JOURNAL OF VIRoLOGY, Feb. 1977, p. 565-578 Vol. 21, No. 2Copyright © 1977 American Society for Microbiology Printed in U.S.A.

Infectious Linear DNA Sequences Replicating in SimianVirus 40-Infected Cells

PETER GRUSS AND GERHARD SAUER*

Institut fiir Virusforschung, Deutsches Krebsforschungszentrum, 69 Heidelberg, Germany

Received for publication 9 July 1976

A new class of linear duplex DNA structures that contain simian virus 40(SV40) DNA sequences and that are replicated during productive infection ofcells with SV40 is described. These structures comprise up to 35% of the radioac-tively labeled DNA molecules that can be isolated by selective extraction. Thesemolecules represent a unique size class corresponding to the length of an openSV40 DNA molecule (FO III), and they contain a heterogeneous population ofDNA sequences either of host or of viral origin, as shown by restriction endonu-clease analysis and nucleic acid hybridization. Part ofthe FO III DNA moleculescontain viral-host DNA sequences covalently linked with each other. They startto replicate with the onset of SV40 superhelix replication 1 day after infection.Their rate of synthesis is most pronounced 3 days after infection when superhe-lix replication is already declining. Furthermore, they cannot be chased intoother structures. At least a fraction of these molecules is infectious whenadministered together with DEAE-dextran to permissive cells. After intracellu-lar circularization, superhelical DNA FO I with an aberrant cleavage patternaccumulates. In addition, tumor and viral capsid antigen are induced, andinfectious viral progeny is obtained. Infection of cells with purified SV40 FO IDNA does not result in FOm DNA molecules in the infected cells or in the viralprogeny. It is suggested, therefore, that these FO III DNA molecules areperpetuated within SV40 virus pools by encapsidation into pseudovirions.

Simian virus 40 (SV40) virus particles are sequences, at least part of which are infectiouscommonly considered to convey superhelical and can be intracellularly circularized to formDNA as the predominant and possibly the sole superhelical FO I DNA containing reassortedmaterial capable of replicating from cell to cell DNA sequences.during the course of infection. As a different It was not possible by infection with purifiedkind of nucleic acid that can be encapsidated FO I DNA to generate SV40 virus pools thatand conferred by SV40 and polyoma virions to induced the synthesis of FO Im DNA structuresother cells, randomly fragmented linear host in infected cells. In this case both the infectedDNA that is contained in pseudovirions has cells and the ensuing viral progeny were devoidbeen described (6, 13, 15, 16, 25, 26, 31). This ofFO III viral host DNA. It appears, therefore,DNA has, however, not been examined for its that we are dealing with linear DNA sequencesability to replicate in the infected cell, although that are capable of replicating and that areit has been shown to reach the nucleus after passaged within pseudovirions from cell to cell.uncoating (10, 16, 17).

In this article we report the isolation of small MATERIALS AND METHODSlinear and nicked-circular DNA fragments Virus and cells. The small-plaque SV40 strainsfrom SV40-infected cells by selective extraction Rh911 and 776 and the large-plaque strain C1-307-L(8) and banding in dye-buoyant density equilib- were used. All strains were propagated in CV-1 cellsrium gradients (18). Closer analysis of these at 0.01 PFU per cell.DNA structures by gel electrophoresis revealed CV-1 cells were grown in Eagle medium with twoa number of discrete size classes, which were times the concentration of Earle amino acids andfurther characterized. In addition to nicked- vitamins. Fetal bovine serum at 10% was used. The

circular SV40 FO II DNA and fragmented host cells were infected at a multiplicity of 1 to 3 PFU/cicularl 4 OLDNA,wfon aewknd ofragmntedr hOstI cell.cell DNA, we found a new kind of linear FO III Preparation of labeled viral DNA. For labeling ofDNA that is capable of replicating in permis- viral DNA with [3H]thymidine (Buchler, Amer-sive cells. These FO III molecules comprise a sham, England; 30 Ci/mmol), the isotope was addedheterogeneous collection of viral and host DNA at 3 to 6 ,uCi/ml for the indicated periods of time.

565

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566 GRUSS AND SAUER J. VIROL.

The DNA was selectively extracted and purified by lowed by centrifugation in a dye-buoyant CsCl-density equilibrium centrifugation in CsCl-ethid- EtBr equilibrium gradient (18). When isolatedium bromide (EtBr) gradients as described (27, 28). 2 days after infection, the mature superhelical

Slab gel electrophoresis. Electrophoresis was per- SV40 DNA FO I banding at the higher densityformed either in vertical 1.4% agarose gels (Seakemagarose, mci biomedical, Rockland, Md.) or in verti- comprised approximately 60 to 70% of the DNAcal polyacrylamide gels (4% acrylamide, 0.2% bisac- m the Hirt supernatant (28).rylamide) in a previously described buffer (23). The The light band, which consists of nicked-cir-size of the analytical gels was 13.5 by 25 by 0.3 cm. cular and linear DNA, contains the DNA struc-Samples (50 to 100 ,ul) were applied to the gels and tures discussed in this work. This material wassubjected to electrophoresis for 18 h at 70 V per gel in isolated and subjected to agarose-gel electro-a cold room. phoresis. When the DNA was extracted 48 h

For preparative isolation ofband II components, a after infection, three prominent bands were1.4% agarose gel (10 by 9 by 0.3 cm) was used. A 30- * '-to 40-,ug sample of DNA was applied to the gel and snaer staiig with EtBr and exminansubjected to electrophoresis for 90 min at 100 V. under UV light (FIg. la). The uppermost bandStaining of the DNA within the gel, excision of the (termed band II) co-electrophoresed togetherdesired gel sections, and further purification of the with marker SV4O FO II DNA. The secondDNA after homogenization of the agarose were per- band (band C) was also detected in mock-in-formed as previously described (27). fected (Fig. lb) and in infected cells up to 24 h

Restriction endonuclease cleavage of DNA. The after infection (Fig. lc). Furthermore, at 48 hcleavage of CV-1 and SV40 DNA with after infection, a class of DNA molecules (bandendo R-EcoRI, HindII/III, and Hpa I/II have been III) could be isolated (Fig. la) that co-electro-described recently (7, 27). Furthermore, treatment phoresed with unit-length linear rods of SV4of SV40 DNA with S, nuclease has been described molecue (FOIll).(2). Cleavage with endo R-Bam was performed in0.01 M Tris-0.01 M MgCl2-0.01 M mercaptoethanol,pH 7.5. The strain Bacillus amyloliquefaciens was agenerous gift of J. Ortin.

