Nonviable mutants of simian virus 40 with deletions near the 3' end of gene A define a function for large T antigen required after onset of viral DNA replication.

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0022-538X/83/090487-08$02.00/0

Nonviable

Mutants

of

Simian

Virus 40 with Deletions Near the

3' End of Gene

A

Define a Function for

Large

T

Antigen

Required

After Onset of

Viral DNA

Replication

JOANNE TORNOWANDCHARLES N. COLE*

DepartmentofHumanGenetics, Yale University Schoolof Medicine,NewHaven, Connecticut 06510 Received 3 February 1983/Accepted 7 June 1983

Deletionmutantsof simian virus40(SV40)withlesionsatthe three DdeI sites

nearthe 3' endof the early region were constructed. Mutantswith deletionsat

0.203 and 0.219mapunits (mu) which didnotchangethelargeTantigen reading

framewereviable. This extendsslightly theupstreamboundary for the location of viablemutantswithdeletions in the 3' end of theAgene. Mutantswith frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable

mutantswith deletions in thisportion of theAgene.Noneof the three nonviable

mutants with deletions at 0.219 mu produced progeny viral DNA. These three

mutantsall used the alternate readingframe located in thisportion of the SV40

early region. Themutant witha deletionat0.193mu, dlA2459, waspositivefor viral DNA replication and was defective foradenovirus helper function. All of thesemutationswerelocated in theportionof theSV40largeTantigenwhich has

nohomologytothepolyomaTantigens.These resultsindicate that thisportionof largeTantigen isrequiredforsomelate stepin theviralgrowth cycle andsuggest

thatadenovirushelperfunction isrequiredforproductiveinfectionbySV40.

Papovavirus large T antigens are multifunc-tional during both productive and transforming

infections. Muchofourknowledge about simian

virus 40 (SV40) largeTantigen derives from the

study of mutants with lesions in the A gene,

which encodes largeT(13). These include both

temperature-sensitive(7, 43) and

non-condition-ally defective deletion (10, 25) mutants. These

studies indicate that large T is responsible for

theinduction of viral DNA synthesis(7, 43), the

stimulation of host DNA replication (20), regula-tion of the level of early mRNAs (3, 36, 45),

induction, and, in some cases, maintenance of

late mRNA synthesis (1, 12), induction of in-creased levels of host cellenzymesinvolved in nucleic acidmetabolism(23),activation of silent

rDNAgenes(40),provision of adenovirus helper

function (9, 19, 34),aswellasinitiation, and, in

most cases, maintenance of the transformed

stateinnonpermissive cells (21, 22, 44).

Comparison of the DNA sequences of SV40

(17, 35) and polyoma (18, 39) indicates that

substantial regions ofhomology exist between

the two genomes. Each genome also contains

unique regions. A sequence of approximately 500base pairs (bp)in themiddle ofthe polyoma

t Present address: Department of Biological Chemistry, CaliforniaCollege ofMedicine, UniversityofCalifornia,

Ir-vine, CA 92717.

tPresent address:DepartmentofBiochemistry,Dartmouth MedicalSchool, Hanover,NH 03756.

earlyregion encodes the unique portion of

mid-dle T antigen and, in another reading frame, a

portion of large T. Thisregion has no

counter-part in SV40. SV40 hasasequenceof approxi-mately 400 bp, between mappositions 0.25 and 0.174 mapunit (mu), that hasnocounterpart in polyoma. This region encodes the C-terminus of SV40largeTantigen. Two properties have been

assignedtothis portion of the SV40genome:(i)

adenovirus helper function, requiring no more

than the C-terminal 37 amino acids oflarge T

(16, 33), and (ii) the62-base small SV40-associ-ated RNA, which is complementary to early

mRNA near 0.21 mu(2, 27). This portion ofthe

SV40early region also containsanopenreading frameof 99 codons which beginsat0.219 mu and

endsat0.169mu.Thetermination codon for this alternate readingframe occurs beyondthe

nor-mal termination codon for large T antigen, but

upstreamfrom the 3' end of the early mRNAs.

