induction of specific chromosomal aberrations by adenovirus type

12
JOURNAL OF VIROLOGY, Dec. 1967, p. 1174-1185 Copyright © 1967 American Society for Microbiology Vol. 1, No. 6- Printed in U.S.A. Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells HARALD ZUR HAUSEN Virus Laboratories, The Children's Hospital of Philadelphia, and School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19146 Received for publication 28 July 1967 Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occur- rence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) syn- thesis, as determined by autoradiography, was seen in metaphases containing adeno- virus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified 3H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation. The mechanisms by which viruses induce chro- mosomal changes within infected host cells are still obscure. Both deoxyribonucleic acid (DNA) viruses (2, 8, 24) and ribonucleic acid (RNA) viruses (9, 15, 16) may cause such alterations. The replication site in the host cell, whether nuclear or cytoplasmic, also seems to be of no importance. Although oncogenic viruses are known to cause chromosomal breaks in infected cells (13, 15, 24, 25), some nononcogenic viruses produce similar effects (8, 9, 16, 28). Studies were initiated to eluci- date some of the causes leading to chromosomal defects within infected cells. For this purpose, labeled viruses seemed to offer an approach to the detection of those metaphases harboring virus genomes (14) and to the localization of viral nucleic acid within the cell. This report deals with chromosomal aberra- tions and the fate of the viral genome in human embryonic kidney cells infected with adenovirus type 12. Special attention has been directed to- wards the question of whether chromosome aber- rations due to adenovirus type 12 depend on the viral DNA and whether the altered loci are possi- bly the sites of viral genome integration (11, 12). MATERIALS AND METHODS Cells. Human embryonic kidney (HEK) cells (obtained through the Tissue Resources Unit, Viral Carcinogenesis Branch, National Cancer Institute, Bethesda, Md.) were used in all experiments. These cells could readily be maintained for up to 20 passages. They were grown as monolayers in Blake bottles containing medium RPMI 1629 (BBL) supplemented with 20% fetal calf serum (FCS), glutamine, penicillin, and streptomycin. Each culture was divided weekly into two parts, with the use of 0.25% trypsin for dislodging of the cells. Ten different HEK "strains" in the 3rd to 15th passages were used in these experi- ments. Tube cultures of primary HEK cells were purchased 1174 on April 13, 2018 by guest http://jvi.asm.org/ Downloaded from

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JOURNAL OF VIROLOGY, Dec. 1967, p. 1174-1185Copyright © 1967 American Society for Microbiology

Vol. 1, No. 6-Printed in U.S.A.

Induction of Specific Chromosomal Aberrations byAdenovirus Type 12 in Human

Embryonic Kidney CellsHARALD ZUR HAUSEN

Virus Laboratories, The Children's Hospital of Philadelphia, and School of Medicine, University of Pennsylvania,Philadelphia, Pennsylvania 19146

Received for publication 28 July 1967

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked inhuman embryonic kidney cells 24 hr after infection with adenovirus type 12. Thesechromosomal changes disappeared in persistently infected cultures. Neutralizationof the virus with type-specific antiviral serum prior to infection prevented the occur-rence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) syn-thesis, as determined by autoradiography, was seen in metaphases containing adeno-virus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virusreduced chromosomal aberrations linearly. This reduction in aberrations wasfourfold slower than the inactivation of viral infectivity. At 24 hr after infection ofcells with purified 3H-labeled adenovirus type 12, the isotope was found to beassociated with the nuclei. The uptake of isotope was reduced ninefold when thelabeled virus was neutralized with type-specific antiviral serum. This difference isconsidered to account for neutralization of labeled virions. In metaphases infectedwith labeled viruses, most of the clustered grains were seen only on one arm of thechromatid, even after 72 hr. Isochromatid labeling was found, however, in a smallpercentage of chromosomes, and increased with time after infection. This increasewas threefold between 24 and 72 hr after infection, whereas the mean grain countsdecreased twofold during the same period. This has been tentatively interpreted tomean that most of the viral DNA molecules or parts thereof are merely attached tocellular chromatin, but a small fraction of them becomes gradually integrated astime proceeds. Certain chromosomal sites appeared to be preferentially labeled whenchromosome 2 was used as a model for evaluation.