Infection of cells with DNA. Cells were washedwith phosphate-buffered saline and infected withDNA in phosphate-buffered saline supplementedwith DEAE-dextran at 1 mg/ml. Approximately 2x 106 CV-1 cells were incubated with the buffer rcontaining DNA and DEAE-dextran 1 day aftertrypsinization for 30 min at 37°C, washed twice withHanks solution, and then fed with fresh medium !containing 10% fetal bovine serum. At 1 day after --infection the cells were trypsinized, mixed with 107 1uninfected CV-1 cells, and seeded at a density thatpermitted at least one to two divisions before con-fluency was reached. At 8 to 12 days after infection,when a cytopathogenic effect developed, [3H]-thymidine was added for 2 days, and then the DNAwas selectively extracted.

Alkaline velocity sedimentation. Alkaline veloc-ity sedimentation was performed as described (27).

Nucleic acid hybridization. DNA-DNA hybridi-zation and RNA-DNA hybridization were as previ-ously described (20, 28). The SV40 complementaryRNA (cRNA) was kindly provided by W. Waldeckand A. Fried. It was synthesized as described byWestphal (29) and further purified as described by FIG. 1. Agarose slab gel electrophoresis pattern ofFried (4). small relaxed and linear selectively isolated DNA

from SV40-infected cells. DNA was selectively ex-

RESULTS tracted from SV40-infected and mock-infected CV-1cells. The DNA in the light band of a CsCl-EtBr

Isolation and purification of small relaxed gradient was subjected to electrophoresis in 1.4%and linear DNA sequences by gel electropho- agarose (100 V, 90 min) and photographed using UVresis. Permissive CV-1 cells (9) were infected light after staining with EtBr (5 pg/ml). (a) DNAwith SV40 at low multiplicities and, after var- isolated 48 h after infection (2.5 pig); (b) DNA iso-ious periods of time after infection, the DNA lated 24 h after mock infection (0.5 pg); (c) DNAious peertiodseo etimeafter Te ml N

isolated 24 h after infection (0.8 pg). The symbols II,was selectively extracted (8). The small DNA C, and III are used to identify the three major bandsmolecules contained in the Hirt supematant ofDNA that will be referred to as bands II, C, and IIIwere further purified by phenol extraction fol- in this paper.

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 567

The existence ofdifferent discrete size classes bands II and III contained any prelabeled hostof nicked-circular and linear DNA in the Hirt DNA, whereas band C displayed, in addition tosupernatant is not a peculiarity of the SV40 3P label, extensive presence of [3H]thymidinestrain Rh911 (5), since the same phenomenon (Fig. 3). We conclude from these data that bandhas been observed when large-plaque strain Cl- C represents both preexisting and newly syn-307-L (11) or strain 776 (24) was used for infec- thesized host DNA, whereas bands II and mtion. Neither does the appearance of such struc- are newly replicated.tures depend on the multiplicity of infection. Analysis of relaxed and linear DNA se-Both at low (9 x 10-4 PFU/cell) and at high quences by velocity sedimentation, restrictionmultiplicities (undiluted, five times serially nuclease digestion, and nucleic acid hybridi-passaged SV40), the patterns obtained were zation. For further structural analysis, bandssimilar to that depicted in Fig. 1. It was of II, C, and III were isolated by homogenizationinterest, therefore, to investigate the kinetics of of the respective excised agarose gel sections asappearance and to test the metabolic stability previously described (27). The structural integ-of these different size classes of DNA. rity of the DNA preparations after isolation

Kinetics of synthesis of nicked-circular and from the gels was ascertained by subjecting tolinear DNA structures. Pulse-labeling and electrophoresis again. Bands II, C and Il werepulse-chase experiments using [3H]thymidine not sheared to smaller fragments and retainedand an excess of unlabeled thymidine were per- their characteristic electrophoresis patternfonned (Fig. 2). In mock-infected cells, when after purification (Fig. 4). However, the homog-labeling took place between 0 and 28 h only the enization procedure introduced some single-labeled band C was found (Fig. 2a). The same strand nicks, which became apparent only afterresult was obtained when infected cells were alkaline velocity sedimentation. Band II DNAlabeled between 12 and 24 h after infection (Fig. revealed two components sedimenting at 18 and2b). Band C could not be chased into other 16S (Fig. 5a). Single-strand nicks that werestructures by a 6-h chase (Fig. 2c). present in some molecules may account for theOne day after infection, after the onset of slower sedimenting material. As was to be ex-

viral DNA replication, in addition to band C pected from its diffuse distribution after elec-there appeared bands II and III, which could be trophoresis, band C displayed a broader patternlabeled between 24 and 30 h (Fig. 2d). When the of sedimentation ranging from structurespulse-labeling (24 to 30 h after infection) was larger than 24S to approximately 6S (Fig. 5b).followed by a 6-h chase, the amount of Finally, most of the material from band Ill[3H]thymidine in the labeled bands did not sedimented at 16S; in addition, some fonn IIIchange markedly (Fig. 2c). The rate of synthe- DNA sedimented more slowly, probably be-sis ofband C, on the other hand, appeared to be cause of the presence of single-strand nicksreduced 30 to 36 h after infection (Fig. 2f). At (Fig. 5c).later stages after infection the synthesis of As a further test for the presence of circularband II was curtailed, whereas, in contrast, relaxed structures, endo R-Bam (30) andband III was still actively synthesized. This is endo R-EcoRI restriction nucleases and also S,shown by its increase when the label had been nuclease, which cut circular SV40 DNA onlyadded either between 75 and 81 h (Fig. 2g) or once (1, 2), were employed. Endo R Bam com-between 0 and 81 h (Fig. 2h). pletely converted 3H-labeled band II DNA asThus we observed, depending on the individ- well as 32P-labeled FO I DNA, which had been