Several SV40 mutants contain deletions near

the 3' endof theAgene(see Fig. 1; 10, 15, 31,

32,33). All ofthese mutants areviable andhave

lesions distal tomap position 0.217 that do not

changethe largeTantigen reading frame. One,

d1265 (Cole), has a 39-bp deletion (47) which

results in thereplacementofthe six C-terminal

amino acid residues with four encoded

down-stream from the normal termination codon for

large T (14). Although some of these mutants have slightly slower rates of DNA replication

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(33), both the lytic and the transforming

infec-tioncyclesproceed normally. Much oftheSV40

A gene distal to map position 0.217 has been

deleted inone oranother of thesemutants. Since

many of these viable deletion mutants fail to

produce the SV40-associated RNA (2; M. Pol-vino-Bodnar and C. N. Cole, unpublished data), it cannot be essential for productive infection.

Although some of the mutants exhibit reduced

levels of adenovirus helper function (9), none

are absolutely defective for this function. No

essential functionprovided bythisportion ofthe

Agenehas been described.

In thisreport,wedescribe thepreparationand

characterizationofadditionalmutantswith

dele-tions in thisportion ofthe SV40Agene. These

mutants were prepared as recombinant DNA

molecules. The method used allowed the

isola-tion ofmutantswithsmall deletions(3to22bp),

incontrasttothelargerdeletions(20to1,000 bp)

isolated inpreviousstudies(10, 25, 33).

Wefound thatmutantswithdeletionsat0.203

and 0.219 mu, which did not alter the large T

antigen reading frame, were viable. This result

extends slightly the upstream limits for viable

deletionmutants. Mutants with frameshift

dele-tionsat0.193and0.219mudid not formplaques.

The nonviable mutant with a lesion at 0.193,

dlA2459, was positivefor viral DNA synthesis,

whereas no viral DNA was produced in cells

infected with nonviable mutants with deletions

at0.219mu. Inaddition, dlA2459wasdefective

for adenovirus helper function. Therefore, the

C-terminus ofSV40 largeTantigen is essential

forproductiveinfection andprovidessome

func-tion requiredafter the onset ofviralDNA

repli-cation. This late function ofSV40 largeT

anti-gen may be related to adenovirus helper function.

MATERIALS ANDMETHODS

Cells,cellculture, and viruses.ThegrowthofCV-1 andCV-lp cells has been described previously (33). All cellsweremaintainedinDulbecco modifiedEagle mediumcontaining5%fetalbovine serum. Our

wild-type strainofSV40wasthesmallplaquestrain

orig-inally characterized byTakemoto etal. (42). Mutant d1BC865wasoriginallydescribedbyCarbonetal.(6) and hasadeletionattheEcoRIsite. Mutant d1A1209 (10) lacks 329 bp (Polvino-BodnarandCole, unpub-lisheddata)between 0.65 and 0.587 mu. Viable dele-tionmutantsdl1263 andd11265,lacking 33 bpat0.20

muand 39bpat0.18mu,respectively,wereoriginally

describedbyColeetal.(10);viablemutantviral DNAs

werepreparedasdescribedpreviously (10).

Preparationofmutants. EcoRI-cleavedSV40DNA

wasinserted into the EcoRI site ofpBR322.ThisDNA wasdigestedwithDdeIendonuclease in thepresence

of180,ugofethidium bromideperml(30, 38)at37°C for 45min.Under theseempiricallydetermined condi-tions, mostmolecules werecleavedonce. ThisDNA

wasextracted withphenol, precipitated withethanol, suspendedinS1buffer(280mMNaCl,50 mM sodium

acetate [pH4.6], 1 mMZnCl2), and digested withSi

nuclease, using conditions which led to the removal of asmall number of nucleotides from each end (5). After electrophoresis in a 1.0% agarose gel in Tris-borate-EDTA buffer (89 mM Tris-hydroxide, 89 mM boric acid, 2.5 mM EDTA [pH 8.3]), the largest class of linears was isolated by electroelution in E buffer (40 mMTris-hydroxide, 12.5 mM sodium acetate, 2.5 mM EDTA). These were ligated at a concentration of 5 ,ug/ml, using T4 DNA ligase, and were used to trans-fect Escherichia coli HB 101 (26). Although pBR322 contains eightDdeI sites, most of these occur within thegenesfor resistance to ampicillin and tetracycline or within the pBR322 origin of replication. Since transfected bacteria were plated on agar containing both ampicillin (20 ,ug/ml) and tetracycline (12.5