The mechanisms by which viruses induce chro-mosomal changes within infected host cells arestill obscure. Both deoxyribonucleic acid (DNA)viruses (2, 8, 24) and ribonucleic acid (RNA)viruses (9, 15, 16) may cause such alterations. Thereplication site in the host cell, whether nuclear orcytoplasmic, also seems to be of no importance.Although oncogenic viruses are known to causechromosomal breaks in infected cells (13, 15, 24,25), some nononcogenic viruses produce similareffects (8, 9, 16, 28). Studies were initiated to eluci-date some of the causes leading to chromosomaldefects within infected cells. For this purpose,labeled viruses seemed to offer an approach to thedetection of those metaphases harboring virusgenomes (14) and to the localization of viralnucleic acid within the cell.

This report deals with chromosomal aberra-tions and the fate of the viral genome in humanembryonic kidney cells infected with adenovirus

type 12. Special attention has been directed to-wards the question of whether chromosome aber-rations due to adenovirus type 12 depend on theviral DNA and whether the altered loci are possi-bly the sites of viral genome integration (11, 12).

MATERIALS AND METHODS

Cells. Human embryonic kidney (HEK) cells(obtained through the Tissue Resources Unit, ViralCarcinogenesis Branch, National Cancer Institute,Bethesda, Md.) were used in all experiments. Thesecells could readily be maintained for up to 20 passages.They were grown as monolayers in Blake bottlescontaining medium RPMI 1629 (BBL) supplementedwith 20% fetal calf serum (FCS), glutamine, penicillin,and streptomycin. Each culture was divided weeklyinto two parts, with the use of 0.25% trypsin fordislodging of the cells. Ten different HEK "strains"in the 3rd to 15th passages were used in these experi-ments.Tube cultures of primary HEK cells were purchased

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VIRUS-INDUCED ABERRATIONS IN KIDNEY CELLS

either from Flow Laboratories, Rockville, Md., orfrom Microbiological Associates, Inc., Bethesda, Md.KB cells (kindly supplied by Z. Gilead, Department

of Microbiology, School of Medicine, University ofPennsylvania, Philadelphia) were used for preparingstock virus. These cells were grown in Eagle's MinimalEssential Medium (MEM) for suspension cultures(Microbiological Associates, Inc.), complementedwith 10%o FCS, glutamine, penicillin, and strepto-mycin.

Virus. Adenovirus type 12 was furnished by J. J.Trentin, Division of Experimental Biology, BaylorUniversity School of Medicine, Houston, Tex. Thevirus was passed three times in primary HEK cellsbefore use in these experiments. The cells were col-lected 2 to 3 days after infection, sonically treated for10 min (Disintegrator-Forty, Ultrasonic IndustriesInc., Plainview, N.Y.), shell frozen in ampoules, andstored at -70 C. Titrations were performed in primaryHEK cells, with the use of four tubes per dilution,and the titers were calculated by the method of Reedand Muench. Cultures were infected at an inputmultiplicity of 1 unless stated otherwise.

Partially purified adenovirus type 12 was foundfree from adeno-associated viruses when examinedelectron microscopically by K. Hummeler. No evi-dence for simian virus 40 (SV40) contamination orhybridization was found in immunofluorescence testswith an anti-SV40 T antigen conjugate (kindly sup-plied by A. J. Girardi, The Wistar Institute, Philadel-phia, Pa.).