ual size classes of DNA, either a steady de- added as an internal marker, to linear FO IIIcrease in synthesis during the course of infec- structures (Fig. 6a). The same results were ob-tion (band C) or an initial increase that leveled tained with endo R EcoRI and with S, nu-off by 2 to 4 days (band II) and, finally, a clease (data not shown), thus confirming thatsteadily increasing rate of synthesis up to 3 band II DNA consists of circular SV40-sizeddays, which was noticed in the case of band III. DNA. In contrast, the original sizes of bands CTo detennine whether preexisting host DNA and III were largely retained despite digestion

sequences participate in the formation of the with endo R Bam (Fig. 6b and c). Only parts oflabeled structures described above, cells were both structures were cleaved to smaller, faster-labeled with [3H]thymidine for 1 day and then moving fragments. It is of particular interestinfected, and between 24 and 48 h after infec- that 60% of the band III DNA molecules weretion double labeling with [32P]orthophosphate devoid of the endo R Bam recognition site andwas performed. The DNA was then extracted, could not be cleaved despite reaction conditionsand the nicked-circular and linear DNAs con- that were sufficient to attain a complete digest.tained in the Hirt supernatant were analyzed This was proven by conversion of the added 32P-in an agarose gel. Neither of the 32P-labeled labeled SV40 FO I DNA to FO III. Treatment of

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568 GRUSS AND SAUER J. VIROL.

E c I.. . . _ . . ._ ..... ~~~~~~~~~ ... . w .. .... = ~~~~~~. ^.

A d 4 e

Muo1k pulseInfected 24--30hr . 36.,~~~

epuise LeEQl12 24 hr

A~~~~~~~~~~~~~~~~

pulse24 -30 hrchasea30 36 hr

XeT -..~ ~ ~ ~ ~4 h iln.->

t 24 36 hf ;!,

DISYrANCE MIGRATEtE MiM -1

FIG. 2. Pulse-labeling of small relaxed and linear DNA bands II, C, and III. SV40-infected and mock-infected CV-1 cells were pulse-labeled for the indicated periods of time with [3H]thymidine. Pulse-chaseexperiments were carried out using medium with 100 Mg of unlabeled thymidine per ml. After selectiveextraction, the DNA contained in the light bands ofCsCl-EtBr gradients was subjected to electrophoresis in1.4% agarose gels (100 V, 120 min). The gels were sliced, and the radioactivity in each slice was determined.The arrows indicate the positions of marker bands II, C, and III (see also photographs). (a) Mock-infected,pulse-labeled from 12 to 24 h post-mock infection; (b to h) SV40-infected, pulse-labeled as indicated in thepanels.

bands C and III with the single-strand-specific For a more detailed restriction nuclease anal-S, nuclease did not affect the size of the DNA ysis, endo R-HindII and -III (21) was em-samples (data not shown). Thus, single- ployed. Band III, when digested withstranded regions are not present in the above- endo R Hind, produced a cleavage patternmentioned DNA structures. that is indistinguishable from the pattern ob-

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 569

II c m Most interestingly, digestion of band IIIDNA with endo R Hind nuclease led to acleavage pattern (Fig. 7c) that is only in part

C? 4 locN reminiscent of the cleavage pattern generated0x from SV40 DNA. There were typical SV40EQ E peaks, although present in inappropriate molar

o ratios, that were superimposed on an increased0.

co -8 background. The fragment Hind A appeared tobe overrepresented, whereas, for example, frag-

3- ment B was present in lower amounts com-pared with fragment A. Although the reactionconditions were chosen such that a complete

6 digestion would be accomplished, there re-mained band III DNA molecules that were

2- either not attached at all or that contained only

-4 ~ 2~JFol[FoX FoI

1 o_U ~~~~~~3x

10 20 30 40 50 _DISTANCE MIGRATED [MM] i

20- i 2FIG. 3. Agarose gel of double-labeled preexisting

and newly replicated DNA in bands II. C. and III.CV-1 cells were labeled with [3H]thymidine (0) 24 hbefore infection with SV40. Between 24 and 48 h 10-after infection, [32P]orthophosphate (a) was added inphosphate-free medium. The selectively isolated 6-DNA, which was contained in the light band of aCsCl-EtBr gradient, was subjected to electrophoresis 2-in a 1.4% agarose gel at 100 V for 90 min (specificactivity ofthe DNA: 3H, 2.55 x 104 cpm/pg; 32p, 3.18X 104 cpmlpg)* 10

tained by digestion of marker SV40 DNA FO I(3) (Fig. 7a). Hence, in the case of band II wemust be dealing with SV40 DNA FO II, inagreement with the results obtained by the 5-other methods described above. In contrast,band C showed a diffuse pattern afterendo RNHind digestion (Fig. 7b). The bulk of 1_the DNA remained unattached, whereas part of ,it was cleaved to smaller fragments with pro- 10 20 30 40nounced fast-moving peaks that coincided with DISTANCE MIGRATED [MM]the positions of endo R HindIll-generated FIG. 4. Isolated bands II, C, and III subjected tomonomers and dimers of the repetitive compo- repeat electrophoresis. The DNA (specific activity,nent ofCV-1 DNA (7), This result suggests that 17.6 x 104 cpm/plg) contained in bands II, C, and IIIband C represents host DNA and is compatible was isolated as described in Materials and Methodswith the notion that band C also occurs in from a preparative agarose gel and again subjected tomock-infected cells. Furthermore, the results of electrophoresis in 1.4% agarose at 100 V for 2 h. (a)th N-DNAecells.bridtioneroe,xtneriments OIa Band II DNA with 32P-labeled (0) SV40 DNA asthe DNA-DNA hybridization experiments (Ta- internal marker. The marker DNA was partiallyble 1), which revealed almost exclusively ho- digested with endo R *EcoRI to generate, in additionmologies between band C and CV-1 DNA, cor- to residual FO I, FO II and FO III (see arrows). (b)roborate the cellular origin of band C DNA. Band C; (c) band III.

