Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome.

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NOTES

Nucleotide

Sequence Analysis of Two Simian Virus

40 Mutants with Deletions in the Late Region of the

Genome

ROLAND CONTRERAS,'* CHARLES COLE,2 PAUL BERG,3 ANDWALTER FIERS'

Laboratoryof Molecular Biology, State University ofGhent, B-9000 Ghent, Belgium'; Department of

HumanGenetics, School of Medicine, Yale University, NewHaven, Connecticut065102;andDepartment of

Biochemistry, Stanford Univesity Medical Center, Stanford, California 943053

Received for publication9August1978

Twomutantsofsimian virus 40, dl-1261 and dl-1262, have deletions thatmap

between coordinates 0.90 and 0.95 (Coleetal., J. Virol. 24:277-294, 1977). Both affect thestructureof thetwominorproteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence

analysis. Mutant dl-1261 is deleted of 54 base pairs, istemperaturesensitive for theprotein defined by the D complementationgroup,andpromotesthe synthesis

ofshorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of

temperatureand hasadeletion of 36basepairs, 23 of which overlap the deletion

indl- 1261.Since thesemutantsproduce normalVP1, the deleted regions probably havenofunction in the splicing ofprecursorRNAtothe VP1 mRNA.

Deletion mutants ofsimian virus 40 (SV40) have been generated by enzymatic cleavage of the genome (9, 13) orby infection of cells with linear viral DNAs (1, 16). Complementation analysis and mutation site mapping have

re-vealed the presence of five genes on the SV40

genome:theearlygenesforsmall-t andlarge-T antigens and the three late genes B/C,D, and E. TheB/Cgene codes forVP1 (10), the main structuralprotein of thevirion, and theDand E

genescode for the minorstructuralproteins VP3

andVP2, respectively (3). Nucleotidesequence

analysisofSV40DNA (5, 14) has allowedus to

define theprecise location of thesegenes onthe

genome. Cole et al. (3) isolated a number of viable deletionmutantsby cleaving the circular SV40DNAwith

Si

nuclease.Twoof thesehave adeletionwhichmaps inHindfragmentE(Fig. 1), an internalsegment of theVP2/VP3 genes;

the mutant with the larger deletion, dl-1261, showed the tsD group phenotype at elevated

temperature, whereas the mutant with the

smallerdeletion, dl-1262,wasviableat temper-aturesup to41°C.

To map the deletionin dl-1261, we digested

the mutant DNA with the restriction enzyme

AtuI, which has thesamerecognitionsequence

asEcoRII(15;H. VanHeuverswyn,J.Seurinck, andM. VanMontagu,personalcommunication).

The 5' ends of the resulting fragments were

dephosphorylated with bacterial alkaline

phos-phatase andlabeled with T4polynucleotide

ki-naseand[y-32P]ATP, and theDNA wasfurther digested with the restriction endonuclease HaeIII. The nucleotide sequence of the AtuI/ HaeIIIsubfragment encompassing thedeletion

was determinedby the partial chemical degra-dation procedure of Maxam and Gilbert (12). The chemical degradation pattern is shown in Fig. 2a; it allows the reading of the sequence

fromnucleotide1339 tonucleotide 1257,as

sum-marized inFig.3.Acomparison of thissequence

information with the knownsequence of SV40 DNA revealed that a continuous stretch of54

nucleotideswas absentfrom thegenome of

dl-1261. Since this number is a multipleofthree, the deletion should not disturb the translation of the carboxyl-terminal part of the VP2 and

VP3 proteins. Thenucleotides deleted from

dl-1262 werealsodefined by digesting themutant DNAwith AtuI and labelingthe 5' ends of the resulting fragments with

[y-32P]ATP

and T4

polynucleotidekinase.Again, the restriction

en-donuclease HaeIII was used for the second

digestion to separate the labeled ends of the

fragments. The partial chemical degradation

patternof theAtuI-HaeIIIsubfragment

encom-passingthe deletion is shown in Fig.2b. A

36-basepair stretch is absent fromthe mutant DNA ascomparedwiththe wildtype.

The 54-base pair deletion in dl-1261 should shorten VP2 and VP3polypeptides byonly2,040

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790 NOTES

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D daltons instead of the

6,000

daltons which was

estimatedbyelectrophoresisof themutant

pro-teinon sodium dodecylsulfate-polyacrylamide gels (3).Thiscould beexplained in severalways: a differential stability of the mutant and

wild-typeCOOH-terminal portion of the polypeptide

to proteolysis; or a change inthe residual

sec-ondarystructure (perhaps duetotheverybasic COOH-terminal

tail),

giving

adistortedestimate

of the change inpolypeptide molecular weight resultingfromthe deletion.Studies of the struc-tureof themutantandwild-type COOH-termi-nalportion of the VP2 and VP3 proteinswould

clarify thisanomaly. Inmutantdl-1262 the

dele-tion of 36 base

pairs

corresponds

toareduction

of1,450daltons ofprotein, whichcompares

rea-sonablywell with the decrease of2,000daltons

estimated onsodium dodecyl sulfate-polyacryl-amidegels. Thisdeletion also remains inphase

and allows normal translation of the carboxyl terminus of VP2 and VP3 (an aspartic acid

res-idue isreplaced byaglutamicacid residue, and 12further aminoacidsare deleted).