Viruis purificationi. Adenovirus type 12 was purifiedby the method of Gilead and Ginsberg (4) with minormodifications. About 109 KB cells in monolayerculture were infected at an input multiplicity of 1.After an adsorption period of 4 hr, the cells werewashed and fresh medium was added. The cells wereharvested 72 hr after infection and treated for 30 minat 37 C with sodium deoxycholate. This was followedby digestion with deoxyribonuclease (NutritionalBiochemical Corp., Cleveland, Ohio) for 30 mmn at37 C in the presence of Mg+. The suspension wastreated twice for 5 min with fluorocarbon (freon 113)by shaking and was centrifuged each time for 20 mmnat 1,000 X g. The supernatant fluid was collected, andthe virus was sedimented at 100,000 X g for 60 min.The sediment was resuspended in phosphate-bufferedsaline (PBS) and layered on top of a preformed CsClgradient. After 3 hr of centrifugation in a Spinco SW25.1 rotor at 22,500 rev/min, the virus band was col-lected, dialyzed against PBS, and stored at -70 C inampoules.

Labelinig of adentoviruls type 12 with 3H-thlymidine.KB cells were infected as described. After the 4-hradsorption period, the cells were washed three timeswith Hanks balanced salt solution (HBSS) and in-cubated in MEM containing 10%c dialyzed FCS. Afteran additional 4 hr, 20 uc of 3H-thymidine (specificactivity, 6 c/mmole; Schwartz BioResearch, Inc.,Orangeburg, N.Y.) was added per ml of culture fluid.The cells were harvested 72 hr after infection. Thevirus was then purified as described above.

Part of the labeled virus was neutralized by specificantibody. For this purpose, 1 ml of purified labeled

virus suspension (106 TCID50/ml) was incubated for 2hr at 37 C with 1 ml of type-specific antiserum with aneutralizing titer 1:120 versus 103 TCID,o. The otherpart of the purified virus was handled in the samemanner except that HBSS containing 2% FCS wasadded instead of antiserum. These virus preparationswere allowed to adsorb onto HEK cultures for 2 hr inthe presence of 500 mg/ml of "cold" thymidine.Thereafter, the cell sheets were washed three timeswith HBS, fresh medium was added, and chromosomeslides were prepared at varying time intervals.

Immunofluorescence. Adenovirus type 12 T-antigenproduction in infected cells was demonstrated byusing fluorescein-isothiocyanate (FITC)-coupled ham-ster T antibodies (purchased from Flow Laboratories)in a final dilution of 1:5. Rabbit antiserum againsttype-specific adeno-12 virion antigen, obtained fromthe Virus Research Resources Branch, National In-stitutes of Health, Bethesda, Md., was used with goatanti-rabbit -y-globulin-conjugate (Microbiological As-sociates, Inc.) for indirect staining.

For immunofluorescence tests, the cells were grownon narrow slides (0.5 X 7.5 cm). The fixation andstaining procedures conformed with those describedby Henle and Henle (10). The preparations wereexamined with a Zeiss-Ultraphot-Microscope underultraviolet illumination.

Chlromosome preparations. HEK cells were culturedin milk-dilution bottles by seeding a total of 5 X 105cells in 12 ml of medium. Colchicin (1:200,000) wasadded 1 to 4 hr before harvest of the cells. The cultureswere shaken vigorously, and the supernatant fluidswere removed and centrifuged. The sedimented cellswere treated as reported earlier (26). One hundredmetaphases were studied in each experiment unlessmentioned otherwise.

Autoradiograplty. Tritiated thymidine (specific ac-tivity 6 c/mmole) was added at concentrations indi-cated in the Results. After the exposure period, thecells were washed three times with HBSS containingexcess cold thymidine and prepared for autoradiog-raphy immediately, or after further incubation for 24to 48 hr. The coating of the slides, exposure, anddevelopment have been described previously (27).

Ultraviolet inactivationt. Amounts of 6 ml of virus

17 1

106L

w 105I-.

=) 104>,

404 8 12 16 20 24 28 32

TIME IN HOURS

FIG. 1. Cell-bowzid infectivity ofadenovirus type 12.

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ZUR HAUSEN

suspension were exposed in open petri plates (10-cmdiameter) to a General Electric germicidal lamp at adistance of 18 cm. The plates were mechanically tiltedup and down 90 times per minute with 2.5-cm ex-cursions.