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570 GRUSS AND SAUER J. VIROL.

cules, in accordance with the data presented ini? 18S*416S Fig. 6c.o5O 0o The DNA sequences which resisted attack by

-x 4- -04xOA the restriction enzymes could either be of viralorigin and might have lost the respective recog-

I2\ a2 nition sequences, or else they could represent2- et \ I IX cellular DNA.1e0.1 To decide between these alternatives, two

DNA-DNA hybridization experiments wereIs rQ performed (Table 1). As expected, DNA from

band II, which represents SV40 FO II DNA,I.5- -3

hybridized extensively with SV40 DNA (60%,1.5- I \ 3 48.8%). For comparison, self-hybridization ofwild-type SV40DNA revealed 68.9% (56%) hy-bridization, whereas there was very little ho-

1- I2 mology with cellular DNA in the case of bothSV40 FO II and FO I DNA. Band C DNAshowed 4.2% homology with SV40 (in one case

Q5 -1

Foul20 X O 4L4X~~~~~0FRACTINUMBER 3I~~x6 -6x

bandsI,C,andII. Porions o the solate DNA4- 1 \ -4

V,)

36 6x

FRACTIONNUMBER36

FIG. 5. Alkaline velocity sedimentation ofisolatedband8 II, C, and III. Portions of the isolated DNA 2- 4bands described in the legend to Fig. 4 were sedi-mented through alkaline sucrose together with32p_llabeled (0) FO I and FO II, which were added as 1l 2sedimentation markers. (a) Band II; (b) band C; (c)band III. Ja few endo R Hind recognition sites. In Fig. 7cthese sequences comprise the fractions that are 2 [ 4larger than the reference SV40 Hind A frag-ment (see also Fig. 9 for comparison). Figure 8 3shows the electrophoresis pattem resultingfrom cleavage of band III DNA with 1 -2endo R*Hpa I (19). SV40 DNA FO I had been

1

added to monitor the degree of digestion and to 1serve as an internal position marker. It may beseen that despite ofa large enzyme excess there 10 20 30 40remained molecules that were lacking some of DISTANCE MIGRATED [MM]the endonuclease recognition sites normally FIG. 6. Endo R -Bam digestion of bands II, C,present in SV40 DNA. In addition, there were and III. Isolated band II (a), band C (b), and bandthree typical endo R Hpa I SV40 DNA peaks III (c) DNA was mixed prior to digestion with endothat were superimposed on an elevated back- R -Bam with 32P-labeled SV40 DNA FO I (0) as anground. These results show that band Im DNA internal position marker. Electrophoresis in 1.4%comprises a heterogeneous population of mole- agarose was carried out for 2 h at 100 V.

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 571

4

3

2

8

6 ..3x

04

4. .2sI~~~~~~~~~2- cm~~~~~~~~~~~~~~~~a

08 0-

6-2

2 I

10 20 30 40 50 60 70 80 90 100 110 120 130DISTANCE MIGRATED [MM]

FIG. 7. Endo R -Hind digestion of bands II, C, and III. Isolated band II (a), C (b), and III (c) DNA wasdigested with endo R -Hind. 32P-labeled SV40 DNA FO I (-) was added prior to digestion to band III DNA.Electrophoresis was carried out for 18 h at 100 V in polyacrylamide.

12%), which may have been brought about by cates that repetitive cellular DNA sequencescontaminating SV40 FO II DNA, and 12.4% that can form a hybrid under conditions offilter(10.5%) hybridization with CV-1 DNA. The lat- hybridization participate (in addition to uniqueter value equals the value reached by self-an- sequences, as will be shown below) in the for-nealing of CV-1 DNA (15%, 8.5%). Band III mation of band III DNA molecules.DNA displayed, apart from 54% (40%) hybridi- Further structural analysis of band IIIzation with SV40 DNA 3.5% (4.5%), hybridiza- DNA. As described above, treatment ofband IIItion with CV-1 cellular DNA. This result indi- DNA with various restriction endonucleases

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572 GRUSS AND SAUER J. VIROL.

c~ 6 6

x ~~~~~~~~~~~~~~~x

U C~~~~~~~~~~~~~~~~~~~~~.)4,,4 lV

3- 3

2- 2

1020 30 40 50 60 0 80 90 100DISTANCE MIGRATED [MM]

FIG. 8. Endo R *Hpa I digestion ofband III. 32P-labeled SV40 DNA FO I (0) was added prior to digestionof isolated band III with endo R *Hpa I. Electrophoresis was carried out in 1.4% agarose for 18 h at 60 V.

TABLE 1. DNA-DNA hybridization between DNA from bands H, C, and III and SV40 or CV-1 cell DNA

DNA-DNA hybridization

DNA on IIa Ca Ila SV40a CV-laDNAte fl Exptfilter ( no. Input Input Input/re Input Input/ Input Input/re- Input

jug) Input/re- bound Input/re- bon nu/3- bound Inu/ bound Iptre onaction3H b action3H bound 3H reaction b action3H toultCpMb to filter b to filter CpMb to filter 3H CpMb to filter cpmb to filtercp MY) cpm (%) cp MY) cp MY) (%)

SV40 i 17,608 60.0 14,332 4.2 17,800 54.0 33,301 68.9 45,360 0.5ii 12,210 48.8 12,992 12.0 2,136 40.0 10,734 56.0 43,300 1.5

CV-1 cell i 16,304 0.6 15,200 12.4 17,272 3.5 31,836 1.2 41,384 15.0ii 15,890 1.3 7,609 10.6 7,472 4.5 19,466 0.5 50,951 8.5

a DNA in solution.b The specific activity of the DNA from bands II, C, and III as calculated from the specific activity of the total DNA

contained in the light band of the CsCl-EtBr gradient was either (i) 7.85 x 104 cpm/,ug or (ii) 1.3 x 104 cpm/Ag. The specificactivity of the SV40 DNA was 4.54 x 104 cpm/,ug, and that of CV-1 DNA was 4.58 x 104 cpm/ ug.