,ug/ml),mostof the deletion mutants isolated mapped

within the SV40 portion of the recombinant. Minily-sates were prepared by the method ofBirnboim and Doly (4) and were screened for the presence of mu-tantswith small deletions by digestion separately with

HindlIl andDdeI.Plasmid DNA was prepared by the

method of Clewell and Helinski (8) and was purified by equilibrium density centrifugation in gradients of cesi-umchloride-containing ethidium bromide (300 ,ug/ml). DNA tobe usedfor sequence analysis was rebanded onceinacesium chloride-ethidium bromide gradient. DNA of SV40 mutants dIBC865 anddIA1209 was digested withBamHIand was inserted into theBamHl site of pBR322. These plasmids were designated pdlBC865 andpd1A1209,respectively.

Plaque assays. The titers of wild-type SV40 and viable deletion mutant virus stocks were determined

by plaque assay on monolayers of CV-lp cells as

previously described (29). All recombinant mutant

DNAs wereprepared for complementation analysis by

digestionwithEcoRIor BamHI (toseparate viral and

bacterial plasmid sequences) and were ligated at low concentration (5 ,ug/ml) with T4 DNA ligase. Gel electrophoresiswasused to insure that both digestion and subsequent ligation were complete. Genetic com-plementation analysis, using viral DNAs, was per-formed as described previously (29). Some mutants were testedfor temperature sensitivity by incubation of transfected CV-lp cells at 32, 37, and 41°C.

Analysis ofDNA replication. DNA replication was

analyzed bytransfection of cultures of CV-1 cells,in

60-mmdishes, as describedpreviously (33), but with thefollowing modifications. Mutant viral DNAs were separated fromplasmid sequencesasdescribed above. Each60-mmdish received 0.2mlofasolution contain-ing Tris-buffered saline(TS;25 mMTris-chloride [pH

7.5]-137 mM NaCl-5 mM KCI-0.6 mM

Na2HPO4-0.05mM

MgClz-0.7

mMCaC12),DNA(1

jig/ml),

and DEAE-dextran(Pharmacia Fine Chemicals, Inc., Pis-cataway, N.J.;500jig/ml) for45min.Cellswerethen washedoncewithTS and were incubated in Dulbecco modified Eagle medium containing 100 ,uM chloro-quine phosphate (Sigma Chemical Co., St. Louis, Mo.) and2% fetal bovineserumfor4h(G. Magnus-son, personal communication). This treatment in-creased the percentage of cellsexpressingTantigento 50 to 60%, as measured by immunofluorescence (J.

Tornow, unpublished data). After chloroquine

treat-ment,this mediumwasreplaced with medium lacking chloroquine andcontaining2% fetal bovine serum.

Analysisof Tantigens.Tantigenswerelabeled with

[355]methionineand wereanalyzed by

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489 tation with anti-T serum, followed by electrophoresis

aspreviously described (33).

Chemicals andenzymes. Restriction endonucleases were purchased from either P-L Biochemicals, Inc., Milwaukee, Wis., or New England Biolabs, Beverly, Mass. DNA polymerase I and the Klenow fragment of DNApolymerase I were obtained from New England Biolabs; T4 DNA ligase was from Collaborative Re-search, Inc., Waltham, Mass., or New England Bio-labs; calf intestinal alkaline phosphatase andS1 nucle-ase wereobtained from Boehringer Mannheim Corp., New York, N.Y. Proteinase K was from Beckman Instruments, Inc., Fullerton, Calif. or Bethesda Re-search Laboratories, Bethesda, Md. [cx-32P]dNTPs (800Ci/mmol) and[35S]methioninewere obtained from Radiochemical Centre, Amersham, England. Chloro-quine phosphate was purchased from Sigma. Cesium chloride was obtained from Gallard Schlessinger, CarlePlace, N.Y.

