Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

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0022-538X/83/010408-12$02.00/0

Expression of Adenovirus Type 12 ElA Gene in Monkey

Cells, Using

a

Simian Virus 40

Vector

KINICHIRO ODA,* HIROYUKI KATO, IZUMU SAITO,t SUMIO SUGANO, KAZUO MARUYAMA,

MIYUKIMASUDA, KAZUKO SHIROKI, ANDHIROTO SHIMOJO

DepartmentofTumor VirusResearch, InstituteofMedical Science, UniversityofTokyo,4-6-1, Shirokanedai,

Minato-ku, Tokyo 108,Japan

Received 24 June 1982/Accepted 27 September 1982

Simian virus 40(SV40) recombinants carrying the adenovirustype12ElAgene were constructed. The SV40 expression vector was constructed by removing mostof the VP1geneandaninternalpartof the interveningsequencefor late 16S

RNAand by joining the 5' and 3' splice sites intoasmallsegment.The adenovirus

type 12 ElA gene with or without its own promoter was inserted downstream

from the SV40 latepromoterand the splicing junctions. The recominant DNAwas

propagated and packaged in monkey cells by cotransfection with an early

temperature-sensitive mutant(tsA58)DNAashelper. Immunofluorescent staining

of themonkey cells infected with the resulting virus stocks showed thatupto20% of the cellsoverproduced the ElAgeneproducts in the nuclei. Two-dimensional

gel electrophoresis of the products indicated that the products werevery similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The ElA mRNAwasinitiated atthe SV40 latepromoterirrespective of thepresenceof the EIApromoterand terminatedat

either the ElA orthe SV40polyadenylation signal. These hybrid mRNAs were

correctly spliced in the ElAcoding region.

The early adenovirus genes ElA and E1B, located in the leftmost11% of the viralgenome, are involved in both the transformation of sus-ceptible cells in culture andthe lytic growth of virus in permissive cells (34). The ElA gene alonecantransformthecells but isnotsufficient to completely transform them (11, 30). During lytic infection, the ElA gene is expressed first (21) and promotes the accumulation of tran-scripts from theotherearlygenes,E1B, E2, E3, and E4 (pre-early function), since the amounts ofthesetranscripts aregreatly reduced in cells infected with host range and deletion mutants defective inthe ElAgene (1, 12). Two alterna-tive mechanisms have been proposed for this accumulation ofearly transcripts, one acts at a transcriptional level (20) and the other at a posttranscriptional level (25).

The ElA gene encodes several related pro-teinsby synthesizing multiple speciesof mRNA which receive differentpatterns of RNAsplicing (8, 27, 29). In the case of adenovirus type 12

(Adl2),

a clusterof severalproteinswith molec-ular weights of 35,000 to 40,000 have been identified (29). These proteins werelocalized in thenuclei of infectedandtransformed cells and were designated as T antigen g based on their

tPresentaddress: Department of Microbiology,Facultyof

Medicine, UniversityofTokyo, Tokyo113, Japan.

granular appearance revealed by immunofluo-rescence. In spite of the interesting multiple functions ofthe ElAgene, no definitebiological function of the ElA gene products has been characterized in vitro, mainly due to a low amountoftheproductsproducedin adenovirus-infected cells.

Simian virus 40 (SV40) has been used as a

vectorforcloningandforexpression ofavariety ofeucaryotic genes in monkey cells (3, 5-7, 9, 15, 18, 24, 32), owing to its simple, well-ana-lyzed genomic structure. In the present study, SV40recombinantscarryingtheAd12 ElA gene wereconstructedtohyperproducethe ElA gene products.The SV40expression vector was con-structed by removing almost all of the coding sequence of the major capsid protein VP1 and most ofthe intervening sequence for late 16S RNAandby joiningthe 5' and 3'splicesitesinto a small segment. By usingthis vector, DNA of up to about 1,900 basepairs (bp)canbeinserted, irrespective of the presence of its own splicing junctions. The Adl2 ElA gene inserted in this vector was efficiently expressed in monkey cells.

MATERIALS ANDMETHODS

Cells and viruses. Established cell lines of African green monkey kidney, CV1 and GC7 (36), and KB cellswerecultivatedinEaglemedium with10%calfor

408

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SV40 RECOMBINANTS CARRYING Adl2 ElA GENE 409

fetal calfserum. Adominant-selection vector,

pSV2-gpt(16, 17) was kindly provided by Paul Berg,

Stan-ford University, StanStan-ford, Calif. 3YgElAis a cell line

established after transfectionof a clonal line of Fischer

rat embryo fibroblast 3Y1-K with pSV2-gpt-EIA

DNA.pSV2-gpt-ElA wasconstructed by ligation of

the leftmost 4.5% ofthe Adl2 DNA with the large

EcoRI-BamHI fragment from pSV2-gpt by using

EcoRI and BamHI linkers. Adl2 (Huie strain) and

SV40 early temperature-sensitive mutanttsA58 were

propagated and titrated in human embryo kidney

(HEK)and CV1 cells, respectively.MutanttsA58 was

kindly providedby Peter Tegtmeyer, State University

ofNewYork at Stony Brook.

