Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the region coding for the carboxyl terminus of the T antigen.

Vol.30, No. 3 JoURNAL OFVIROLOGY, June 1979,p.936-941

0022-538X/79/06-0936/06$02.00/0

Nucleotide

Sequence Analysis of

Two

Simian Virus

40

Mutants

with

Deletions

in

the

Region Coding for the Carboxyl

Terminus

of the T

Antigen

HUGO VANHEUVERSWYN,' CHARLES COLE,2 PAULBERG,3AND WALTER FIERS'*

LaboratoryofMolecular Biology,State University of Ghent,B-9000Ghent,Belgium';Departmentof

HumanGenetics, SchoolofMedicine, Yale University, New Haven, Connecticut($5102;andDepartment of

Biochemistry,Stanford University Medical Center, Stanford, California,943053

Received forpublication 28 November 1978

Nucleotidesequenceanalysis oftwo si virus40earlymutants,dl1263 and dl1265, which lack a DNA segmentaroundmap positions0.21 and 0.18,

respec-tively (C. Cole, T.Landers, S. Goff, S.Manteuil-Brutlag, and P. Berg, J. Virol. 24:277-294, 1977), revealed in-phase deletions of33 nucleotidepairsfordl1263 and39nucleotidepairsford11265.The33-base-pairdeletion indl1263doesnot

correspond to anapparentshortening by 6,000 daltons observed for themutant Tantigen. Indl1265the normal terminationsignalaswellasmostof the proline-richterminal tryptic peptidehas beenremoved, and thecarboxylterminus of the

mutantTantigen isaseries of threecysteine residues.

Thesimian virus40(SV40)DNAearly region

is at least 2,574 base pairs long and extends counterclockwise from approximately 0.65 to 0.17 mapunits. Theearlyregion codes fortwo proteins,small-t andlarge-Tantigen (11,14,15), whichareinitiatedatthesameAUGstartcodon

corresponding to map position0.649 (12). The

correspondingATGsequence onthe DNA fixes areadingframe that isopen up tothe informa-tionfor a termination codon at position0.547, and a polypeptide with molecular weight of

20,503, whichpresumablycorrespondsto

small-t, can be deduced from the DNA nucleotide

sequence (19). Large-T hasamolecular weight

of 94,000, and thelarge-T gene comprises two noncontiguoussegmentsof theearly region:the first extends from 0.649 to about 0.59 andthe secondsegment runsfrom about0.535 to 0.174

(1,5, 6, 12, 18,19). Atleasttwoadditional

virus-induced early antigens, U antigen and tumor-specifictransplantation antigen, have been

de-tected both inlyticinfections and intransformed

cells; theyarecoded forby the distalandmiddle portion, respectively, of the SV40 early region (ref.20and referencestherein). Moreover, SV40

expresses afunction whichhelpshuman

adeno-virus to growinmonkeycells. By isolationand

characterization ofadenovirus-SV40hybrids,it wasshown that thishelper function corresponds tothecarboxyl-terminalpartofthelarge-T an-tigen (8,17),of which thecodingDNAsequence wasrecentlypublished (16, 18).

Twoviableearly-regionmutants, dl1263and

d11265,lackaDNAsegment inthisC-terminal

region. The position of the deletion in d11263 wasestimatedaround0.21 mapunits (theexact mapposition of thisdeletion is in fact0.20),and its large-T antigen is apparently 6,000 daltons lighter than wild-type large-T; the deletion in

dl1265 maps around position 0.18 and

appar-entlydoesnotaffect the size oflarge-Tantigen (4), although in the absence of alkylation the

latter seems to be more prone to aggregation effects (C. Cole, L. V. Crawford, and P. Berg, submitted for publication). Both dl1263 and

dl1265arederived from the viablemutantd1861

(4).

Tocharacterize these deletionsatthe nucleo-tidelevel,eachmutantDNAwas digested

sep-arately with restriction endonucleases HindII

+ III, HaeIII, and AluI (Fig. 1 and 2). d11263

gave rise to a shorter (relative to wild type)

HindII + III fragment B and a shorter AluI

fragmentA.The characteristic bands for HaeIII fragmentsC and D weremissinginthe restric-tion cleavage pattern. The resulting newband

was notdetectedon the gel,but itpresumably

comigrated with the HaeIII fragment B. We conclude thatdl1263lacks the HaeIIIcleavage

site atmapposition0.195.

