Lebowitz and Ghosh, submitted for

publica-tion).Thepositionof theHogness-Goldberg

se-quence and thesurroundingsequences with

re-spect to the upstream 5' termini bears some

http://jvi.asm.org/

(14)

resemblancetotheposition of RNApolymerase

III promoters for 4S and 5S RNAs. In these

systems,thepromoters arelocated within

inter-nalportions of the genes downstream from the

5'termini ofthe RNAs (10, 27, 43).Whetherthe

Hogness-Goldberg sequence or adjacent

se-quences or both might act as a promoter or

portion ofapromoterfor theupstream5' termini of the SV40 earlymRNA'sin asimilarfashion is unknown. However, itseems morelikely that initiation from theupstream sites is underthe

control of a more upstream promoter, and it

seems possible that atleast a portion ofearly

transcription in vivo insystemslacking the Hog-ness-Goldberg sequence may be initiated at

these upstream sites. Based upon our findings that theearly5'terminiareheterogeneous, that

the most upstream termini lie within or just

downstream from thepair of72-basepair

tan-dem repeats mentioned above (residues 25

through 96 and 97 through 169 [Fig. 1]), and

thatmost,ifnotall,5'termini ariseby

transcrip-tioninitiation,ahypothesisfor initiation ofearly transcriptionatmultiplesitesmaybeproposed.

Inthis schemeweenvisionanobligatoryinitial interaction of DNA polymerase II with

se-quences at or upstreamfrom residues9through

13(themostupstream 5'termini whichwehave

identified) and thensomeform ofmovementof

the enzyme in a downstream direction, with

initiation of transcription at multiple specific sites thataredeterminedby either local

nucleo-tidesequences, melting of the DNAhelix,or a

combination of these factors. Of interest is the

factthat the two most upstreaminitiation sites

which we identified (at residues 9 through 13

and 5,231 through 5,235) lie at identical sites within a pair of 21-base pair tandem repeats

(from residue 1 toresidue 21and fromresidue

5,223 to residue 5,343), illustrating the

impor-tancethatlocal nucleotidesequencesmayhave

intranscription initiation. Dependingupon

spe-cificlocalfactors,initiationatspecificsitesmay varyfrom weaktoveryefficient.Inthisscheme,

theHogness-Goldbergsequence wouldserve as

suchalocal factorspecifying efficient initiation oftranscription from specific downstream sites.

The early SV40 Hogness-Goldberg sequence isunlikethat ofmost eucaryoticgenes studied

inthat it isflankedonits 5' side by another 11 adenine-thymine base pairs. It is conceivable

that initiation ofearly transcription occurs at

three major downstreamsites rather than at a

single site as a result of interaction of RNA

polymerase II with this elongated Hogness-Goldbergsequence.However,it seemsdoubtful

thattheelongatedsequenceplaysarole in het-erogeneous initiation from other genomic sites.

Early-late shift in5' termini. Perhaps the most significant finding ofour study from the

standpoint of regulation ofSV40 early

transcrip-tion is that there is a shift in the principal 5'

terminiused by the earlymRNA's fromresidues

5,150through5,155,downstreamfrom the

Hog-ness-Goldberg sequence, early in infection to

residues5,190through5,194,upstream from this

sequence,lateinthelytic cycle.Furthermore, in

studies with atsA mutant wehave found that this shift is mediated by large T antigen. In

agreementwith thelatterfinding,wehave also

foundthatthe shift canbeblocked, atleastin

part, byblocking protein synthesis with

cyclo-heximide (unpublished data). Sincemost,ifnot

all, early5'termini ariseby transcription initia-tion, the shiftmustbe mediatedatthislevel. We offer thefollowingtwohypothesestoexplain the role ofT antigen in the early-late shift. First, sinceTantigen is essential for the initiation of

DNAreplication and since the shift is blocked by the suppression ofreplication, it is possible

thata newDNAtemplate which isaproduct of

thereplicatoryeventisrequired for initiation of transcription at the upstream sites. Second,

sinceTantigen binds to two sitesdownstream fromtheHogness-Goldbergsequence(with high affinityto asitebetween residues5,103and5,130

[Fig. 1,site 1] and with moderate affinityto a

site between residues 5,150 and 5,175 [site 2] (49), itseemsplausible that transcription initia-tion from residue 5,150through5,155 andfrom other downstream sites decreasesasincreasing

quantities ofTantigenaccumulate and bindto

the downstreamtemplate sites and,as aresult,

that transcription is shifted to upstream sites,

where there is less interference by T antigen (Fig. 8). If the latter mechanism is operative, there wouldstillbe arequirement for initiation

ofDNAreplication, since theshift is blockedin

ara-C-treated cells. Indeed, transcription on a newtemplate and alterations in available initi-ation sites due to T-antigen binding to DNA

may both be involved in the early-late shift.