Constructionof recombinant viruses.Theprocedure

for the construction of the recombinant viruses is

diagrammed in Fig. 1.

(i) Construction of anSV40 DNA segment containing thesplice sitesfor late 16S RNA.Cloning ofthe 383-bp DNA segment containing both 5'and 3' splice sites for

SV40 late 16S RNA in pBR322 (Fig. la) was carried

outin PaulBerg's laboratoryatStanford University in

collaboration with Hiroto Okayama.

The pBR322-SV40 recombinantDNAcontaining the

SV40 latesequences from thePvuII site (253)to the

Hindlil site(1,476)(BBB numberingsystem)(34)was

partially digested with Avall, and full-length linear

DNA waspurified by extraction by the glass powder

technique (35), modified byPaterson,afteragarosegel

electrophoresis. The DNA was treated with the

Klenow fragment of Escherichia coli DNA

polymer-ase) I to convertthe cohesive termini toblunt ends,

ligated to synthetic BamHI linkers with T4 DNA

ligase, and cleaved with BamHI. TheDNAwasused

to transform E. coli HB101 afterligation. An Ampr

transformantcontaining theplasmid(no. 3-2inFig.la)

with a BamHI linker at the Avall site (571) was

selected.

The pBR322-SV40 recombinant DNA (positions

253-1,476)wascleaved withPvuII,ligatedtoHindlIl

linkers, cleaved withHindIll,and introducedintoE.

coli HB101 after ligation. The resultingplasmidDNA

was cleaved with HindIII and electrophoresed in ar,

agarosegel. The largestDNAfragmentofpBR322was

ligated to a small SV40 DNA fragment (positions

1,029-1,476) after extraction and introduced into E.

coli HB101. The plasmid DNA was then partially

digested with BstNI (which recognizes the same

se-quence as EcoRII), and full-length linear DNA was

extracted afteragarosegel electrophoresis. TheDNA

wastreated withE.coliDNApolymerase I, ligatedto

BamHIlinkers, andcleaved withBamHI. The DNA

wasintroducedintoE.coliHB101afterligation,and a

transformantcontaining theplasmid (no. 1-1-2in Fig.

la) with aBamHIlinkeratthe EcoRIIsite(1,391) was selected.

Plasmid 3-2 DNA was cleaved with HindIll and

BamHI,and thelargefragmentcontainingthepBR322

sequences wasisolated. This fragmentwasligatedto

the85-bp fragment isolated from plasmid 1-1-2 DNA

aftercleavage withHindlll and BamHI. A

transform-antcontainingplasmidno.3-2-1(Fig.la)wasselected.

The plasmidDNAwascleavedwithBamHI,treated

withE.coliDNApolymeraseI,andligatedtodestroy

theBamHI site. Atransformant containingthis

plas-mid (no. 3-2-1* in Fig. la) was selected, and the

generation ofaClaIsiteattheoriginalBamHI sitewas

confirmed. The 383-bp fragment containing both 5'

and 3' splice sitesforSV40late 16S RNA splicing was

isolated after cleavage oftheplasmidDNAwithKpnI

andHindIll.

(ii)Cloning of the Adl2 ElA gene.The Adl2SalIC

fragment (positions 0-3,497) was cloned in pBR322.

Thisrecombinant plasmid(PASC in Fig. lb)DNA was

cleaved with either HaeII and AccI orTaqI and AccI

(Fig. lb). The former cleavage generates a 1,100-bp

Adl2DNAfragment covering thecodingregion of the

ElA gene and its transcription termination signal,

whereas thelattercleavagegenerates a1,257-bp

frag-mentwhich alsocoversthepresumptiveElA

promot-ersignal. TheseDNAfragmentswereligatedtoa1:1

mixtureofHindIll and BamHI linkers after the

forma-tionof blunt ends byE. coliDNApolymerase I and

were cleaved with HindIll and BamHI. The digests

wereelectrophoresedon a 1.2% agarosegel, andthe

Adl2 DNA fragments were extracted. These DNAs

were ligated to the largeHindIII-BamHl fragment of

pBR322and introduced intoE.coliHB101. The

trans-formants containing the recombinant plasmid with

HindlIl and BamHI linkersatthe5'and3' sides of the

ElAgene wereselected.

(iii) Construction ofSV40-Adl2 EIA recombinants.