As shown in Fig. 3, the deletion in dl-1262 overlaps the deletion of dl-1261by 23basepairs.

Since dl-1261 is defective at elevated tempera-tureinthe Dcomplementationgroupfunction, it isinvitingtospeculate that the10amino acids codedby the nonoverlapping DNA stretch, from nucleotide1269 tonucleotide1299,contributeto

thecorrectthree-dimensionalfolding of the

pro-tein or that these amino acids are directly in-volved inaninteraction betweenthe protein and

other viral components. The temperature

sen-go

sitivity ofmutantdl-1261 and the fact that the

basic carboxyl terminus of the VP2/VP3

pro-teinsmayinteractdirectly with theDNA (2, 4,

0 8, 10)favor thelatterpossibility.

Fromnucleotidesequencestudiesonthe late

WCFF

mRNA's, Haegeman and Fiers (7) have shown

thatthepresumptive site for the splicing ofthe

nuclear (or cytoplasmic) precursor to formthe

late, 16S mRNA is located between nucleotides

L 1431 and L 1458 (Fig. 3). A more precise localization of this site has been reported

re-cently by Ghosh et al. (6). Secondarystructure

modelsfor thenucleotide sequence

encompass-ing this regionhave been proposed (4, 6). The

deletion mutants described here do not affect these hairpinstructures. Both mutants

synthe-size normal amounts of VP1 protein (3) and

probablyof the late, 16SmRNA. It is therefore

unlikely that the signal for the processing or

FIG. 1. HindIII digestion pattern of wild-type

SV40 DNA and the DNAof the deletionmutants

dl-1261anddl-1262. Thelengthofthe deletion ineach

mutant was estimated from this gelpattern as 50

basepairsfordl-1261 and 40basepairsfordl-1262.

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Gin-Ile-Ser-Phe-Gly-His-Thr-Tyr-Asp-Asn-Ile-Asp-Glu-Ala-Asp-Ser-Ile-Gln-Gl n-Val -Thr-Glu-Arg-Trp-Glu-Al a-Gln-Ser-Gl

n-5: * *CMA.ATA.TCA .TTT .GGG .CAC .ACC .TAT .GAT .AAT .ATT .GAT GAA.GCA .GAC .AGT .ATT .CAG.CMA.GTA.ACT .GAG .AGG .TGG GAA.GCT .CAA .AGC .CAA

1260 t 1280 1290 d-1261 t 1310 1320t 1340

dl-1262

Ser-Pro-Asn-Val

-Gln-Ser-Gly-Glu-Phe-Ile-Glu-Lys-Phe-Glu-Ala-Pro-Gly-Gly-Ala-Asn-Gln-Arg-Thr-Ala-Pro-Gln-Trp-Met-Leu-Pro-AGT .CCT.AAT .GTG .CAG.TCA .GGT .GAA .TTT .ATT .GAA .AAA .TTT .GAG .GCT .CCT .GGT .GGT.GCA.AMT.CAA.AGA .ACT .GCT .CCT .CAG.TGG,.ATG.TTG CC;

1350 1360 13?0 1380 1390 1400 1410 1420 1430

Leu-Leu-Leu-Gly-Leu-Tyr-Gly-Ser-Val

-Thr-Ser-Ala-Leu-Lys-Ala-Tyr-Glu-Asp-Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu-Ser-Arg-TTA .CTT. CTA.GGC. CTG.TAC.GGA.AGT.GTT.ACT.TCT .GCT .CTA .AAA .GCT A

j-C

.CCC .AAC.AAAM.AAG.AM .AGG .AAG .TTG .TCC .AGG...3'

1440 1450 1460 1470 -4i14u8 90 1500 1510 1520

HindE K

FIG. 3. Nucleotide sequenceoftheHind-K-proximalpartof HindfragmentE. The sequence shown above

is thecomplementof the chemicaldegradation patterns shown inFig.2, and it has thesamepolarityasthe

SV40 late messenger. Nucleotidesarenumberedaccordingtothe system usedbyFiersetal.(5)forthe total

sequenceof the SV40 genome; the lastdigit ofeach number appears under thenucleotidebearingthe number.

The amino acid sequence codedforby this DNA region is shown above the nucleotide sequence and is part

of the carboxyl-terminalportionofVP2 and VP3. Thedeletionofdl-1261 is54basepairslongand doesnot

alter thereadingframe ofVP2 and VP3(although18amino acidsarelostfromeachprotein). The deletion

of dl-1262 eliminates36basepairs, which correspondstoalossof12amino acidsfrom VP2 andVP3. This

deletionbeginsaftertheG residue inaGAC codon and ends12codonsaway

(reading

in the5'-* 3'direction)

after the C residueofaCAAcodon,thuscausingtheconversionofanasparticacid(GAC)to aglutamicacid

(GAA) andconserving thereading frameofthecarboxyl terminusofVP2 and VP3. The deletionsofthetwo

mutantsoverlap forastretchof23basepairs. The potentialstartcodonsforVP1areboxed. The dotted line

indicates theregionwherethesplicingthat resultsin theprocessingofprecursorRNAto16S mRNAoccurs

(7);accordingtoGhoshetal.(6), thespliced-out region extendstonucleotides1441through1443.

splicing of the major late mRNA contains

nu-cleotides located between positions 1268 and

1335. The signal present in Hind fragment E that is required for the expression of the VP1

gene(10) may verywell be limitedtothepartof

thisHind Efragment justproximal to the Hind E/K junction.

