Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA.

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JOURNAL OF VIROLOGY, Oct.1976,p.86-95

Vol. 20, No. 1 Printed inU.S.A.

Isolation

and Structural Characterization

of

Monomeric

and

Dimeric Forms of Replicative Intermediates

of

Kilham

Rat

Virus

DNA

M. GUNTHER* ANDP. MAY

Unite deBiophysique, Institut de Recherches ScientifiquessurleCancer, B.P. No.8-94,800Villejuif,France

Received for publication19February1976

Two virus-specific species of newly synthesized DNAwere isolated fromrat fibroblast cellcultures infected with the Kilhamratvirus (RV).ThesetwoDNA specieswere purified; their behaviouron hydroxyapatitechromatography and

theirsedimentation coefficients insucrose gradientswere determined. One of

thetwospeciescorrespondstothe lineardouble-strandedform of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a

fractionof both species showedanintramolecularrenaturation; these molecules arecomposed of viral strandcovalently linkedtocomplementary strand. Models

forthe structure of both speciesareproposed. Both speciesmaybeconsideredas

double-stranded replicative intermediates of the single-strandedRV DNA.

Kilhamrat virus(RV),oneof the small icosa-hedric single-stranded DNA (ssDNA)virusesof theparvovirus group, wasfirstisolated froma rat sarcoma by Kilham (6). (The recognized notationRV will beusedhere, althoughthere areatleast three other knownparvovirusesof therat.) RVDNAhas beendemonstratedtobe linear(15) and singlestranded (8, 11, 13), and

tohave amolecular weight of about 1.6 x 106 (8, 11, 13).

Vertebrate parvoviruses fall into two

subgroups: (i) parvoviruses, including RV, H,

virus, and minute virus ofmice, which have been shown to replicate autonomously in rap-idly dividing cells (21)andtobe dependentfor theirreplicationupon acellular event(s)inthe late S phase (9, 16); and (ii) adeno-associated

viruses, whichcannot replicate in cells unless anadenovirus"helper"ispresent(12). Plus and minusstrands ofadeno-associated virus DNA havebeen showntobecoatedseparately(2).

Thestudy ofparvovirusreplication may pro-videamodel for thereplication of ssDNA ina

mammaliansystemand for thereplicationofa

linearDNAmolecule. It isknown thatssDNA ofseveralparvoviruses isconvertedintoa dou-ble-stranded formafter infection(10, 14,20).

Inthispaper, twospecies ofdouble-stranded forms of RV DNA found in infected cells are

described. Thelength andthe structureof these molecules werestudied, andtheirpossiblerole intheRV DNAreplicationisbrieflydiscussed.

MATERIALS AND METHODS

Cell strains and virus. The KilhamRV used in

these experiments wasobtained from R. Tournier

and originally derived from W. Rowe's strain. RV was produced in Wistar AF rat embryo primary

culture. Rat embryos (17 daysold)weretrypsinized

andseeded inbottles of a tissue culture Rollacell

(New Brunswick Scientific Co.) in 120 ml of Eagle

medium supplementedwith10%tryptose phosphate

broth and 10% calf serum (Sorga). Infected cells

(multiplicity of infection, 0.1 to 1 PFU/cell) were

collected 2 days after infection and frozen and

thawed three times. Cellular debris was then

treatedtwice inhypotonicmedium(double-distilled H20). The supernatantscollectedbylow-speed

cen-trifugation were combined and made isotonic by

addition of double-concentrated culture medium.

This viral suspension wasused as a stock virus after

titrationby the plaque assay technique on

second-arycultures of ratembryocells.

Intracellular synthesis ofviral DNA wasstudied

in aratfibroblast strain(RTcells)obtainedfrom a

rat embryo cell culture. (Some RT cells have been

found [41tobecontaminated byanewparvovirus,

the "RT virus." For the experimentsdescribedhere,

different passages ofroutinely cultured RT cells

weretested by the technique of Hallauer et al. [4;

HAtiter ofglycine buffer extracts] and foundfree of

virus. Insome rare cases a contaminant viruswas

foundindegenerating cultures, but this was always

identified by inhibition hemagglutination tests as

theRVvirus.We cannotruleoutthepossibilitythat

this contaminant virus comes from the RV virus

usedinthislaboratory.) RT cellsweremaintained

intheculture mediumsupplemented with 5% calf

serum inFalconplasticbottles(75 cm2).Studies of

viral DNAsynthesis werecarriedout inpetri dishes

(60-mmdiameter)with thesamemedium.