RESULTSAdenovirus type 12 propagation in serially main-

tained HEK cultures. Cultures of HEK cells inmilk dilution bottles were exposed to virus at aninput multiplicity of 1, and individual cultureswere harvested at intervals thereafter. Figure 1

demonstrates the cell-bound infectivity. Logarith-mic increase in virus started after a latent periodof about 20 hr. Distinct cytopathic effects (CPE)were not seen before 48 hr. At this time, most ofthe cells became rounded, and about 90% re-vealed nuclear inclusions characteristic of adeno-virus infection (7). When lower multiplicities ofinfection (ranging from 10-1 to 10-5) were used,no CPE became evident, but nuclear alterationswere seen in a small number of cells and increasedin frequency with time. The cultures continued togrow and were maintained for more than 10 pas-sages, infectious virus being present during theentire period. No signs characteristic of virus-induced cellular transformation were observed.The general morphological appearance of the cellswas unaltered and T- and virion (V) antigen pro-duction was noted in not more than 3% of thecell populations.T antigen was found preferentially in the nu-

cleus of the infected cell (18) and only occa-sionally in the cytoplasm. The distribution of T-and V-specific fluorescence during the early stagesof infection is demonstrated in Table 1.Chromosomal studies. Chromosomal studies

were performed mainly with one of the HEK cell"strains." Tests with the other nine strains usedindicated similar responses.On karyotyping 25 metaphases of uninfected

HEK cells, all those which were euploid exhibited

TABLE 1. Percentage of cells revealilig T anid type-specific V antigeni formation in the first 3 days

after infectionz at variouis multiplicities

Time after infectioni

Mlultiplicityof infection 24 hr 48 hr 72 hr(TCID 6o/cell)

T V T V T V

antigen aPtigen lantigen antigen antigen antigen

1 .0 50-55 5-7 90-95 90 _0.1 4-6 0.5-1 8-10 3-5 4-6 2-40.01 0.5-1 - 0.5-1 0.5 1-2 0.5-10.001 0.1 0.1

TABLE 2. Development of chromosomal changes inHEK cells after infection by I TCID5o per cell

of adenovirus 12

'NormalTime after rmeta

infection (hr) p -s

468

20242848a

Controls48

2448

9798922821224.

96989897

Meta-phases

with chro-mosomalbreaks

328

6873724

4123

Frag-mentedmeta-phases

3

Extremepyknosisof chro-mosomes

466

29

1

a Only 40 metaphases counted.

TABLE 3. Inivolvement of chromosomes inchromatid and chromosome aberrations

(50 metaphases)

Chromosome No

Chromosome 1 ................. 22Chromosome 2 .................. 3Chromosome 3....... 3Group4-5... 10Group 6-12,XX.... 22Group 13-15................... 8Chromosome 16.. 1Chromosome 17 .... ... 70Chromosome 18.................. 2Group 19-20.3............. 3Group 21-22 .............. 0

a Number of abnormal chromosomes found in50 metaphases.

a female karyotype. Twenty-one contained 46chromosomes without any structural abnormali-ties. Of the remaining four metaphases, three had45 chromosomes and one had only 42. In thesemetaphases, an apparently random loss of chro-mosomes had occurred.HEK cells infected with adenovirus type 12 at

an input multiplicity of 1 revealed a high incidenceof chromosomal aberrations 24 hr after infection.These aberrations began to appear 8 hr after in-fection (Table 2). Neutralization of the inputvirus by type-specific antiserum reduced theoccurrence of chromosomal changes to the levelof uninfected controls.

Fifty well-spread metaphases showing chro-mosomal aberrations 24 hr after infection were

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VIRUS-INDUCED ABERRATIONS IN KIDNEY CELLS

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Localization of Breaks in Indicated Chromosomes

FIG. 2. Involvement of chromosomal sites in chro-matid and isochromatid breaks 24 hr after infection withadenovirus type 12.

photographed and karyotyped. The chromosomesinvolved in aberrations (predominantly singlechromatid breaks and isochromatid breaks) areshown in Table 3. Chromosome 17 was by farmost frequently involved in these changes.