Upon digestion with therespective enzymes, dll265 DNA also resulted in a shorter HindII

+IIIfragmentBand in ashorter AluIfragment A. Furthermore, digestionwithHaeIII enzyme revealed a shorter fragment C, thus localizing thed11265deletionbetween theHindII+IIIG/ Bjunction (map position0.169) andtheHaeIII cleavage siteatmapposition0.195(Fig.2).

936

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1 2 3

FIG. L. Mappingofthedeletionsinmutantsd11263

and d1ll. ThepurifiedDNA ofeach mutant was

digestedwithrestriction endonuckasesHindII+III

(A),HaeIII(B), orAluI (C), andelectrophoresedon

a4%polyacrylamideslab gelovernightat180V. (1)

Wildtype;(2)dll263; (3) d11265.

Tantigen

le.

--;; x =_

S_aa _;E 7_'a ';

1<41 Ia

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'if

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11

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0

G B

HindII. m

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AluI ---I aA

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FIG. 2. Location ofsome restriction enzyme cleavagesites in the SV40DNA region encompassingthe

regioncoding forthecarboxyl-terminalpartofTantigen.Mapunits arebasedonthecompletenucleotide sequence(6), with theunique EcoRI cleavage siteaspointzero.

937

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938 NOTES

To mapmoreprecisely the deletion ind11263

DNA, the HindII + III restriction fragments

were5'-terminally labeled with [y-32P]ATP and T4polynucleotide kinase.AluI digestion of the

Hind fragment B generated a singly 5'-termi-nally labeled subfragment, Hind-B-Al, which

spannedthedeletion(Fig. 2).Thissubfragment

was partiallydigestedwithHinf.Separation of the products showed the absence of the Hinf restriction site atmapposition0.200 (data not shown). Hence, because d11263 also lacks the

HaeIIIsite atposition 0.195, the deletionmust extend at least fromposition0.195 tothe Hinf

site at position 0.200. To determine the exact size of thedeleted DNA segment, the Hinf

frag-mentbetween mappositions0.992and 0.204was

5'-terminally labeled and cleaved with HindII

+ III. Partial chemical degradation of the

smallersubfragment bytheprocedureof Maxam

and Gilbert (9) revealedanin-phasedeletionof 33 base pairs (Fig. 3A and 4). This deletion

correspondstothe absence ofnucleotides E2413 toE 2445 inthenewnumberingsystemadopted

forthecompleteSV40 DNA sequence (6).

The exact extent of the deleted DNA segment in mutant d11265 was determined as follows.

HindII + III fragment B was 5'-terminally la-beled and cleaved withHaeIII; the nucleotide sequence of theresulting subfragment contain-ing the deletion, Hind-B-E3, was then deter-mined bypartial chemicaldegradation. A dele-tionof39basepairswasfound

(Fig.

3B and4),

which corresponds to nucleotides E 2523 to E 2561. For d11263, several gels were run

inde-pendently,whereas ford11265concurrent infor-mation was obtained from both strands (not

shown).

The 33-base-pair deletionindll263 doesnot accountfor theapparent6,000-daltonreduction insizeobserved for thelarge-T antigeninduced by this mutant(4).Consideringthisdiscrepancy,

and also because theoriginalmutantDNAstock showedsomeheterogeneity, the d11263mutant wasrecloned. The DNAobtainedfrom the

sin-gle-plaque-derived

stockwas

perfectly

homoge-neous, and nucleotide sequence analysis

com-pletelyconfirmed the aforementioned in-phase

deletion. This reclonedd11263produced againa major87K inadditiontoaminor 93K Tantigen (Cole et al.,submitted forpublication). There-fore,it isunlikelythat thediscrepancybetween theexpectedand theobservedweightreduction

for thelarge-Tproteinsynthesized byd11263is

due to thepresenceof acontaminant. Theminor

93K polypeptide could correspond to the ex-pected Tantigen,whichis shorterby11amino acids compared to the predicted sequence of wild-typelarge-T (Fig. 4).The fact thatd11263

J. VIROL.

retains 32% of the wild-type helper capacity for growth of human adenovirus in monkey

cells,

whereasd11265, whichlacks only the nine

car-boxyl-terminal amino acids (Fig. 4), retains

merely 6.5% of the helper ability (Cole et al., submitted for publication), also supports the

idea that there is no out-of-phase deletion in

d11263. Shortening the large-T antigen by 11 amino acids may affect the proper folding such that the carboxyl-terminal tail becomes more

susceptible to proteolytic removal. Recent

re-sults obtained by pulse-labeling with

[3S]me-thionine, however, argue against a

precursor-product relationshipbetween the 93K and 87K

large-Tproteins (Cole et al., submitted for

pub-lication). Also, protein synthesis in vitro pro-grammedwith

d11263

early mRNA mainly

pro-ducesthe 87Kpolypeptide(10, 13). Theseresults suggestthatthe latter is not forned by

proteol-ysis. Alternatively, the 87K polypeptide might result from premature chain termination at a leaky stop codon, but at least 60 nucleotides in the region preceding the deletion have been checked and were found to be identical to the wild-type sequence. Stillotherexplanations are

possible, such as an additional splicing event.