RESULTS

Deletion mutants with lesions in the

C-termi-nalportion oflarge Tantigenhave beenisolated

previously in this(10, 33) and other (15, 31, 32)

laboratories. The location of the deletions in

these mutants is shown in Fig. 1. They include

d11263, dl1265, d12491 through d12495, d12194,

and theviable (temperature-independent)

rever-tantoftsA1499, d11499(N. Bouck, A. Pater, C.

Chang, and G. diMayorca, personal

communi-cation). All ofthese mutants contain deletions which aremultiples of3 bp, ranging from 15 to

81bp, leaving the largeTantigen reading frame intact distalto thedeletion. Withthe exception

ofnucleotides 2,721 to2,797, all of the SV40A gene distal to nucleotide 2915 (the 5' boundary of the deletion in d11499(31; numberingsystem as defined by Tooze [46]) has been deleted in

one oranotherof these viable mutants.

As partofaproject to prepare a setofSV40

0.645 0.59 0.54 0.5

2 a

N

N I C t Ag

C.

Dj52

de I DdeIo DdeI 0.l

A1499

Start of alternate 2 TAA-termination openreading frame 12491 I codon for T-antigen

_24931

2495 TAA-terr

I codonfor

I92

SI

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SASRNA AdenovirusHelperFunction

TABLE 1. Propertiesofmutantswithdeletionsin

the 3' end of theA gene

MuatDdel

site

DeletionNuloie

Mutant deleted size

Ndcletede

Viability

no.

(mu) (bp) d

2413 0.219 22 2,925-2,946

-2414 0.219 10 2,927-2,936

-2415 0.219 10 2,928-2,937

-2423 0.219 6-9 +

2431 0.219 3-6 +

2440 0.219 3-6 +

2429 0.203 6-12 +

2451 0.203 6-9 +

2459 0.193 14 2,785-2,798

-a Sizes of deletions of nonviable mutants were de-termined by DNA sequence analysis. The deletion sizes of viablemutants wereestimated from restriction endonucleaseanalysis (datanotshown).

b Nucleotide sequences of themutants were deter-mined by the method of Maxam and Gilbert (28).

Nucleotidenumbering followed thenumberingsystem of Tooze (46).

cViability is definedasthe abilitytoformplaques onmonolayers ofCV-lpcells.

mutants with small deletions throughout the

SV40 early region, we prepared mutants with

small deletions atDdeI cleavage sites, mostof

whicharelocated within theearlyregion.Inthis

report, we describe the properties of nine

mu-tants with deletions at the three DdeI sites

(0.193, 0.203, and 0.219 mu) in the C-terminal

portionof the SV40genome. The nine mutants

are listed in Table 1. The characterization of

mutantswithdeletionsatother DdeIsites will be

described elsewhere (J.TornowandC.N.Cole,

Proc. Natl. Acad. Sci. U.S.A.,in press).

CV-lp cells were transfected with mutant

DNAs,using plaqueassayconditions. Both

mu-0.4 0.3 0.2 0.16 Map Units

I.. I

Tag

16 Map Units An-3'

nination alternate idingframe

FIG. 1. Map oftheearly region of theSV40 genome. Map coordinates are expressed as the fractional length ofwild-type SV40, where 0.00 is the map position of the singleEcoRIsite. The 3' end of early mRNA is shown enlarged in the lower part of the figure. Beneath this are shown the positions of previously described viable deletion mutants (10, 15, 31-33), as well as the regions in which the SV40-associated small RNA (2) and adenovirus helper function (9, 16) map.

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TABLE 2. Genetic complementation analysis of nonviablemutantsa

DdeIsite Complementing mutant

Mutant no. deleted (PFU4Lg)

(mu) dIA1209 dIBC865

2413 0.219 0 5 x 104

2414 0.219 0 4 x 103b

2415 0.219 0 8 x 104

2459 0.193 0 5 x 104

a Monolayers of CV-lp cells were exposed to dilu-tions of mutant DNA and 0.01 ,ug of complementing mutantDNA.

bThis value is lower than values obtained with other mutants because the dl2414 DNA was not ligated before infection of the CV-lp cells.

tants with deletions at 0.203 mu (d12429 and

d12451) and three of the six with the deletionsat

0.219mu wereviable(d12423, d12431, dl2440).In

general, plaquesappearedatthe sametime and

enlarged at the same rate as wild-type SV40 plaques. These mutantsextend slightly the

up-stream boundary for viable deletion mutants in

the A gene of SV40. The sole isolate with a

deletionat0.193 mu(d12459)and theother three

mutants with deletions at 0.219 mu (d12413,

d12414, and d12415)were notviable. Plaques did

not appear when dl2459-transfected cells were

incubatedat32, 37,or41°C, indicating that this

mutant was nonviable rather than temperature

sensitive. These represent the first nonviable

mutantsisolated thathavelesions in thispartof

theSV40genome.