ThepBR322-SV40recombinant (no. VIII in Fig. lc) in

which whole SV40 DNA wasinserted at theBamHI

site ofpBR322 DNA was cleaved completely with

KpnIandpartially with BamHI. Afterelectrophoresis,

the larger fragment containing the whole pBR322

sequences and the SV40 early region, including the

origin, was isolated (Fig. lc). The DNA was mixed

with an equimolar ratio ofthe 383-bp fragment

con-taining the SV40late16SRNAsplicingjunctions(Fig.

la) and the Adl2 DNAfragment covering the ElA

coding sequences (Fig. lb) and was ligated with T4

DNA ligase. The DNA was introduced into E. coli

HB101, and thetransformantscontaining the plasmid

with theexpectedsequencearrangement were

select-ed(Fig. ld).Amoiety of theSV40-Adl2 recombinant

couldeasily beseparated fromthepBR322sequences

bycleavage of thepBR322-SV40-Adl2double

recom-binant (pSAdElA or pSAdElAp in Fig. ld) with

BamHI and AccI, since AccI does not cleave the

recombinant sequence but cleaves the pBR322

se-quences at two sites.

Transfection. DNAtransfectionwascarriedoutwith

DEAE-dextranasafacilitatoressentially accordingto

themethod ofSompayrac andDanna(31). Confluent

culturesofCV1 orGC7 cells (ca. 1.5 x 107 and 5 x

106, respectively) were washed twice with Dulbecco

modified Eagle medium and incubatedwith 4 to 8 ,ugof

linear recombinant DNA plus0.2 ,ug of SV40tsA58

helper DNA in Dulbecco modified Eagle medium containing 0.05 MTris-hydrochloride(pH 7.3) and 200 ,ugofDEAE-dextran per ml at37°Cin aCO2incubator

for8h.Themonolayerswerethen washedtwicewith

Dulbeccomodified Eagle medium and fed with 50 ml ofthe same medium containing5% fetal calfserum.

The cellswereincubatedateither 41°C(CV1cells)or

39.5°C(GC7 cells) for12to16days untilthe

cytopath-ic effect was well developed and then were

freeze-thawedfivetimes. Thelysatewascentrifugedat3,000 rpm for 10 min, and the supernatant was used for

infection in allsubsequentexperiments.

Spot hybridization. Intracellular viral DNAs were

extracted from theinfected cellsby the procedure of

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410 ODA ET AL.

(a)

Hind111( 1476)

EcoRII(1391) HindIII(1029)

Pvu II Hindlillinkers

Pv i ligase

(253) aII (571 )

iAvaII partial IDNA polymerase

BamHIlinkers Iligase

3-2

BamHI(Avail1)

Hind 111+BamHI Hind III

BamHI

ligase

HindI1l(1476)

EcoRII( 139 )

Hind1II(1029/253)

BstNI,partial

DNApolymerase BamHI linkers

Iligase

HindllI

BamH I(EcoR II)

01-1-2

t HindIIl+BamHI

KpnI +Hind11l

HindIll KpnI

FIG. 1. Construction of SV40 recombinants carrying the Adl2ElAgene.(a)Constructionof therecombinant

plasmid containingthe 5'and 3' splice sites for SV40late16SRNA in a small DNA segment. (b)Cloning ofthe

Adl2 ElA gene. (c) Preparation of the linear pBR322 sequences containing the SV40 early region. (d)

Construction ofpBR322-SV40-Adlfdouble-recombinantDNA.TheDNAfragments preparedinpanels a, b, and

cweremixed at anequimolarratio and ligatedasdescribedin the text.Solid lines, pBR322 sequences; openbars, SV40 orAdl2sequences;0, origin ofSV40 DNAreplication;shadedregions, SV40sequencescontaining the 5'

(striped regions) and3'(crosshatched regions) splice sites forlate16S RNA (caretsymbolsrepresent the sitesof

splicing); stippled regions, Adl2 sequences containing the ElA gene; P, Adl2 ElA promoter; T, Adl2

polyadenylation signal; A,ATG; A, TAA.

Hirt (10). Aliquots (0.5 ,ug) of heat-denatured DNA were spottedonto acirculararea markedon a

Milli-pore membrane filteras previously described (14) to determine the ratio ofrecombinant to helper DNA.

Reconstructionspotsweremade by mixingthe

recom-binant DNA with salmon sperm DNA. The immobi-lized DNAs were hybridized with 32P-labeled Adl2

DNApreparedbynicktranslation(26).Theamountof

recombinant DNAwas estimatedby the intensityof

thespots in theautoradiogram.

Si

nucleasemappingofhybridmRNAs. Thehybrid

mRNAcontainingthe Adl2 ElAsequence was

ana-lyzed by S1 nucleasemapping (2, 28).Thecytoplasmic

RNAsprepared from the infected cellswere

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VOL. 45, 1983

(b)

Taq 1 (338) _{Hae II +}_Acc_I

3amH I (Acc I )

HindIllIaamHt I HindllBaBamH I

(c) BamH 1(2516)

(d)

BamHI

pSAdEl Ap

orn

P KpnI nd IIll

ligase

BamH I

II

ligase

FIG. 1. Continued

ized with uniformly 32P-labeled Adl2 EcoRI DNA

fragment C at 52°C for 3 h. Aftertreatment with SI

nuclease, the hybrids wereprecipitated with ethanol

and subjected to either alkaline or neutral 1.4%

ag-arose gel electrophoresis. The gels were dried and

autoradiographedwith Fuji X-ray film.