Characterization of the 87K protein and the

correspondingmRNAmight shed morelighton

thisproblem.

The large-T antigenproduced byd11265has

lostmostof thepeculiar,

proline-rich

carboxyl-terminal segment as deduced from the DNA sequence (Fig. 4). The low helper ability of

d11265

suggeststhat the carboxyl-terminal

re-gion of large-T is indeed involved in the helper process but is not required for SV40 viability.

Also,

since both mutants were shown to

trans-form ratfibroblasts for growth in soft agar al-mostaswell aswildtype (2), the carboxyl-ter-minal tail appears to be inessential for transfor-mation. It may be noteworthy that in

d11263

the nucleotidesborderingon the deleted region are

locatedwithin two loops of apossible secondary structure thatcanbe constructed for the DNA segmentencompassingthedeletion (Fig. 5).

Be-cause

d11263

wasprepared by

S1

nuclease

cleav-age ofamixtureofSV40 I and II DNAs (3), the aforementioned observation may be related to thehypothesis that supercoiling of a DNA mol-eculeproduces regions of interrupted secondary structure whichmigrate until they reach a seg-ment where sufficient intrastrand complemen-tarityis present (7, 21). No such model can be proposed for

d11265,

because several possible secondary structurescanbeconstructed for the relevantregion.

We thank A. Van de Voorde and Jose Van der

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FIG. 3. Sequence analysis ofthe relevant DNA segmentsofmutants d11263 and d11265. (A) TheHinf

fragmentbetweenmappositions0.942 and 0.204wasi-terminallylabeled andcleaved withHindII+III.The

resulting singly5i-terminallylabeledsubfragmentcontainingthedeletionwaspartially degraded according

totheprocedureofMaxam and Gilbert(9).Thereactionproductswerefractionatedon a12%polyacrylamide

slabgelcontaining7MureaandTris-boratebuffer (pH83).Thenucleotidespecificity ofeachreaction is

indicated above each lane. The siteofthedeletion is indicated by adeltasymboL (B) Thesubfragment

containingthedektioninmutantd11265 and labeled atonlyone5'endwasgenerated bykinationofthe

HindII+IIIfragmentBandsubsequentcleavagewith the restrictionendonuclease HaeIII. Itssequencewas

determinedasdescribedin(A).

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940 NOTES J. VIROL.

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1263

(0.20)

A

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G*A

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A-T A-T G-C T-A A G C-G 0-C G-C 0-C C T C C T-A C-G A-T G-C A C A-T G-C G0*

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FIG. 5. Possible secondarystructure of the DNA

segmentencompassing thedeletion inmutantdll263.

The deletedsegmentis 33nucleotidepairs long and

ismarked with aheavyline. Thebordering

nucleo-tides(residuesE 2412 and E2446)areboth located

within aloop. Thereadingframe ismarkedoff by

dots, and base-pairing is indicated by dashes.

Heyden for help with the biological aspects of this

work,and L.Crawford for keepingusinformedabout

his results. This research was supported by grants

fromthe Algemene Spaar-en Lijfrentekas and from

theGeconcerteerde Akties of the Belgian Ministry of

Science.

LITERATURE CITED

1. Berk, A. J., andP. A.Sharp.1978.Spliced early

mes-sengerRNAsofsimianvirus 40. Proc. Natl. Acad. Sci. U.S.A. 75:1274-1278.

2. Bouck, N., N. Beales, T. Shenk, P. Berg, and G. diMayorca. 1978. New regionofthe simianvirus 40

genomerequired for efficientviral transformation. Proc. Natl. Acad. Sci. U.S.A.75:2473-2477.

3. Carbon, J.,T. E.Shenk, and P. Berg.1975.Biochemical procedure for production ofsmall deletions in SV40

DNA.Proc. Natl.Acad. Sci. U.S.A. 75:1392-1396. 4. Cole, C., T. Landers, S. Goff,S. Manteuil-Brutlag,

and P.Berg. 1977. Physical and genetic characteriza-tion of delecharacteriza-tion mutants of simian virus 40 constructed invitro. J.Virol. 24:277-294.