The DNA sequences of all four nonviable

mutants wereanalyzed.The resultsareshown in

Table 1. Mutant d12459 lacked 14 bp, which

shouldcauseashift in thereadingframe oflarge

Tantigen. The deletions in d12413, d12414, and d12415were22, 10, and10bp, respectively, and were overlapping. Theselesions shouldcause a

shift into the alternate readingframe of99

co-dons located between 0.219and 0.168 mu. The DdeIsiteat0.219mu(CTAAG)containsthelast

termination codon (TAA) upstream from this

alternate reading frame. The approximate sizes

of the deletions in the viable mutants,

deter-minedfrom restriction endonucleasedigestions,

arelisted in Table1.

Genetic complementation analysis. All four

nonviable mutants weretested bygenetic

com-plementation analyses, using dlBC865 and

dlA1209. MutantdlA12O9 contains a deletion of

329 bpbetween map positions0.587 and 0.656,

including the cap site for early mRNAs, the

initiation codon for both large and small T

antigens, and the donor splice site for large T

mRNA(10,33; C.ColeandM.Polvino-Bodnar,

unpublished data). MutantdlBC865 has a small

deletion at the EcoRI site (6) and produces a

small amino-terminalfragment of VP1 (C. Cole and S. P. Goff, unpublished data). All four

mu-tants formed plaqueswithdlBC865butnotwith

dlA1209 (Table 2). Therefore, all four belonged

tothe Acomplementationgroup.

Large T antigens produced by the deletion

mutants. Cultures ofCV-1 cells, transfected by

each mutant or wild-type SV40 DNA, were

labeled with [35S]methionine for1 h, 30h after infection. Large T antigens were examined by

immunoprecipitation, using hamster antitumor

(anti-T) serum and Formalin-fixed protein

A-bearing Staphylococcus aureus, followed by electrophoresis in sodium dodecyl sulfate

(SDS)-polyacrylamide gels. Mutant and

wild-type largeT antigens are shown in Fig. 2. The

threeviable mutantswithdeletions at0.219 mu

produced large T antigens (Fig. 2, lanes 7to 9) indistinguishable in electrophoretic mobility from wild-type T antigen (lane 1). The large T

antigen produced in cells transfected by dl2429

(lane 10) migrated slightly more rapidly than wild-type large T (lane 11). The large T antigen

produced in cellstransfected bydl2459 (lane 3)

migrated morerapidlythanwild-type, in agree-mentwithareading frame shift and termination

of translation at 0.189 mu, just downstream from

the deletion at 0.193 mu. The large T antigens

_,C ._

a 1

FIG. 2. Autoradiogram of [35S]methionine-labeled Tantigens immunoprecipitated from cytoplasmic ex-tracts. CV-1 cells (in35-mm dishes) were transfected withwild-type or mutant DNAs. Cultures were labeled for1hbeginning 30hafter infection, using75 ,uCiof

[35S]methioninein 0.25 mlof medium lacking

methio-nine. Extracts were immunoprecipitated, alkylated, and subjected to electrophoresis on 7 to 20%

SDS-polyacrylamide lineargradient gels. Gels werefixed,

fluorographed, dried, and exposed to Kodak XAR-5 film withanintensifying screen for 3 days at -70°C. The positions of the large and smallT antigens are shown. Lanes: (1 and 11)wild-type SV40; (2) mock-infected; (3) 2459; (4) 2413; (5) 2414; (6) 2415; (7) 2423; (8) 2431; (9) 2440; (10) 2429. Lanes 10 and11arefrom the sameautoradiogram and werealigned with each otherby matching the positions forsmallt.