In a repeat experiment, the localization ofbreaks in 50 metaphases was analyzed by dividingchromosomes into regions (21). The results withidentifiable chromosomes are given in Fig. 2. Theno. 17 chromosomes revealed obvious nonrandombreaks at the site of normally occurring secondaryconstrictions (17, 20). The no. 1 chromosomesalso showed nonrandom, near-terminal breakswhich occurred at a lower frequency than thoseseen in the no. 17 chromosomes. Figure 3 showsa metaphase plate and a karyotype with typicalchanges in these chromosomes. Figure 4 presentsa collection of altered no. 1 and no. 17 chromo-somes. At input multiplicities of infection above 1,mitoses were markedly inhibited. Among themetaphases observed, many revealed pyknosis ofchromosomes which did not permit an evaluationof these specific chromosomal lesions. At multi-plicities of infection low-er than 1, the chromo-somal aberrations at 24 hr decreased in frequencyas the dose of virus was reduced. The viral infec-tion persisted in these cultures, but chromosomalaberrations were rarely seen and breaks in the no.

17 chromosome were no longer found.Effect of ultraviolet (UV) irradiation on various

properties of the adenovirus type 12. UV-irradiatedadenovirus type 12 was tested for infectivity andits capacities to induce T antigen and breaks inchromosome 17. The results are shown in Fig. 5,

with control values as 100%. All three curvesreveal linear declines in the activities indicativeof single hit events. In contrast to results obtainedby others with adenovirus type 12 (5) and SV40(3), there was no significant disparity in the inac-tivation of infectivity and the capacity to induceT antigen. The capacity of the virus to inducebreaks in chromosome 17 also decreased withtime of irradiation but at a markedly slower ratethan the loss of infectivity. Alterations in all otherchromosomes diminished at similar rates on UVirradiation of the virus.

Autoradiographic studies. HEK cells were ex-posed 24 or 48 hr after infection with adenovirustype 12 to 1 Mc/ml of 3H-thymidine for 1 hr andprepared for autoradiography immediately there-after. The silver grains were located predomi-nantly over the crystalline nuclear inclusionbodies known to be the replication site of adeno-virus (8). The feulgen-negative central nuclearinclusion was found to be unlabeled (Fig. 6c-f).Metaphases with chromosomal aberrationsshowed no labeling. Cells labeled for 24 hr with0.05 ,uc/ml of 3H-thymidine prior to infection, andexamined 1 or 2 days after infection, showed aneven distribution of the grains throughout inclu-sion-bearing nuclei (Fig. 6a, b).

Studies with 3H-labeled virus. Purified 3H-labeled adenovirus type 12, untreated or neu-tralized by specific antiserum, was used to infectHEK cells at a multiplicity of 0.1 to 1, as de-scribed in Materials and Methods. Metaphasepreparations from these cultures were obtained atvarying intervals after infection. These slides werecoated with NTB-3 emulsion and exposed for 14days in the refrigerator.

In preparations made 24 hr after infection,silver grains were found only within nuclei andnot in the cytoplasm. Nuclei with adenovirusinclusions demonstrated relatively heavy labeling(Fig. 7a, b). The mean grain count in these nucleiwas 36.4 silver grains (100 nuclei counted), ascompared with 22.86 for nuclei without morpho-logical changes. In inclusion-bearing nuclei, thelabel was predominantly localized within thecrystalline body. In metaphases, the label was as-sociated mainly with chromosomes, either in smallclusters or as single grains (Fig. 7c-f). By treat-ment of the slides with deoxyribonuclease beforeautoradiography, all label was removed. Exposureof the labeled virus to type-specific antiserumprior to infection of HEK cells significantly re-duced the uptake of labeled material (Table 4). In100 metaphases, the average grain counts werereduced to about one-ninth of that obtained withnon-neutralized virus. A preferential localizationof the grains in the centromeric region and in

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VIRUS-INDUCED ABERRATIONS IN KIDNEY CELLS ~~~Ii81I

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FIG. 4. Collection ofno. I and no. 17 chromosomes, demonstrating nonrandom breaks after adenovirus type 12infection. X 4,500

100

1o

x.--x Chromosome #17 oberation

o---.o T-ontigen producing cells

&____ Infectivity

0.1

0.01\

0.001 .