5. Crawford, L. V., C. N.Cole,A. E.Smith, E. Paucha, P.Tegtmeyer, K.Rundell, and P. Berg. 1978. Or-ganization and expression of early genes of simian virus 40.Proc. Natl. Acad.Sci. U.S.A. 75:117-121. 6. Fiers, W., R. Contreras, G. Haegeman, R. Rogiers, A.

Van de Voode, H. VanHeuverswyn, J. Van Her-reweghe, G. Volekaert, and M. Ysebaert. 1978. Complete nucleotide sequence of SV40 DNA. Nature (London) 273:113-120.

7. Lebowitz, J.,C. G. Garon, M. C. Y.Chen, and N. P. Salzman.1976.Chemical modification of simian virus 40DNA by reaction with awater-solublecarbodiimide. J.Virol. 18:205-210.

8. Lebowitz,P., T. J.Kelly,D.Nathans,T. N.Lee,and A. M. Lewis. 1974. A colinear map relating the simian virus 40(SV40) DNA segments of six adenovirus-SV40 hybrids to the DNA fragments produced by restriction endonucleasecleavage of SV40 DNA. Proc. Natl. Acad. Sci. U.S.A. 71:441-445.

9. Maxam,A.M., and W.Gilbert.1977.A new method for sequencingDNA. Proc.Natl. Acad.Sci.U.S.A. 74:560-564.

10. May, E., M. Kress,and P. May. 1978. Characterization of twoSV40 early mRNAs and evidence for a nuclear "prespliced" RNA species. Nucleic Acids Res. 5:3083-3099.

11.Paucha, E., R.Harvey, R. Smith, and A. E. Smith. 1977.Thecell-free synthesis ofSV40 T antigens, p. 189-198.In P. May, R.Monier, and R.Weil (ed.), Early proteins of oncogenic DNA viruses. INSERM Collo-quium, vol. 69.INSERM,Paris.

12.Paucha, E., A.Meilor,R.Harvey, A. E. Smith, R. M. Hewick, andM. D. Waterfield.1978.Largeandsmall tumor antigen from simian virus 40 have identical aminoterminimappingat 0.65mapunits. Proc. Natl. Acad.Sci. U.S.A.75:2165-2169.

13. Paucha, E., and A. E. Smith. 1978. The sequences between 0.59 and 0.54 map units on simian virus 40 DNAcode for theunique regionof small-tantigen.Cell 15:1011-1020.

14. Prives, C., E. Gilboa,M. Revel, and E. Winocour. 1977. Cell-free translation of SV40 early messenger RNAcoding for viral T-antigen. Proc. Natl. Acad. Sci. U.S.A.74:457-461.

15.Tegtmeyer, P.,M. Schwartz,J. K. Collins,and K. Rundell. 1975.Regulationoftumorantigensynthesis bysimian virus40gene A.J. Virol. 16:168-178. 16.Thimmapaya, B., B. S. Zain, R. Dhar, and S. M.

Weissman. 1978. Nucleotide sequence of the DNA template for the 3' ends of SV40 mRNA. J. Biol. Chem. 253:1613-1618.

17.Tjian,R.,G.Fey,and A. Graessmann.1978.Biological

activityofpurifiedsimian virus40T-antigen proteins. Proc.Natl. Acad. Sci. U.S.A. 75:1279-1283.

18.Van Heuverswyn, H., A. Van de Voorde, and W. Fiers. 1978. Nucleotide sequence of the SV40 DNA regioncodingfor thecarboxyl-terminalpart of the T antigen.Eur. J. Biochem. 86:335-344.

19.Volckaert, G.,A. Van deVoorde, andW. Fiers. 1978. Nucleotidesequence of the simianvirus 40 small-tgene. Proc.Natl.Acad.Sci. U.S.A.75:2160-2164.

20. Walter,G., and H. Martin.1975.Simianvirus40-specific proteinsinHeLacells infected with nondefective ade-novirus 2-simianvirus 40hybridviruses. J.Virol. 16: 1236-1247.

21. Woodworth-Gutai, M.,and J. Lebowitz.1976. Intro-duction ofinterrupted secondary structurein super-coiled DNA as afunctionofsuperhelixdensity: consid-eration ofhairpinstructures in supercoiled DNA. J. Virol. 18:195-204.

A,

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