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produced by mutants dl2413 (lane 4), dl2414 (lane 5), and d12415 (lane 6) should be slightly

larger than wild-type large T due to the use of

the alternate reading frame distal to 0.219 mu.

The T antigens produced by these mutants

mi-grated slightly morerapidlythanwild-type large

Tantigen.

DNAreplication inmutant-infectedcells. CV-1

cells were transfected with either wild-type or

nonviable mutant DNAs (dlA2413 or dlA2459)

and were analyzedfor the presence ofprogeny

DNA molecules (Fig. 3). Proteinase K-treated

Hirt supernatants were extracted with phenol, precipitated with ethanol, and digested with MboI. Plasmid, butnotprogeny, DNAcontains methylated adenosine residues in the MboI

rec-ognition and cleavage sequence, 5'-GATC-3',

and is resistant to digestion by MboI. After

digestion, DNAwas subjected to

electrophore-sis inagarosegels,transferredtonitrocellulose,

and hybridized with radiolabeled SV40 DNA.

Progeny viral DNA was synthesized in cells infected by d12459 (Fig. 3C), but not in cells

infected bydIA2413, a nonviable mutantwith a

deletionat0.219mu(Fig.3B). About one-fourth

as much progeny DNA was formed in

dl2459-infected cells asinwild-type SV40-infectedcells (Fig. 3A). No progeny DNA was detected in cells transfected with dlA2415, another

nonvia-ble mutantwithadeletionat0.219mu(datanot

shown). We conclude that mutants with dele-tionsat0.219 mu wereblocked atthe initiation of viral DNA replication, the first step in the

productive infection cycle which is known to

require large T(43). Mutant dlA2459, however, was blockedat alater stage.

Effect of mutantcoinfectiononadenovirus late

protein synthesis. The ability of dlA2459to

en-hance thelevelsof late adenoviruspolypeptides

synthesized in CV-1 cells was examined by

transfecting cells with wild-type SV40 or

dele-tionmutantsand superinfecting them 48 h later

withadenovirus type 2. [35S]methionine-labeled

whole cell extracts were prepared 72 h after

superinfection and analyzed by

SDS-polyacryl-amide gel electrophoresis (Fig. 4). Cells

coin-fected bywild-type SV40 and adenovirus (lane

d) had substantially higher levels of adenovirus polypeptidesII, III, and IV thandidCV-1 cells

infectedonly by adenovirus (lane b). The level

of polypeptide IIincellscoinfectedby dlA2459

was slightly lower than that in cells coinfected

by d11265 (lanef) and much lowerthanin cells coinfected with either wild-type SV40 (lane d)or

d11263 (lane g). The level of adenovirus helper function ofd11263 and d11265 isonly 30 and6% of that ofwild-type SV40, respectively,as

mea-sured by plaque assays (9). Fiber polypeptide

(IV)was notdetectable in cells coinfected with

dlA2459. We conclude that dIA2459 provides

little or noadenovirus helper function.

DISCUSSION

In this report, we have described the

con-struction and characterization of nine mutants

with deletions in the C-terminal portion of the

SV40Agene.Fourofthese were not viable and

were the first nonviable mutants with lesions in

this portion oftheSV40genome. The five viable

deletion mutants isolated, with lesions at 0.203

and0.219 mu, were similar to deletion mutants

which we have described previously (10, 33).

Theisolation of viablemutants with deletions at

0.219 muextends slightly the upstream bound-ary for viable deletion mutants. We have

con-structedsix mutants with deletions at 0.243 mu

(Tornow and Cole, unpublished data), and one

of these had an in-phase deletion. All were

nonviable, suggesting that the boundary for the

location of viable deletion mutants lies

some-where between0.219 and 0.243 mu. To date, all

viablemutants with lesions in this portion of the Agene havein-phase deletions.

UrO

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FIG. 3. Autoradiograms ofSouthernblots(41),showingDNAreplication inculturesof CV-1 cellstransfected withwild-type(A),dIA2413(B), ordIA2459(C)DNA.Cultureswereharvested after 0, 1,2, 3, 4, and5days. The marker lanes contained20 ngofwild-type SV40DNAdigested with MboI.