Immunofluorescent staining ofAdl2 T antigen g.

GC7 andCV1 cellson coverslipswereinfected with

the recombinantvirusstockandfixedwith acetone at

34 and 58 h after infection, respectively. Indirect

immunofluorescence wascarried outby using

anti-T-antigen-g serum from rats bearing HY cell tumors

followed by rabbit anti-rat immunoglobulin G

conju-gated withfluoresceinisothiocyanate (29).

Two-dimensional gel electrophoresis. The infected

cells werepulse-labeled with 200,uCi of

[35S]methio-nineperml for2 h in methionine-free mediumatthe

times indicated in the legend to Fig. 6. The cell

extracts were prepared by lysing the cells in lysis buffer containing 0.5% Nonidet P-40, 50 mM

Tris-hydrochloride (pH 8.0), 120mMNaCl, and150pLgof

phenylmethylsulfonyl fluoride per ml.

Two-dimen-sional electrophoresiswasperformed accordingtothe

methodof O'Farrell (23)with thefollowing

modifica-tions.Intheisoelectrofocusing gel,2%ampholine(pH

3.5 to 10) was used withoutmixing with the ampho-lines in the other pH ranges. Isoelectricfocusingwas for 12 h at 350 V. Thegelswereequilibratedand then

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applied to 15% polyacrylamide slab gels. The gels were dried andfluorographedwith Fuji X-rayfilms for 10days.

RESULTS

Construction of SV40 recombinants carrying theAdl2 EIA gene. SV40 recombinants carrying theAdl2 ElA gene were constructed and cloned inE. coli aspBR322-SV4O-Adl2 double-recom-binant plasmids (Fig. 1). From the published sequence data (33), the coding region of the Adl2 ElA gene resides between positions 502 and 1,374from the left end of the genome. The main RNA initiation site is located about 30 bp downstream fromthe TATA box at position 414. TwotypesofAdl2ElADNA wereinserted into theSV40vector;theHaeII-AccIDNAfragment (positions 495-1,595) contains a whole coding region together with the polyadenylation signal at position 1,436, and the TaqI-AccI fragment (positions 388-1,595) contains the main RNA initiation site inaddition to the sequence men-tioned above (Fig. lb). The latter fragmentwas used todeterminetheefficiencyof theutilization of the Adl2 ElA promoter in the SV40 vector. These fragments were cloned in pBR322 by using HindIll and BamHI linkers (Fig. lb).

To construct an SV40 vector which still re-tainsthelate mRNAsplice junctionsinaddition tothe initiation andtermination signals forlate mRNA, a small DNA segment in which the 5' and 3' splice sites forlate 16S RNAwerejoined by removing most ofthe intervening sequence was cloned in pBR322 (Fig. la). The DNA fragment containing the splice junctions was generated by cleaving the plasmid DNA with KpnI and HindIll.

For the construction of the pBR322-SV40-Adl2 double-recombinant plasmid,the pBR322-SV40 recombinant (no. VIII in Fig. lc) was

cleavedcompletelywithKpnIandpartiallywith BamHI; thelargeDNAfragment containingthe whole pBR322 sequences and the entire SV40 early region with thereplication origin (Fig. lc) was then mixed with an equimolar ratio of the Adl2 ElA DNA generated by cleavage with HindlIl and BamHI and the small fragment containing the splice junctions and was intro-duced intoE. coliafterligation. The

transform-antscontainingthedouble-recombinant plasmid wereselected aftercleavageof theplasmid DNA withanappropriate restriction enzyme (Fig. ld). These plasmids were designated as pSAdElA andpSAdElAp; theformercontained the Adl2 E1Acodingregionandpolyadenylation site,and the latter contained the main promoter for the ElA gene in addition to the former sequences. The precise structures of pSAdElA and pSA-dElApwereconfirmedbydigestionwith

AvaIl,

which cleaves these DNAsatmultiple sites(Fig. 2). An electrophoretic analysis ofthe products showed all of the DNAfragments larger than 500

bp

as having the expected lengths, suggesting that the recombinants were made exactly

ac-cordingtotheschedule. Amoiety ofSV40-Adl2 recombinant DNA, SV-AdElA (4,470 bp) or

SV-AdElAp (4,672 bp), was obtainedby diges-tion of the recombinant plasmids with BamHI and AccI followed by gel electrophoresis. The latter enzyme, which cleaves the pBR322

se-quences at two sites but does not cleave the recombinant sequence,facilitated theseparation ofrecombinant DNA from pBR322 DNA (Fig. ld).