Viruspurification.Infected cellswerelysedwith

1%sodium deoxycholate and treated for18hat37°C

with50,ug of DNase I per mland5 ,ug of RNaseIll

(Sigma)per ml in10mMMgCl2. Themixture was

adjustedto0.01%trypsin(Choay)andincubated for 86

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REPLICATIVE INTERMEDIATES OF RAT VIRUS DNA

30 min at 370C. The virus was then purified by

isopycniccentrifugationinCsCl (1.4g/cm3)at35,000

rpm for 18h at 25°C in aSpinco SW50.1rotor.

Viral DNA extraction. Viral DNA was extracted

by sodium dodecyl sulfate (SDS) and purified

ac-cordingtotheprocedureofMayandMay (8). Viral

DNA was alsoeasilyprepared byextraction in 0.1 M

NaOH for 20 min at room temperature.

Purification of DNA from infected cells.Growing

RT cells (1.5 x 105 to 1.8 x 105 cells/cm2) were

infected with RV at amultiplicityof infection of 5

PFU/cell. At various timespostinfection (p.i.),cells were labeled with tritiated thymidine ([3H]Tdr, 5

,uCi/ml, 20Ci/mM; Saclay) forperiods of 1 to 3 h.

Low-molecular-weight 3H-labeled DNA was then

extracted byan SDS-selective procedure according

to Hirt(5),exceptthat thelysatewasincubatedin

the presence of 100 yg of pronase per ml for 30 min at

370C. Similar results were obtained when pronase wasaddedbefore or afterremovingthe high-molec-ular-weightDNAbycentrifugation. The 3H-labeled

DNAfrom the Hirt supernatantwasdeproteinized

bytwochloroform treatmentsandthenpurifiedin an equilibrium cesium chloride density gradient

(CsCl,1.72g/cm3)at35,000 rpm at250C for48h in a

Spinco65rotor, eitherdirectlyor afteran ethanol

precipitationovernightat-20°C.Fractionsfrom the

majorpeak ofradioactivity (Fig. 1) were collected

and dialyzed against

tris(hydroxymethyl)amino-methane (Tris)-EDTA buffer (0.01 MTris,0.001 M

EDTA,pH 8). The purification wascompletedbya

phenol extraction at room temperature, and DNA wasdialyzedagainst the samebuffer.

Alkaline denaturation of DNA. The 3H-labeled

DNA wasincubatedfor 10 min at roomtemperature

in 0.1NNaOHandthenneutralizedwith 0.1 NNa

H2PO4 unlessspecifiedotherwise.

Hydroxyapatite chromatography.

Hydroxyapa-tite chromatography of purified 3H-labeled DNA,

either native ordenatured,wasperformedaccording

tothemethodofTapieroetal. (19). Portions of

3H-labeledDNA preparations weredilutedto4ml with 0.01Mphosphate buffer, pH7.85, and mixed with 0.25g ofhydroxyapatite (DNAgrade;Bio-Rad) and

incubated for 5 min at room temperature.

Alkali-denaturedDNA (100 ,ul) wasdiluted with4 ml of

0.01Mphosphate buffer before neutralization with

NaH2PO4. The hydroxyapatitewaspelletedby

low-speed centrifugation,and the supernatantwas

col-lected. Hydroxyapatite was then washed by the

sameprocedure with 0.01 Mphosphatebuffer, pH

7.85.Stepwise elutionofthe DNA wasperformed by

resuspending the hydroxyapatite pelletin 4 ml of

phosphate bufferof increasingmolarities (from 0.1

to0.7M). The tubes were incubated in a water bath

at560Cfor 8 min with intermittent agitation.

Hy-droxyapatite was againpelleted by low-speed

cen-trifugation at room temperature, and the

superna-tants were collected. Carrier DNA (calf thymus;

Choay)wasadded to eachstep-elutionfraction, and

coldtrichloroacetic acid was added to a final

concen-tration of 5%. After an incubation for 10 min in an

ice-waterbath, the acid-precipitable material was

collected on glass fiber filters (Whatman GF/C).

Eachtube was washed with 4 ml of 5%

trichloroace-tic acid, which was poured onto the filters. The

filters weredried, andtheradioactivity was

deter-minedin anIntertechniqueliquid scintillation spec-trometer.