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FIG. 4. Effect of dIA2459preinfection on adenovi-rus lateprotein synthesis. Cultures of CV-C monkey

orA549 humancells, in 35-mm dishes,were transfect-edwithwild-typeormutantDNAs(100ngperplatein

avolumeof 0.2 mlof TS containing 500,ug of

DEAE-dextran per ml) or mock-infected and subsequently exposed to 100 ,uM chloroquine diphosphate as de-scribed inthetext.Whenrecombinant DNAplasmids

were used, the viral DNA was separated from the

plasmidsequences asdescribedinthetext.After48h, thecultureswereinfected with adenovirustype2(2x

107 PFU per plate, approximately 10 to20 PFU per

cell). After an additional 48 h, the cultures were

washedoncewith TS, incubated for 30minwith 1 ml ofDMEMlacking methionineperplate, and thenwere

incubated for 2 h with (per plate) 0.3 ml ofDMEM lacking methionine and containing 80 ,uCi/ml of [35S]methionine and 2%dialyzed fetal bovine serum.

Thecultureswerewashed twicewithTS,andextracts

were prepared by the addition of 0.2 ml of sample buffer (1% SDS-1.4 M 2-mercaptoethanol-0.25 M EDTA-15%glycerol-0.1% bromphenol blue).The

ex-tractswereboiledfor 10min, andequal quantities of acid-precipitable radioactivitywereloadedonto17.5% SDS-polyacrylamide gels,asdescribedby Klessigand Anderson (24). The gels were fixed, dried, and

ex-posedtoKodakXAR-5 film for18h. Lanes:(a)CV-C cells, infectedwithadenovirus; (b) CV-C cells, mock-infected; (c) A549 cells, infectedwith adenovirus; (d) CV-Ccells,preinfected with wild-type SV40; (e) CV-C cells, preinfectedwith dlA2459;(f)CV-C cells, prein-fected with d11265; (g) CV-C cells, preinfected with d11263. The positions of adenovirus polypeptides II, III,and IV areshown.

The large T antigens encoded by dIA2413,

dIA2414, and dlA2415 (Fig. 5) should exist as a fusion product between the N-terminal 629

ami-noacids of large T and the residues encoded by

the alternate readingframe at the 3' end of the

early region (Fig. 5), resulting in a polypeptide

slightly larger than wild-type large T antigen. The polypeptides detected (Fig. 2) migrated

more rapidly than large T. There are two

possi-ble explanations for this observation. (i) This new C-terminal domain could cause a major

change in the physical properties oflarge T, so

that the mutant large T migrates more rapidly

than wild-type large T. (ii) This domain of the

mutantlarge Tantigens may be unstable,

result-inginproteolysis. SV40 largeTmigrates with an

apparent molecular weight of90 kilodaltons in

ourSDS-polyacrylamide gels,whereasitsactual size, determined from the DNA sequence, is

81.6kilodaltons (17, 35). It has been shown that

deletions in this portion of large T cause it to

migrate more rapidly than would be expected

from thesizeofthedeletion,suggesting that this

portion of large T contributes to its aberrantly

slow electrophoretic migration (10, 33).

There-fore, the mutant large T antigens would be expectedtomigrateneartheobserved positions

in the absence ofproteolysis.

Mutants dlA2413 and dlA2415, with deletions

at 0.219 mu, were defective for replication of

viral DNA. Mutant dIA2459, with a lesion at

0.193 mu, was not viable but was positive for

viral DNA replication. Therefore, dIA2459was

blocked at a later step ininfectionthan were all

previously described nonviable mutants with deletions in the A gene (10, 25; Cole,

unpub-lisheddata) and most tsA mutants (7, 21, 22, 43).

Amino acids encoded distally to 0.193 mu are

notrequired forthe viral replication function of

large T antigen. We cannot determine whether

amino acids encoded distally to 0.219 mu are

required for replication, because the large T

antigens of dlA2413, dIA2414, anddIA2415 con-tain at their C-termini approximately 95 amino acids encoded by the alternate reading frame.

The loss of the replication function of large T

mayresult from this substitution. The

substitut-ed residues differ substantially in both charge

andhydrophobicity from the wild type(Fig. 5).