Propagation of SV40 recombinants carrying the Adl2 ElA gene. To increase the efficiency of infection, the recombinant DNA was packaged intovirionsby transfection of GC7 or CV1 cells with a mixture of the recombinant DNA and SV40 tsA58 DNA as helper at molar ratios of 20to40. The cells wereincubated at the nonper-missive temperature, and the virus stocks were prepared when the cytopathic effect was well developed. These stocks were used for the fol-lowing experiments. The amounts of recombi-nant and helper DNA in monkey cells infected with the virus stocks were first analyzed by ethidium bromide staining of the intracellular viral DNA after electrophoresis in an agarose

gel. However, owing to a low amount of the recombinant DNA, an exact estimation was

difficulttoperform. A quantitation of the

recom-binant DNA was, therefore, performed by spot hybridization with 32P-labeled Adl2 DNA as a

probe. Intracellular viral DNA(0.5pLg)washeat denatured and spotted onto acirculararea ona nitrocellulose filter. After hybridization, the amount of recombinant DNA wasestimated by comparing the intensity of the spots in the autoradiogram with that of reconstruction spots (Fig. 3). The results showed that the virus stocks prepared with SV-AdE1A and SV-AdE1Ap DNA in GC7 cells contained approximately 15 and 4% of recombinant genomes, respectively. This percentage was variable from preparation to preparation; however, the same difference between the amountofrecombinant genomes in SV-AdElA and that inSV-AdElApwas repeat-edly observed. The DNA sequence present only inSV-AdElAp(positions338-495)mayhave an inhibitoryeffect onthereplicationor encapsida-tion of the recombinant genome, or on both. Preparation of the virus stocks in CV1 cells alwaysresulted inareduction in the percentage ofrecombinant genomes.

Si nuclease mapping of Adl2 ElA mRNA

transcribed fromrecombinant viruses.The

struc-ture ofAdl2 ElA mRNA transcribed from the

recombinant DNA wasdeterminedbySI

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A

Bam-lHl (2516)

BarHI 1 45

(Acc

I

1595)

FIG. 2. Restrictionanalysis ofpBR322-SV40-Adl2double-recombinant DNA. (A) TheAvaIIrestrictionsites

inpSAdElADNA are shown by arrows. The numbers in boxes are the expected lengths of restriction fragments.

Thesamefragmentsof indicatedlengths must be generated from pSAdElAp DNA. Symbols are as described in

thelegend to Fig. 1. (B) pSAdElA and pSAdElAp DNAs digested withAvaIl andelectrophoresed on a1.4% agarosegel. Lanes: a,HindIll-digestedplasmidgABA DNA; d, BstNI-digested plasmid VIII (see Fig. 1)DNA;

b, pSAdElADNA; c,pSAdElApDNA.

ase

mapping

ofthe cytoplasmic RNA

prepared

from the infected cells at the late phase of infection. The RNA was hybridized with uni-formly

32P-labeled

Adl2EcoRI DNA

fragment

C (0-16.5 map

units).

After

S1

nuclease

treat-ment, the DNA-RNA hybrid was electropho-resed in both alkaline and neutral agarose gels (Fig. 4). When the hybrid formed with SV-AdElAp RNA(the cytoplasmic RNAprepared from the SV-AdElAp-infected cells) was elec-trophoresed at alkaline pH, four segments of 730, 640, 450, and 310 basesweredetected

(Fig.

4, laneb).Theformertwosegmentscorrespond

to the

5'-coding

segments of the Al and A2

mRNA species, respectively. The 5' termini of these segments correspond to the end of the insertedAdl2 DNA in therecombinant, indicat-ingthattranscription is initiated attheSV40late promoter rather than at the ElA promoter, which is located 110 nucleotides downstream from the end of the inserted Adl2 DNA. The faint band of 310 bases corresponds to the 3'-codingsegmentof theElAmRNAspecies from the 3' splice site to the ElA

polyadenylation

site,and the 450-base segmentof much stronger

intensity corresponds to the region from the3' splice site totheotherendofthe inserted Adl2 DNA.The resultsindicate that mostofthe ElA transcript extends pastits own polyadenylation site and is polyadenylated at the downstream SV40 polyadenylation site. Judging from the intensity of the 730- and 640-base segments, almost equalamountsofAl and A2 mRNAwere

formed after processing of the primary

tran-script. An analysis of SV-AdElA RNA gave essentially the same results (Fig. 4, lane a). S1 nuclease treatment ofthe hybrid yielded three bands of570, 480 plus 450, and 310 bases. The former two bands correspond to the 5'-coding segment of the Al and A2 mRNA species,

re-spectively. The intensity of these bands was

strongerthan that ofbands generated from the SV-AdElAp hybrid, indicatingthat moreofthe hybridmRNAwaspresentin SV-AdElA-infect-ed cells thanin SV-AdElAp-infectedcells. The

amount was, therefore, proportional to the

amount of the recombinant virus in the virus

stocks.