2 4 6 8 10 12

UV Irradiation in minutes

FIG. 5. Decrease of infectivity, T-antigen inducingcapacity and chromosome no. 17 aberration after UVirradiation of adenovirus type 12.

to 72 hr after infection. Isochromatid labeling,however, increased during this period by a factorof 3, examples of which are given in Fig. 10. Thenumber of cells approximately doubled withinthis 48-hr period.

DIscUSSIONHuman embryonic kidney cells could be main-

tained in culture for up to 20 passages and were

capable throughout of producing infectious ade-novirus type 12 progenies. In this cell system,infection at an input multiplicity of 1 (based uponTCID50) destroyed the culture, but at multiplicitiesof less than 1 the cultures survived and persistentinfections resulted. Such carrier cultures could bemaintained for more than 10 passages.

After 24 hr of exposure at a multiplicity of 1,and to lesser extents at lower multiplicities,chromosomal changes in infected HEK cells werenoted involving at a high frequency the long armof chromosome 17 at a site where a secondaryconstriction may normally occur (17, 20). Sim-ilarly, a near-terminal, nonrandom break occurredin the no. 1 chromosome at a lower frequency.Although other viruses are known to producenonrandom chromosome aberrations (22, 23),changes induced by adenovirus type 12 werepreviously reported to be random (13, 24).

Autoradiography with tritiated thymidinerevealed heavy labeling of the crystalline inclusionbodies, known to be the sites of adenovirusreplication (7). When the cells were labeled priorto infection, an even distribution of grainsthroughout the nucleus was observed 24 to 48hr after infection. Metaphases revealing chromo-somal aberrations 24 hr after infection did notshow any uptake of isotope when the cells hadbeen exposed to 3H-thymidine 1 hr before harvest.This indicates that at least during this 1-hr periodno viral DNA replication occurred within thesecells. Adenovirus DNA replication begins, at theearliest 10 hr after infection (6), whereas somevirus-induced chromosome changes were foundto be present already 8 hr after infection. Thus,viral DNA replication is apparently not necessaryfor induction of chromosomal aberrations. In thisrespect, infection by adenovirus type 12 differs

VOL. 1, 1967 1179

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FiG. 6. (a-b) Nuclei ofprelabeled cells (labeledfor 24 hr prior to infection) 24 hr after infection with adenovirus12. Note the even distribution of the grains throughout the nucleus. (c-f) One-hour labeling 24 hr after infection;c and d show an early stage of viral replication, and e andfshow a late one. Note the unlabeled central inclusionand the heavy labeling of the crystalline inclusion in a andf. X 4,500

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FIG. 7. Infection ofHEK cells with purified 3H-labeled adenovirus 12. (a and b) Nuclei revealing inclusionstypical for the adeno infection. Note that all grains are within the crystalline inclusion body. They are partly ob-scured by the heavy staining of the inclusion (b). (c-f) Metaphases revealing clusters of grains and single strainsassociated with chromosomes. X 4,500

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TABLE 4. Mean grain count in 100 metaphases ofHEK cells infected with labeled adeniovirus

type 12

Time after Cells infected Cells infectedinfection with labeled with neutralized with unlabeled

(hr) virus virus virus

24 28.23 3.34 1.8848 23.50 2.72 1.90

50

40

35

MEAN GRAIN COUNT PER METAPHASE(100 METAPHASES COUNTED)

PERCENT OF METAPHASESCONTAINING OR MORECHROMOSOMES WITH ISO-CHROMATID LABEL

30-

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-I..,M. ,LOCALIZATION OF GRAINSIN CHROMOSOME No.2