The determination of the importance for viral

DNA replication of residues encoded between

0.219and 0.193muwillrequiretheisolation and

analysisof SV40mutantswhichproduce large T

antigens truncated at different sites between

0.219 and 0.193 mu.

In dlA2459-infected cells, DNA replication

occurred, although only one-fourth as much

progeny DNA was synthesized (Fig. 3). No

plaques were produced when CV-lp cellswere

transfected with dlA2459 DNA. In these proper-.,I

=

!R

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[image:6.492.51.242.82.356.2]

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A. 630

SV4.LeuArg AsnSerAspAsp Asp Asp Glu Asp

iR

Gln Glu Asn Ala Asp dlA2459... l

650

Lys Asn Glu Asp Gly Gly Glu Lys Asn Met Glu Asp-,-,r"Gly His GluThr Gly

B.

670

leAspSe.r.Gin Se.rGin Gly

,S;e

Phe Gln Ala Pro Gln Se,SerGinete Vol Val CysSer-COOH

690

His Asp His Asn GinPro Tyr His Ile Cys Arg Gly Phe Thr Cys PheLys Lys

706

Pro ProrE Pro Pro Pro Glu Pro Glu Thr -COOH

Glu Thr Val Met Met Met Met Lys Thr Ala Arg Lys Met Leu Ile Lys Met LysMetVal Gly Arg Arg Thr Trp Lys Thr Gln Ala Met Lys Gin Ala Leu Ile His Ser Pro Lys Ala His PheArg Pro Leu Ser Pro His Ser Leu Phe Met Ile Ile Ile Ser His Thr Thr Thr PheVal Glu Val Leu Leu Ala LeuLysAsn Leu Pro His Leu ProLeu AsnLeuLysHis Lys MetAsn AlaIle Val Val Val Asn Leu PheIle Ala Ala Tyr Asn Gly Tyr Lys-COOH

FIG. 5. (A) Amino acid sequence of the carboxy terminus of large T antigen. Amino acid residues are numberedaccordingtoTooze(46).Phosphorylatedresiduesareboxed;residues which may bephosphorylated

areenclosedby a dotted box (37). Ser676,Ser677, andSer679arephosphorylated inasmallfraction oflargeT molecules present in the infected cell(G. Walter, personalcommunication).Thecarboxyterminus of thelargeT

antigen produced bydlA2459isalsoshown. TheopenboxrepresentsthatportionofdIA2459largeTidentical to

wild-type antigen. (B) Amino acid sequence of thealternateopen readingframe.

ties, this mutantresembles tsA1642 (11),which

has a single base alteration at 0.31 mu. The

lesions in tsA1642 and dlA2459 affect different functions of large T, since tsA1642 is able to

complementdlA2459 forplaque formation

(Tor-nowand Cole, in press).

Adenovirus helper function is the only known property oflarge T associated with the portion of the polypeptide that is absent from dIA2459 largeT.Since dlA2459largeTlacks the residues associated with adenovirus helper function, it

was not surprisingtodiscoverthatdlA2459had

littleornoabilitytoenhancethesynthesisofthe

lateadenoviruspolypeptides (Fig.4).Therefore,

ourresults suggest thatadenovirus helper func-tion isrequired foraproductive SV40 infection. The large T antigen produced by dlA2459 also lacksat least one site of phosphorylation (Fig. 5).

MutantdlA2459 defines an additional T

anti-gen function that is required for a productive infection. Although SV40 and polyoma largerT

antigens show substantialhomology,each

poly-peptide has regions which have no homology

with the other large T antigen. The entire

C-terminal one-sixth ofSV40large T antigen is not

homologous toanyportion ofpolyoma large T.

The studies described in this report show that

thefunction defectivein dlA2459requires some

of thisportion oflargeT.

ACKNOWLEDGMENTS

We thankGeorge Santangelo and Maryellen

Polvino-Bod-narfor usefuldiscussions,suggestions,andcritical reading of

the manuscript. We also thank Kathy Miceli for excellent technical assistance.

This work was supportedby grant PCM80-21805 from the National Science Foundation. J.T. wassupported by a Public Health Service predoctoral training grant from the National Institutes of Health.

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