c Corrected for unspecific binding (1.1%) to heterologous bacteriophage fd DNA (15 ,ug) and adenovirus type 2 DNA (5gg), respectively. The data represent the mean value of two experiments.

generated molecules that were eith-ier com- SV40DNA (insert, Fig. 9). The DNA from frac-pletely devoid of the respective recognition se- tions 22 to 52 hybridized to a larger degree withquences or contained a reduced number of 3H-labeled SV40 cRNA than, for example, CV-1recognition sites. To test whether there are DNA or calf thymus DNA. There was, how-nevertheless viral DNA sequences present in ever, a greater rate of hybrid formation whensuch molecules, we isolated those DNA se- pure SV40 DNA was reacted with cRNA. Thisquences that proved to be larger than the Hind result suggests the presence of both viral andA SV40 segment after electrophoresis in aga- host DNA sequences within the large-sizedrose (see bar in Fig. 9). Unlabeled SV40 DNA fragments that have lost all or most of theirisolated from the same region of a preparative endo R -Hind recognition sequences.gel run in parallel was denatured, immobilized Heteroduplex analysis in the electron micro-on nitrocellulose filters, and hybridized with scope was performed to check whether cellularsynthetic complementary 3H-labeled SV40 and viral DNA sequences were covalentlycRNA. This method indicated the presence of linked or whether they existed separately in

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 573

different molecules. After denaturation and an- natant medium was harvested and used fornealing with endo R EcoRI-generated SV40 infection of CV-1 cells, which were then incu-FO III DNA at a Cot value that ensures rena- bated for 2 days with [3H]thymidine. Thereafterturation of the SV40 DNA sequences, there the DNA was selectively isolated and centri-appeared molecules with only partially base- fuged in dye-buoyant density gradients. Itpaired double-stranded regions and with un- turned out that in each case superhelical DNApaired long single-stranded tails on both ends. had been synthesized, apparently owing to in-The nonhomologous regions indicate the pres- tracellular circularization of the inoculatedence of cellular DNA sequences that are cova- DNA. This result also shows that band III DNAlently linked with SV40 DNA sequences (man- had been capable of giving rise to infectioususcript in preparation). viral progeny. To determine the properties ofThis conclusion is corroborated by the results the newly generated superhelical DNA, a com-

in Table 2. Sequential hybridization of band III plete digestion with endo R *Hind was carriedDNA first to SV40 DNA on filters and, after out with 32P-labeled wild-type SV40 DNA FO Ielution, back hybridization to either CV-1 DNA as an internal marker. The resulting cleavageor SV40 DNA revealed 11.4% homology to cellu- pattern was aberrant, with fragments J and Klar DNA and 42.5% homology to SV40 DNA, apparently missing (Fig. 10). The size of therespectively. new FO I was, however, equal to wild-type-size

Biological properties of band III DNA. To FO I when compared in agarose gels, whichtest its biological activity, band III DNA was shows that fragments Hind J and K are presentused to infectCV-1 cell cultures in the presence in the genome and probably co-electrophoreseof DEAE-dextran (14). As a control, infections with the adjacent Hind fragments G and/or thewith both SV40 FO I DNA and endo R EcoRI- unresolved fragments EF. This could comegenerated SV40 FO III DNA were carried out. about by loss or alteration of the Hind cleavageWhen a cytopathogenic effect had developed sites between fragments J-G or J-F and K-F or(usually 1 to 2 weeks after infection), the super- K-E.

'. 10-0x

6-1 4/ o SVx40DN o 15 I,Z

C FRACTKONS 22-52 (015p)0 3- CVX IDNA (1vg)x 2- 0. CALF TWMUS DNA 1op)E5 1 . CALF THYMUS DNA (015pq)

14 Fo 0t *o203040so 607080cV) 4- (VI][ 3Hc RNA added

3

2

A B CDEF G HIJK10 20 30 40 50 60 70 80 90 100 110 120

DISTANCE MIGRATED [MM]FIG. 9. Identification of reassorted SV40 DNA in band III using SV40 cRNA. Isolated 3H-labeled and

unlabeled band III DNA was digested with endo R -Hind to completion and subjected to electrophoresis inparallel tracks in 1.4% agarose for 18 h at 60 V. The pattern ofthe radioactively labeledDNA was determined.The positions of endo R -Hind-cleaved marker SV40 DNA FO I are indicated by the letters A to K. Thosefractions of the unlabeled band III DNA that were larger than the largest SV40 Hind fragment (A)(corresponding to fractions 22 to 52 ofthe labeled DNA) were isolated from the gel. The DNA was denaturedand immobilized on nitrocellulose filters. Furthermore, SV40, CV-1 and various amounts of calf thymusDNA (as indicated in the insert) were immobilized and hybridized with increasing amounts of 3H-labeledSV40 cRNA (specific activity: 2 x 107 cpmlpg, 2 x 104 cpm/p,). The hybridized radioactivity is indicated inthe insert.

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574 GRUSS AND SAUER J. VIROL.