S1 nuclease mapping ofthe DNA-RNA hy-brids in neutral agarosegels confirmed the

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2(ngi

100) 5 \/

0 41 A3

50 10

\ 20 / 44 /

O*

FIG. 3. Spot hybridization of intracellular viral

DNAfrom recombinant-virus-infected cells with32p_ labeledAdl2DNA.The virusstocks(Al, A2, A3, and A4) were prepared independently by transfection of

GC7cells withSV-AdElAandSV40 tsA58DNAs (Al

andA2)orwithSV-AdElApand tsA58 DNAs (A3 and

A4) at aweight ratio of40.Intracellularviral DNAs

were prepared from CVl cells infected with these

virus stocks. The intracellular viral DNAs (0.5

[Lg)

were spotted onto the filteras indicated and

hybrid-ized witha32P-labeled Adl2 KpnIfragment (1.7-16.0

mapunits). Reconstructionspots weremade with the

indicated amounts of SV-AdElA DNA plus salmon

spermDNA(total, 0.5 pLg).

mation described above. The hybrids formed with SV-AdElA RNA (Fig. 4, lane c) were

separated into four major bands of 1,020 (570 +

450), 930 (480 + 450),880 (570 + 310), and 790 (480+310) bp. The largertwobandscorrespond

to the Al and A2 species with their 3' termini extendedtothe end of the inserted Adl2DNA, and thesmallertwocorrespondtothe Al and A2 species polyadenylatedattheirown

polyadenyl-ation site. Similarly, the hybrids formed with SV-AdElAp RNA (Fig. 4, lane d)were

separat-ed into four major bands of1,180 (730 + 450), 1,090 (640 + 450), 1,040 (730 + 310), and 950 (640 + 310) bp. The lengths of these hybrids confirmed the initiation of transcription at the SV40 late promoter. The bands of 570 and 730 bp (Fig. 4, lanes candd) seem tobe generated by cleavage of the DNA-RNA hybrid at the splice site. The origin of the other faint bands hasnotbeendetermined.

Synthesisof Adl2 ElAgeneproductsin

recom-binant-virus-infectedcells.Theexpression of the ElA gene in the recombinant-virus-infected

monkey cellswasanalyzed by indirect

immuno-fluorescence. CV1 cells on cover slips were

infected withSV-AdElAorSV-AdElAp

recom-binant virus stocks at 41°C, and the cells were

stained with immunofluorescent antibody at 48 to60h after infection. The products (T antigen g) were detectedinthe nuclei(Fig. 5) and hada

morphology similar to that ofproducts synthe-sized in Ad12-infected HEK cells (29). The percentage of positive cells in SV-AdElA-in-fected cells varied between 10 and 20%, depend-ing on the virus stocks used, but did not exceed 20%, presumably due to a small amount of the recombinant virus in the virus stocks. The strongintensity ofimmunofluorescence, howev-er,indicates that T antigen g was overproduced in most of these positive cells. In SV-AdElAp-infected cells only 5% of the cells exhibited T antigen g, as could be expected from a smaller amountof recombinant virus in the virus stocks. The ElA gene products synthesized in the recombinant-virus-infected cells were labeled with [35S]methionine and analyzed by two-di-mensional gelelectrophoresis followed by auto-radiography. The amount produced was com-pared with that synthesized in Adl2-infected and -transformed cellsbyelectrophoresisof the cell extracts directly without immunoprecipita-tion(Fig. 6). A cluster ofpolypeptides (molecu-lar weight, 35,000 to40,000; pl, 5.0 to 5.5) was clearly visible in the extracts prepared from SV-AdElA-infected GC7 and CV1 cells(Fig. 6a and b). A much greater amount of thepeptides was detected in the infected GC7 cells than in the infected CV1cells,presumablydue to the differ-ence in the susceptibility of the cells to SV40 infection as stated above. These peptides were detectedat apositionsimilar tothat of the ElA polypeptides immunoprecipitated from the ex-tractofAdl2-infected HEKcells (Fig. 6d). No significant amount of the polypeptides was de-tected in the extracts from Ad12-infected KB cells(Fig. 6e) or from3YgE1Acellswhich were transformed with an Ad12 DNAfragment con-taining the ElA gene (Fig. 6f). No polypeptide wasdetected inuninfectedGC7 cells (Fig.6c).

DISCUSSION

The SV40vector used in thepresentstudy is

useful forcloning and forexpressionof aforeign gene of up toapproximately 1,900 bpinmonkey cells. Since the vector not only contains the initiation and termination signals for transcrip-tion butalso retains thesplicingjunctions down-stream from the late promoter, the gene to be inserteddoes not have topossess its own splic-ing junctions for expression. The Adl2 ElA gene with or without its own promoter was

insertedinto this vector toseewhether theElA

promoter is recognized in the vector. The

recombinant DNAs were propagated and pack-aged bycotransfection withSV40tsA58 DNAas

a helper in monkey cells. The ElA gene

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VOL.45,1983~SV40RECOMBINANTS CARRYING Adl2 ElA GENE 415

a b

-130

640

510-3

450- ~

310-c d

-1180

-1090

1020- -1040

930-

- 950

790-0 (730)

(570)-310

B

-:'-- ,. / _:

[image:8.489.102.388.76.518.2]

I

FIG. 4. Si nuclease mappingofAdl2 EtAmRNAfromrecombinant-virus-infected cells. (A)Cytoplasmic

RNAspreparedfromSV-AdElA(lanesaandc)-orSV-AdElAp(lanesb andd)-infectedCV cellsat56 hafter

infection. The RNAswerehybridizedwitha32P-labeled Adl2 EcoRIDNAfragmentC(0-16.5mapunits).The

hybridsweretreated withSinuclease andanalyzed byalkaline(lanesaandb)orneutral(lanescandd)agarose

gelelectrophoresis. The numbers indicate thelengthsof thefragmentsestimatedfrom thepositionsof the size

marker DNAfragments. (B)Expected lengths of the DNAfragments protected bythe RNAs.

ucts (T antigen g) were efficiently produced in

monkey cells after infection with the resulting

virus stocks,judgingfrom thestrongintensityof immunofluorescence in the nuclei. However,

theproportionofT-antigen-positive cells didnot exceed more than 20% of the cells, owingto a

small amount of the recombinant virus in the

virus stocks. Thegeneproducts werevery simi-lar or identical to those synthesized in Adl2-infected HEK cells, since they migrated to the

same spot upon two-dimensional electrophore-sis.

Si nuclease mapping of the hybrid mRNA transcribed from the recombinant genome

indi-A

I

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45,

1983

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1- --1-.1 mmmmmmmmir

.k. ?

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ODA ET AL.

FIG. 5. Indirectimmunofluorescent staining ofrecombinant-virus-infected cells for T antigen g.

cated that the transcripts were initiated exclu-sively at the SV40 late promoter. The ElA promotercould not beutilized, presumably

ow-ing to its promoter activity being weaker than thatofthe SV40 late promoter under the condi-tionsused. Theexpression of thispromotermay be restricted to the early phase of infection or

may need more base sequences upstreamfrom the presumed ElA promoter, or both. In

con-trast,theElApolyadenylation

signal

was recog-nized. Althougha

majority

of the

hybrid

mRNA

was terminated at the SV40

polyadenylation

site, a small but

significant

amountofRNAwas

terminatedatthe ElApolyadenylationsite. The hybrid RNA was correctly spliced in the ElA coding region, and nearly equalamounts ofAl and A2 mRNAspeciesweregenerated,aswould be the case in Adl2-infected human cells.

Translation of these hybrid mRNAs

tran-scribed from the SV-AdElArecombinant must

be efficient, since the ATG codonfor the ElA polypeptides is located

only

7

bp

downstream from the 5' endoftheinsertedAdl2 ElA DNA. Thedistance between the upstream 3'

splice

site and the ATG codon is therefore 40

bp

and is almost thesame asthat between the 3'splicesite and the ATG codon for the VP1 (36bp) in 16S

mRNA. This distance in the recombinant

SV-AdElAp was about 100 bp longer than that in SV-AdElA; however, the

efficiency

of transla-tion of the hybridmRNAs may notbeas

differ-ent between SV-AdElAp and SV-AdElA

re-combinants, since the amount of the ElA polypeptides synthesized seemed tobe propor-tionaltothatof thehybridmRNAssynthesized.

Thehigher level of the E1A gene products in the SV-AdElA-infected cells as compared with the SV-AdElAp-infected cells may therefore be as-cribed to the higher proportion of the recombi-nantgenomein thevirus stocks. The level of the ElA gene products in the SV-AdElA-infected cells was also compared with that in KB cells infected withwild-type (WT) Adl2 and with that intransformed3YgElA cellsby two-dimension-al electrophoresis of [35S]methionine-labeled

cell extracts followed by autoradiography.

Un-der the conditions used, the dense spot of the ElAproducts was visible in the autoradiogram of the extract from SV-AdElA-infected cells, whereas no clear spot was detected in the

ex-tracts from KBcells infected with WTAdl2 or

in the extracts from transformed cells. The re-sults suggest that the level of the ElA gene products in SV-AdElA-infected cellsis at least 10-foldhigherthan that in the latter two types of cells.

The ratio of the recombinant to the helper virus in the virus stocks may be influenced by thefollowing factors. First, the sequence of the inserted DNA is critical for the replication or packagingof the recombinant DNA.SV-AdElA (4,470 bp) andSV-AdElAp(4,627bp)differ only in the sequences upstreamfrom the ElA coding sequence, whereas the amount of recombinant genome in the virus stocks was consistently lower with SV-AdElAp DNA than with SV-AdElA DNA. The base sequences presentonly in SV-AdElAp may therefore be inhibitory to replication or packaging of the recombinant DNA. It has been reported that a specific