We thankJose Van derHeyden for preparing the viral DNAand J.Seurinck and M. Van Montagu for gifts of AtuI endonuclease.

Wealso thank the Kankerfonds of the Algemene Spaar-en Lijfrentekas forfinancial support. R.C. holdsafellowshipfrom theNFWO ofBelgium.

LITERATURE CITED

1. Carbon, J., T. E. Shenk, and P. Berg. 1975. Biochemical

procedure forproduction of small deletions in simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 72: 1392-1396.

2. Christiansen, G.,T.Landers,J.Griffith,and P.Berg. 1977. Characterization ofcomponents released by alkali disruption of simian virus40. J. Virol.21:1079-1084. 3. Cole, C.,T.Landers,S.Goff,M.Manteuil-Brutlag,

and P.Berg.1977.Physicalandgenetic characteriza-tionofdeletionmutantsofsimian virus40constructed invitro. J.Virol. 24:277-294.

4. Contreras,R.,R.Rogiers,A. Van deVoorde, andW.

Fiers.1977.Overlapping of theVP2-VP3gene and the VP,genein theSV40genome.Cell12:529-538. 5. Fiers, W., R.Contreras,G.Haegeman,R.Rogiers, A.

Vande Voorde, H. Van Heuverswyn, J. Van Her-reweghe, G. Volckaert, and M. Ysebaert. 1978. The complete nucleotide sequence ofSV40 DNA. Nature

(London)273:113-120.

6. Ghosh, P. K., V. B.Reddy,J.Swinscoe, P. V.

Chou-dary,P.Lebowitz,and S.Weissman.1978.The 5'-terminal leadersequenceoflate16S mRNA from cells infected with simian virus 40. J. Biol. Chem. 253: 3643-3647.

7.Haegeman, G., and W. Fiers.1978.Evidence for splicing ofSV40mRNA.Nature(London) 273:70-73. 8. Huang, E. S.,M. K.Estes, and J. S. Pagano. 1972.

Structure and function ofthe polypeptides in simian virus. I. Existence of subviral deoxyribonucleoprotein complexes. J. Virol. 9:923-929.

9. Lai,C.-J., and D. Nathans.1974.Deletion mutantsof SV40 generated by enzymatic excision of DNA frag-mentsfrom the viral genome. J. Mol. Biol. 89:170-193. 10. Lai, C.-J., and D. Nathans. 1976. The B/C gene of

simian virus40.Virology 75:335-345.

11. Martin, R. G. 1977.Onthenucleoprotein coreof simian virus40.Virology 83:433-437.

12.Maxam, A., andW.Gilbert. 1977.Anew method for sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 74: 560-564.

13. Mertz, K. E.,J.Carbon,M.Herzberg, R. W. Davis, andP.Berg. 1974. Isolation andcharacterization of

FIG. 2. Partialchemicaldegradation pattern of the DNA restriction fragmentencompassing the deletion ofeach mutant: (a) fragmentAtuI1392-HaeIII,218fromdl-1261 and (b) fragmentAtuI1392-HaeIII1218from dl-1262. Thenumbering of the nucleotides is according to Fiers etal. (5) and isbased on thenumbering of the

totalsequenceoftheSV40 genome. The reaction mixtures were run on12%polyacrylamide gels containing 7

Murea, asdescribed earlier (4). Thenucleotide sequence isreadfrom the bottomof the gel (5'endofthe

fragment)tothe top(3' end of the fragment); the sequence read from the gel pattern is thecomplement of the

sequenceshowninFig.3. Thepointofthedeletion isindicatedbyan arrow.

792 NOTES J. VIROL.

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individual clones of simian virus 40 mutantscontaining deletions, duplications and insertions in their DNA. ColdSpring HarborSymp. Quant.Biol. 39:69-84. 14. Reddy, V. B., B.Thimmappaya, R. Dhar, K. N.

Sub-ramanian, B. S. Zain, J. Pan, P. K.Ghosh, M. L. Celma, and S. M. Weissman. 1978. The genome of simian virus 40.Science200:494-502.

15.Roizes,G.,M.Patillon,and A. Kovoor. 1977. A restric-tion endonuclease from Agrobacterium tumefaciens.

FEBSLett. 82:69-70.

16. Shenk, T. E., J.Carbon, and P. Berg. 1976. Construc-tion andanalysis of viable deletionmutants of simian virus 40. J. Virol. 18:664-671.

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