TABLE 2. Detection of covalently linked host viral-DNA sequences by DNA-DNA hybridizationInput

Hybridization step DNA on filter DNA in solution cpm eluted cpmihyd filter(%)b

Hybridization and elution of SV40 (10 ,g) Band III (3H la- 70,000DNA specifically bound to beled)dSV40 DNAC

Hybridization of eluted DNA to SV40 (5 jug) Eluted band IIIf 15,000 42.9CV-1 cell DNA and to SV40DNAe

CV-1 (5 ,ug) Eluted band III' 4,000 11.4a Filters were washed in 6 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and the DNA was

eluted with formamide as described (28).b Corrected for nonspecific binding of SV40 DNA to filters with heterologous T4 DNA.c Hybridization was performed for 48 h in 6x SSC at 65°C. For binding of CV-1 DNA to SV40 DNA and

vice versa see Table 1.d Band III DNA (6.8 ,ug; 3 x 105 cpm) was isolated as described in Materials and Methods.e Hybridization was performed for 48 h in 3 x SSC and 50% formamide at 370C.'35,000 cpm.

c 020 290

a.0:r15- B 3 c~

CD EF x

a.0

10- -2a.Cv,

G

5-

016 00056 6i O40 60 70 80 90 100 110 120DISTANCE MIGRATED [MM]

FIG. 10. Endo R *Hind cleavage pattern ofFO I DNA progeny obtained after infection with band IIIDNA.CV-1 cells were infected with band III DNA (7 pg) as described in Materials and Methods. One week afterinfection, the 3H-labeled DNA was selectively extracted, and the superhelical DNA was isolated from a CsCl-EtBr equilibrium density gradient and restricted with endo R -Hind . 32P-labeled SV40 DNA FO I was added(-) prior to digestion. Electrophoresis was carried out for 18 h at 60 V in polyacrylamide.

Although band III DNA comprises a hetero- with endo R Hind to completion (Fig. hla andgeneous group of molecules, as shown above, c). At least seven new bands are visible in Fig.the FO I DNA obtained after infection with llc in addition to the typical SV40 wild-typeband III DNA revealed a cleavage pattern that cleavage products. In the digest shown in Fig.is rather similar to endo R Hind cleavage of Ila one of the aberrant fragments was presentwild-type FO I DNA. To test whether band III in large preponderance (see arrow). Further-always gives rise to progeny FO I DNA with more, the small fragments Hind J and K weresimilar cleavage patterns, we repeated the ex- present in both of these DNA preparations,periment shown in Fig. 10. Parallel cultures of unlike the pattern shown in Fig. 10. Thus,CV-1 cells were infected with portions of an- different infections with band III DNA result inother purified band III DNA preparation, and different types of FO I DNA.the resulting superhelical DNA was digested It is interesting to note that the aberrant

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 575

* _ typical for wild-type SV40 DNA (Fig. llb andd).The new FO I DNA generated in vivo from

band III DNA is able to induce both tumor andSV40 capsid antigen (unpublished data). Also,the DNA is encapsidated and gives rise to infec-tious viral progeny, the properties of which arecurrently being studied.To investigate whether band m DNA mole-

cules are generated de novo during each infec-tious cycle or whether they exist as autono-mously replicating linear structures that mightbe conferred to the host cell by pseudovirions,the following experiment was performed. SV40FO I DNA that had been repurified in agarosegels was employed together with DEAE-dex-tran for infection of cells. This procedure ex-cludes rigorously contaminations of the infect-ing FO I DNA with linear DNA and permitsthe detection ofde novo synthesized linear bandHII DNA. Inoculation of FO I DNA did not leadto the appearance of band III DNA (Fig. 12a).There is (besides band C DNA, which is alsofound in mock-infected controls) nearly no dis-cernible radioactivity in the position of bandIII. At the same time FO I DNA displaying awild-type endo R Hind cleavage pattern wasreplicated. After six serial undiluted passagesof the viral progeny that originated from puri-fied SV40 FO I DNA, however, some band IHDNA reappeared (Fig. 12b). This material wasisolated and restricted with endo R Hind. Theresulting cleavage pattern is entirely differentfrom the comparable band III DNA patternsobtained after infection with virions shown inFig. 7c and 9. Besides larger material, there aresome viral peaks (Hind E + F and G) superim-

FIG. 11. Polyacrylamide slab gel electrophoresis posed on a general background fragments Apattern ofendo R -Hind-digested FO I DNA progeny andB arenerreprsentd,und, fragments Jobtained after infection with band III DNA, endo and B are underrepresented, and fragments JR-EcoRI FO III DNA, and FO I DNA, respec- and K are present in large excess. Interest-tively. CV-1 cells were infected with 0.8 Mg of band ingly, these latter fragments are contained inIII DNA, endo R *EcoRI FO III DNA, and FO I the corresponding FO I DNA to a nonnal extentDNA, respectively, as described in Materials and (unpublished data). The evolution of the bandMethods. The resulting superhelical DNA was sub- Im DNA from increasingly passaged FO I DNAjected to electrophoresis (2 pg in each track) after progeny is currently being investigated.digestion in polyacrylamide (18 h, 110 V), stainedwith EtBr, and photographed using UV light. (a) DISCUSSIONBand III superhelical DNA progeny; (b) FO I SV40 A distinct size class of linear DNA moleculesDNA; (c) band III superhelical DNA progeny [sepa- A dscr thatclas s of A heteculesrate experiment from (a)]; (d) superhelical DNA (FOIII)isdescribedthatconsistsofaheterogeprogeny derived from endo R -EcoRI FO III. neous population of SV40 and cellular DNA

sequences. These molecules undergo replica-tion in SV40-infected permissive cells. Analysis

cleavage patterns reflect the heterogeneity of by gel electrophoresis made these structuresthe infecting band III DNA rather than de novo apparent, since they form, like FO II, a discreteoccurring recombination or reassortment proc- band in the gel. They have probably escapedesses, since both wild-type SV40 FO I DNA and detection thus far because the small linear andendo R EcoRI SV40 FO III, which were used relaxed-circular DNA contained in the lightas controls in DNA infections, resulted in FO I band of CsCl-EtBr gradients (which are em-DNA progeny with a cleavage pattern that was ployed for purification of SV40 FO I DNA) were

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576 GRUSS AND SAUER J. VIROL.

lie iii missive cells regardless of the multiplicity of1 1, 1 minfection that had been used. This means, in

turn, that there are some reassorted and recom-bined SV40 and host DNA sequences present inthe form of band III DNA, although the mature

C? 10- llSV40 FO I DNA replicated in the same cellCo10 culture displays a normal wild-type restrictionx l l cleavage pattern. The band III DNA was esti-E mated to comprise 22% of the selectively ex-

tracted labeled DNA (in the "Hirt" supernatantJ l l including FO I DNA) when pulse-labeling was

4- performed from 30 to 36 h after infection. Thisrelative amount of band III DNA increases latein infection up to 34%, as shown by pulse-label-ing between 75 and 81 h. Thus, this class ofmolecules accounts for a substantial amount ofthe replicated, selectively isolated DNA inSV40-infected cells.