Furthernore, the absence ofindependent viral

DNAreplicationmayberesponsible for failure

of efficient utilization of the upstream loci as

initiation sites intransforned cells evenin the

presenceofanabundance ofTantigen.

Since early mRNA's produced by dl 892, which lacks most of the third T-antigen binding site(residues5,197through5,223[Fig.1]),

man-ifest the shift in 5' termini late in infection, it

appearslikelythatsequences within this siteare

not involved in the shift. Studies to elucidate

the mechanism of the shift further are in

prog-ress.

Since the most upstream 5' terminus of the

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238 GHOSH AND LEBOWITZ

TAg#3

Early Lytic Infection

LateLytic Infection

J. VIROL.

TAg #1

i -L

TAg#2

r, A

EmRNAs

~~~~~~~~5'

, E

ITAAATAI IPIP 1 IFiPi E

1 1 IIL

5194 5190 5181 5176 5155 5150 5125 5123

E

mRNAs

(D (9

51 (D OCL)(E00_0 _D 0 0 0 (D

BNITAAATApL

LmRNA 5155 5150 5125 5123 mRNt

5194 5190,89

E Strand

L Strand

E Strand

-L Strand

FIG. 8. Schematicdiagramproposingonepossible mechanism by which(i)principal sites for initiation of earlytranscriptionareshiftedfromsites downstreamfrom theHogness-Goldbergsequenceearly in infection tosites upstreamfrom this sequence late ininfectionand(ii)latetranscriptionfromresidue 5,189 is initiated asaconsequenceof theshiftinearly initiation sites. Details of this mechanism are presented in the text. The darkarrowsindicate theprincipal sites,andthe light arrows indicatesignificant minor sites of initiation of transcription. P, RNA polymerase II molecules; T, large-T-antigen (TAg) molecules. The residues are numberedasinFig.1.

late mRNA's thus faridentified lies atresidue

5,189 (17) and since the sequence at this site

(AUU)conformstothemostabundant latecap

ofSV40(6,20), itseemslikelythatonelocusfor initiation oflatetranscription liesatthis site. It

isstrikingthattheprincipal early5' terminilate

in infection at residues 5,190 through5,194 lie

adjacenttoresidue 5,189.Thus, it is also

tempt-ingtoproposethat thesame

T-antigen-depend-ent events which shift initiation ofearly

tran-scription to the sitesatresidues 5,190 through

5,194 late in infection are also responsible for

diversion ofpolymerase molecules tothe

adja-cent residue 5,189site, where they initiate late transcription (Fig.8).

We have indicated two positions where we

found altemative nucleotides in earlymRNA's obtained from SV40-infected and transformed cells (Fig. 1). Atposition 5,127, early mRNA's

from lytically infected cells contain a guanine

residue, whereas mRNA's from transformed

cells containanadenine residue (Fig. 5); and at

position 5,096,mRNA's frominfectedcells

con-tain anadenine residue,whereasmRNA's from

cellstransformedwithwild-typeSV40or

origin-defective mutants contain a guanine residue (14).Inthelattercase, the viral DNAs used for

infection and transformation have been shown

to contain a thymine residue on the coding

strand. The basis for theuseofalternative

nu-cleotidesatthesetwopositionsisunknown,but

it iscurrently beinginvestigated.

ACKNOWLEDGMENTS

We thankSherman Weissman for helpful advice and criti-calreading of the manuscript and Charlene Ivory for excellent technical assistance.

This researchwassupportedbyAmericanCancerSociety grant1N-31-T7, Public Health Service grant 16038 from the National Institutes of Health, and grants from the Leonard Eckstein Fund forLeukema Research and theSwebilius Trust ofthe YaleComprehensiveCancerCenter.

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