FIG. 8. Localizationi of grains in 200 no. 2 chro-mosomes. Note the accumulation of grainis in thecentromeric region.

from that by herpes simplex virus, in which caseviral DNA replication was regularly observed inaltered metaphases (19).UV inactivation of adenovirus type 12 dimin-

ished its capacity to cause chromosomal aberra-tions. This effect appeared to be a single-hit eventand suggested that viral DNA is responsible forinduction of chromosomal aberrations. The viralinfectivity was inactivated four times as fast asthe capacity to induce chromosomal changes.This indicates that only a part of the virus genomeis responsible for the chromosome damage (3).These results seem to support neither activation oflysosomal enzymes (1) nor inhibition of cellularDNA synthesis (14) as causes of virus-induced

24 48TIME IN HOURS AFTER INJECTION WITH

LABELED ADENOVIRUS TYPE 12

72

FIG. 9. Meaui graini count and isochromatid label atdifferent times after infectioni with labeled adenovirustype 12.

chromosomal aberrations. The action of anenzyme induced by viral genes offers a morelikely explanation.To approach the question of whether viral

DNA is incorporated into, or attached onto, thesite of chromosomal alterations, 3H-labeled viruswas used for infection. It is assumed that most ofthe label represented viral DNA, although itcannot be excluded that some labeled host DNAwas included in the virions. Interpretation of theresults with labeled virus must be considered,therefore, with some reservation. In cells infectedwith labeled virus, the isotope was found to beexclusively associated with the chromosomes asearly as 24 hr after infection. The silver grainsoccurred more often in clusters than singly. Thesites of most of the chromosomal breaks werefree from label, but occasionally single grainswere seen at the breaks of chromosomes 17 and 1.This result suggests that usually no attachmentof the viral genome or part thereof occurs at thesite of the chromosomal aberration.

Certain sites of chromosomes appeared to bepreferentially labeled, but the deviation of somegrains rendered a quantitative evaluation difficult.An accumulation of label was, however, apparentfor example in no. 2 chromosomes in the centro-meric region and at several other sites.

Isochromatid label was found to increase withthe time after infection. Although at 72 hr mostof the clustered label was still seen only on onearm of the chromatid, isochromatid labelingincreased threefold when compared with results

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FIG. 10. Examples of isochromatid label int metaphases ofHEK cells infected with labeled adenovirus type 12at 48, and 72 hr after inifection. X 6,000.

obtained at 24 hr. The total grain count permetaphase decreased twofold during this period.These results might suggest that the viral genome,or parts thereof usually attached themselves, or

lie adjacent to the chromosomes, without beingintegrated into cellular DNA. Were it inte-grated, one would expect isochromatid labelingat the incorporation site after the first cellular

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ZUR HAUSEN

DNA replication. Since this type of labeling was a

relatively rare, but nevertheless increasing, eventwith time after infection, integration of viralDNA into cellular DNA might occur. It probablydepends, in addition, on other factors, such as thenumber of viral genomes per cell or the patho-genicity of the virus. The fact that these cellsundergo several mitoses in spite of the presenceof viral DNA suggests the frequent occurrence ofnonlethal, abortive cellular infections by adeno-virus type 12.

Type-specific antiviral serum considerablyreduced the attachment of labeled material to thechromosomes. The decrease in grain counts wasabout ninefold as compared with labeled virusnot exposed to antibody. This difference is takento account for neutralization of labeled virions.

It cannot be stated that the results reportedhere bear any relevance to the possible onco-genicity of adenovirus type 12. Though somenononcogenic adenoviruses (types 2 and 5) ap-parently do not induce chromosomal aberrations(16; zur Hausen, unpublished data), preliminaryresults indicate that the association between viraland host cell DNA is very similar.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService research grant CA-04568 from the NationalCancer Institute.

Helpful discussions and the review of the manu-script by Werner Henle, Gertrude Henle, and GertrudeKohn are gratefully acknowledged. I am indebtedto Rosemary Makarczyk for excellent technical as-

sistance.

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