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~~~~~~~

e

_

-gal

FIG. 6. Two-dimensional gel electrophoresis of proteins from recombinant-virus-infected cells. Confluent

monolayersofCV1 (7 x 106)and GC7 (2x106) cells were infected with 1.0 ml of theSV-AdElA virus stock at 41

and39.5°C,respectively. The cells werelabeled with[35S]methionine for 2 h in methionine-free medium at the

times indicated below. Ascontrols, KB and HEKcells were infected withAdl2 at 10 and 50 PFU per cell,

respectively, in the presenceof 25 ,ug ofcytosine arabinoside per ml at37°Cand were similarly labeled with

[35S]methionineat 22and 12 hafterinfection, respectively. The cell extracts were prepared as described in the

text and subjectedto two-dimensional gelelectrophoresis. Each electrofocusing is from left (basic) to right

(acidic). Sincethesecond-dimension electrophoresis in panel d was carried out in a 13% gel instead of a 15% gel,

thepolypeptides migrated slightlyfaster than in theother panels. Panels: a, AdElA (GC7 cells, 36 h); b,

SV-AdElA(CV1cells, 56 h); c,uninfected GC7cells; d,Adl2 (HEK cells, 12 h) (proteins were immunoprecipitated

withanti-T-antigen-g serum);e, Adl2(KBcells,22 h); f, 3YgE1Acells.

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418 AL.

pBR322 DNA sequencehas aninhibitory effect onthereplication of pBR322-SV40 recombinant

DNA in monkey cells, and the recombinant DNAwhichefficiently replicated in the cells has

this sequence deleted (13). The EtA coding

sequences mayalsobe inhibitory topropagation

of therecombinant DNA sincean SV40

recom-binantcarrying the Adl2 EtBgene

(SV-AdElB-B) propagates very efficiently in monkey cells, as described in the following paper (22). Ithas

been suggested that 70 to 100% of full-length

SV40 DNAis suitable for packaging (19);

how-ever, the efficiency of packaging may differ

significantly within the lengths in thisrange.The

length of the recombinant SV-AdElB-B DNA (5,307 bp) is 101% of full length, whereas the recombinant SV-AdElA and SV-AdElAp DNAs correspondto85and 88% of full length, respectively. The upperlimit of the size is also

critical for packaging; a length 102% of full

length could notbe packaged (19).

Second, the susceptibility of monkey cells to

SV40 and the efficiency of transfection are im-portantfactorsforobtainingavirusstockwitha

higher ratio of recombinant virus, especially if the recombinant DNA is replicated and pack-aged inefficiently as compared with the helper

DNA. We consistently observed that the virus stocksprepared with GC7 cells contained higher ratios of the recombinant virus than those

pre-pared with CV1 cells. As previously described, GC7cellsare moresusceptibletoSV40 thanare

CV1cells;togetT-antigen-positive cellsinmore

than 90% of the cells by infection withSV40, a

multiplicity of 5 PFU per cell is sufficient for

GC7 cells, whereas CV1 cells require a

multi-plicity of 75 PFUpercell(36).Ahigher

efficien-cyoftransfection of the recombinantDNAwas

always observed by exposure of the cells to

DNA in medium containing 200 ,ug of DEAE-dextran permlfor 8 h thanbytheconventional method in which the cells were exposed in

mediumcontaining 500 ,ug of DEAE-dextranper

mlfor 30 min. Efficient transfection and

infec-tion may minimizethe number of infection

cy-clesrequired forthedevelopmentofa

cytopath-iceffect in the cells and avoid the decreasein the

ratio of recombinantto helpervirus.

Finally, theuseofanearlydeletionmutantof

SV40 instead of a point mutant as helper may

also facilitate the preparation of virus stocks

with higher ratios of recombinant virus. The

plaques formed after transfection of monkey cells with the recombinant plus SV40 tsA58

DNAs containedWT SV40 in aminor

propor-tion. The results suggest that the virus stocks

prepared after development of the cytopathic effect contained a small but significant amount

of WT SV40. A population of WT SV40 may

increase upon successive passages in culture.

The probability ofgeneration of WT SV40 by recombination between the recombinant and helper DNAs may be lowered if the mutant which deleted most of the SV40 early region is usedashelper.Ithas been recently reported that the helper DNApSVL5, in which only the early region between the origin and the TaqI site (about 500 bp) is retained, efficiently replicates in Cosl cells (4), giving rise to mature virions (9).

The ElA gene of adenovirus is involved in both the activation of other early genes in the lytic infection cycle and the transformation of susceptible cells. In spite of these interesting multiple functions, no biological activity of the gene products has been investigated in vitro. Efficient production ofthe

EtA

gene products in monkeycellswill facilitatetheir purificationand theanalysis for biologicalactivities in vitro.

ACKNOWLEDGMENTS

Wethank P.Berg and H.Okayama for valuablesuggestions andadvice.Cloning of the SV40 DNA segment containing the splicing junctions for late 16S RNA was carried out in collabo-ration with H. Okayama when K.O. was at P. Berg's labora-tory atStanford University.

This work was supported by grants from the ministry of Education, Science, and Culture of Japan and by grants from theMitsubishi Foundation.

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