10- An interesting property of these linear struc-tures is their ability to undergo replication.This is concluded from the following observa-tions. Up to 24 h after infection there is no DNAthat can be stained with EtBr in the position ofband III in agarose gels. It should be pointedout, however, that very small amounts ofDNA

5- (less than 0.05 ,ug) would remain undetectedunder these conditions. In fact, when[3H]thymidine-labeled purified SV40 virionswere used for infection, it was possible to findradioactive label in the position of FO III DNA

1 in agarose gels after extraction of intracellularDNA 1 day postinfection. The amount of the

10 20 30 40 radioactively labeled DNA is too small to bevisualized after staining with EtBr (unpub-FIG. 12. Exclusion of band III DNA from replica- visheddafta)Asthnifecti cycl (prcd

tion after infection with purified SV4 DNA FOi,CV-1 cells were infected with purified SV40 DNA FO band III DNA accumulates and can be labeledI (2 ug) as described in Materials and Methods. One with [3H]thymidine. That band III DNA is not aweek after infection the -H-labeled DNA was selec- breakdown product (at least as far as its cellu-tively extracted, and the DNA in the light bands of lar moiety is concerned) that may originateCsCl-EtBr equilibrium density gradients containing from large cellular DNA by cleavage to frag-the small relaxed and linear molecules was subjected ments of unit length is shown in Fig. 3. Cellu-to electrophoresis in 1.4% agarose for 2 h at 100 V. lar DNA sequences that were labeled prior to(a) Small relaxed and linear DNA obtained after infection do not participate in the formation ofinfection with purified FO I DNA; (b) the same as band III DNA, whereas band C DNA consisted(a) except thatFO IDNA progeny virus was used that of substantial amounts of prelabeled material.had been passaged serially and undiluted six times. of rests shount of IIIed molerThe arrows indicate the positions ofmarker bandsII These results show that band III DNA mole-C, and III in a parallel track of the same slab gel. cules accumulate in SV40-infected cells by a

replication process. The mode of replication is,however, still unknown.

commonly considered to consist either of FO II Infection of cells with purified SV40 DNA FOor of randomly fragmented host DNA se- I did not result in significant amounts of bandquences. The uniformity of their size and the III DNA molecules in the replicating pool ofease with which they can be separated from DNA. This result shows that band III DNAother structures (such as FO II or fragmented molecules serve as their own templates for rep-host DNA) by gel electrophoresis enabled us to lication, which, once they are omitted from theinvestigate their structural and biological prop- infecting material, cannot be immediately re-erties. placed by de novo generated templates. It ap-We would like to stress here that these mole- pears, therefore, that band III DNA is perpetu-

cules are regularly found in SV40-infected per- ated within virus pools by encapsidation into

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VOL. 21, 1977 INFECTIOUS LINEAR SV40 DNA 577

pseudovirions. Preliminary unpublished exper- LITERATURE CITEDiments have shown that pseudovirions indeed 1. Beard, P., J. F. Morrow, and P. Berg. 1973. Cleavage ofcontain a size class of DNA that co-electropho- circular, superhelical simian virus 40 DNA to a linearreses in agarose gels with FO III DNA and that duplex by S, nuclease. J. Virol. 12:1303-1313.contains SV40 DNA sequences. 2. Chowdhury, K., P. Gruss, W. Waldeck, and G. Sauer.1975. Action of S, nuclease on nicked circular simian

Several repeated passages of the FO I DNA virus 40 DNA. Biochem. Biophys. Res. Commun.viral progeny led to the reappearance of newly 64:709-716.generated band III DNA. Therefore, this sys- 3. Danna, K., and D. Nathans. 1971. Specific cleavage oftem permits the investigation ofthe recombina- simian virus 40 DNA by restriction endonuclease of'tion between viral and host DNA andit allows Hemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A.tion between viral and host DNA and it allows 68:2913-2917.us to pursue the evolution of various recom- 4. Fried, A. H. 1975. Temperature dependence of strandbined and reassorted band III DNA species as separation of the DNA molecules containing inte-they are being passaged. grated SV40 DNA in transformed cells. Nucleic Acidtheyarebeingpassaged. ~~~~~Res. 2:1591-1608.The notion that the linear band III DNA 5. Girardi, A. J. 1965. Prevention of SV40 virus oncogene-molecules are uniformly of unit length despite sis in hamsters. I. Tumor resistance induced by hu-their composition of various viral and host man cells transformed by SV40. Proc. Natl. Acad.DNA sequences requires an explanation. One 6.Sci. U.S.A. 54:445-451.6. Grady, J., D. Axelrod, and D. Trilling. 1970. The SV40might conceive ofa mechanism that operates in pseudovirus: its potential for general transduction inanalogy to the "headful" mechanism (22), in animal cells. Proc. Natl. Acad. Sci. U.S.A. 67:1886-which, regardless of the base sequences, pre- 1893.cisely unit-length pieces that fit into the capsid 7. Gruss, P., and G. Sauer. 1975. Repetitive primate DNAcontaining the recognition sequences for two restric-are cut from larger precursors. Such large lin- tion endonucleases which generate cohesive ends.ear precursor DNA structures could, for exam- FEBS Lett. 60:85-88.ple, arise by replication along a rolling circle. 8. Hirt, B. 1967. Selective extraction of polyoma DNAThis explanation is atpresent entirely specula- from infected mouse cell cultures. J. Mol. Biol.Thiseplanatonis t presnt entrely secula- 26:365-369.tive since it is not known how band m DNA is 9. Jensen, F. C., A. J. Girardi, R. V. Gilden, and H.produced. Koprowski. 1964. Infection of human and simian tis-We have shown that infection of cells with sue cultures with rous sarcoma virus. Proc. Natl.

linear band III results in the formation of su- Acad. Sci. U.S.A. 52:53-59.10. Kashmiri, S. V. S., and H. V. Aposhian. 1974. Degrada-perhelical FO I DNA with an aberrant restric- tion of pseudoviral DNA after infection ofmouse cellstion cleavage pattern (Fig. 10). This newly gen- with polyoma pseudovirions. Proc. Natl. Acad. Sci.erated DNA resembles the substituted SV40 U.S.A. 71:3834-3838.FO I DNA (12) that was found after serial undi- 11. Kit, S., D. R. Dubbs, P. M. Frearson, and J. L. Mel-nick. 1966. Enzyme induction in SV40 infected greenluted passages of SV40 and that contains reas- monkey kidney cultures. Virology 29:69-83.sorted viral sequences and also sometimes host 12. Lavi, S., and E. Winocour. 1972. Acquisition of se-DNA sequences. We have found (unpublished quences homologous to host deoxyribonucleic acid bydata) that infection with high multiplicities of closed circular simian virus 40 deoxyribonucleic acid.data)tatmfeclonwlt hlgh mltlpllctles OI J. Virol. 9:309-316.SV40 that had been serially passaged undiluted 13. Levine, A. J., and A. K. Teresky. 1970. Deoxyribonu-exerts a drastic influence on the composition of cleic acid replication in simian virus 40-infected cells.band III DNA: sequences accumulate that can- II. Detection and characterization of simian virus 40

notbe cleaved at all or only in part by restric- 1 pseudovirions. J. Virol. 5:451-457.not be cleaved at all or only m part by restrlc- 14. McCutchan, J. H., and J. S. Pagano. 1968. Enhance-tion endonucleases (presumably host DNA se- ment of the infectivity of simian virus 40 deoxyribo-quences). We think it therefore very likely that nucleic acid with diethyl-aminoethyl-dextran. J.band III DNA molecules might serve as the Natl. Cancer Inst. 41:351-357.precursorofthe substituted and reassortedsu. 15. Michel, M. R., B. Hirt, and R. Weil. 1967. Mouse cellu-precursors of the substituted and reassorted su- lar DNA enclosed in polyoma viral capsids (pseudovi-

perhelical DNA described by Lavi and Wino- rions). Proc. Natl. Acad. Sci. U.S.A. 58:1381-1388.cour (12). Since we are now studying the de 16. Ostermann, J. V., A. Waddell, and H. V. Aposhian.novo appearance of band III DNA after infec- 1970. DNA and gene therapy: uncoating of polyomation with serially passaged FO I DNA viral pseudovirus in mouse embryo cells. Proc. Natl. Acad.

Sci. U.S.A. 67:37-40.progeny, we should be able, by comparing the 17. Quasba, P. K., and H. V. Aposhian. 1971. DNA andcleavage patterns of both band III and of the gene therapy: transfer of mouse DNA to human andcorresponding FO I, to test whether band III mouse embryonic cells by polyoma pseudovirions.DNAmoleculesmayrepresent under specific Proc. Natl. Acad. Sci. U.S.A. 68:2345-2349.DNA molecules may represent under specific 18. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dye-

passage conditions the precursors of aberrant buoyant-density method for the detection and isola-FO I DNA. tion of closed circular duplex DNA: the closed circular

DNA in Hela cells. Proc. Natl. Acad. Sci. U.S.A.ACKNOWLEDGMENTS 57:1514-1521.

This work was supported by the Deutsche Forschungsge- 19. Sack, G. H., and D. Nathans. 1973. Studies of SV40meinschaft and by a grant from the Bundesminister fur DNA. VI. Cleavage of SV40 DNA by restriction endo-Forschung und Technologie. nuclease from Hemophilus parainfluenzae. Virology

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51:517-520. pseudovirus. Science 168:268-271.20. Sauer, G., and J. R. Kidwai. 1968. The transcription of 26. Trilling, D., and D. Axelrod. 1972. Analysis ofthe three

the SV40 genome in productively infected and trans- components of the simian virus 40: pseudo-, mature,formed cells. Proc. Natl. Acad. Sci. U.S.A. 61:1256- and defective viruses. Virology 47:360-369.1263. 27. Waldeck, W., K. Chowdhury, P. Gruss, and G. Sauer.

21. Smith, H. O., and J. Wilcox. 1970. A restriction enzyme 1976. Random cleavage of superhelical SV40 DNA byfrom Hemophilus influenzae. I. Purification and S, nuclease. Biochim. Biophys. Acta 425:157-167.geTneral properties. J. Mol. Biol. 51:379-391. 28. Waldeck, W., K. Kammer, and G. Sauer. 1973. Prefer-

22. Streisinger, G., J. Emrich, and M. M. Stahl. 1967. ential integration of simian virus 40 deoxyribonucleicChromosome structure in phage T4. LI. Terminal acid into a particular size class ofCV-1 cell deoxyribo-redundancy and length determination. Proc. Natl. nucleic acid. Virology 54:452-464.Acad. Sci. U.S.A. 57:292-295. 29. Westphal, H. 1970. SV40 DNA strand selection byEsch-

23. Tegtmeyer, P., and F. Macasaet. 1972. Simian virus 40 erichia coli RNA polymerase. J. Mol. Biol. 50:407-deoxyribonucleic acid synthesis: analysis by gel elec- 420.trophoresis. J. Virol. 10:599-604. 30. Wilson, G. A., and F. A. Young. 1975. Isolation of a

24. Todaro, G. J., K. Habel, and H. Green. 1965. Antigenic sequence-specific endonuclease (BamI) from Bacillusand cultural properties of cells doubly transformed by amyloliquefaciens H. J. Mol. Biol. 97:123-135.polyoma virus and SV40. Virology 27:179-185. 31. Winocour, E. 1968. Further studies on the incorpora-

25. Trilling, D., and D. Axelrod. 1970. Encapsidation offree tion of cell DNA into polyoma-related particles. Vi-host DNA by simian virus 40: a simian virus 40 rology 34:571-582.

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