DNA-DNA hybridization. Purified 3H-labeled

DNA wasdenaturedandfragmented into piecesby

boilingfor 15 min in 0.01 x SSC(SSCis 0.15 MNaCl

plus0.015 sodiumcitrate). At this stage, the DNA

sedimented in an alkaline sucrose gradient as a

uniformbandcorresponding toabout 9S. The DNA

wasthenreannealed in 100 ul ofTris-EDTAbuffer

containing 0.3 M NaCl at650Cwithdifferent

quanti-ties ofunlabeledRVDNA (up to 880 ng).After48h

ofincubation,two40-ul portionsdesignatedAand B

were taken and treated separately. Portion A was

diluted to 0.4 ml with Tris-EDTAbuffer, and the

total radioactivity of A was counted in the cold

trichloroacetic acid-precipitable material. For

por-tionB, onlythe radioactivity remaining in a

double-strandedform was counted in the cold

trichloroace-tic acid-precipitable material after digestion of

ssDNAby theS, nuclease fromAspergillus oryzae

preparedaccording to Vogt (22). Portion B was

di-luted to 0.4 ml in abuffer containing 0.03 Msodium

acetate, pH 4.6, 0.3 MNaCl, 0.001 M ZnSO4,and5%

glycerol. Heat-denaturedcalfthymus DNA (20 ,ug/

ml) and 5 U ofS, nuclease per ml were added.The

mixture was then incubated for 1 h at450C. The

results are given as the percentage of the

radioactiv-ityremaining in portion B as compared to that in

portionA.

Velocity sedimentationinsucrose gradients.

Por-tions (up to 0.2 ml) of the purified 3H-labeled DNA

sampleswere layered on 4 ml of a 5 to 20% linear

sucrosegradient. Neutral sucrose gradients in 1 M

NaCl and Tris-EDTA buffer were centrifuged at 200C for 150 min at 54,000 rpm in a Spinco SW56

rotor. Foralkaline sucrose gradients the 5% sucrose

solution was made pH 13 in 0.3 N NaOH, 0.7 M

NaCl, and Tris-EDTA buffer, and the 20% sucrose

solution was made pH 13 in 0.6 N NaOH, 0.4 M

NaCl, and Tris-EDTA buffer. The alkaline sucrose

gradients were thencentrifuged at200Cfor 180 min

at54,000 rpm. Gradientswere fractionated by

col-lectingdrops from the bottom of the tube directly

ontoglass fiber filters. Filters were washed twice

with cold 5% trichloroacetic acid and then with

ethanol. Radioactivitywasdetermined by counting

driedfilters in a liquid scintillation spectrometer.

RESULTS

Presence of two new species of 3H-labeled DNA in RV-infected cells. RV-infected rat fi-broblasts (RT cells) werelabeled with [3H]Tdr (5

ACi/ml)

for1h

p.i.

tovarious

lengths

of time. After the labeling period, they were lysed by SDS.Theradioactivitywasthencountedinthe trichloroacetic acid-precipitablematerial of the SDS-pronase Hirtsupernatants (see Materials andMethods). Undertheseconditions, incorpo-ration of[3H]Tdr was first detected at 6 h p.i. and thenbecame linear as a function of time between 9and 18 h p.i. and reached a plateau (data not shown). Under the same conditions, incorporation of[3H]Tdr in the trichloroacetic 87

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88 GUNTHER AND MAY

acid-precipitablematerial of the Hirtpelletsof infected cells reached a plateau at 6 h p.i. In most ofthe experiments reported here, DNA was labeled at 12 h p.i. for theindicated period, then extracted, and purified as described in Materials and Methods. As shown later, the DNA of Hirt supernatant labeled at 12 h p.i. was essentially virus specific. The last step of the DNA purification was an isopycnic centrifu-gationgradientinCsCl(Fig. 1).The bulk of the radioactivity banded as a single peak slightly

heavier than that of cellular DNA (the

differ-ence in densities was about 0.005 g/cm3),

whereas verylittle radioactivity was found in the region ofthe RVssDNA. Thestudy ofthe

3H-labeled DNA contained in the major peak

will be reported in this paper. Purification of 3H-labeledDNAbyadensitygradient centrifu-gationreducedcontamination

by

cellularDNA. The radioactivity remaining at the top of the gradient corresponded to 3H-labeled DNA linked to viral proteins in spite of the SDS-pronase treatment performed as described in

Materials and Methods. (Progeny ssDNA was

always foundinthisform.)The

study

ofthese DNA-protein complexeswillbedescribed in a

subsequentpaper.

The3H-labeledDNA thuscollected from the preparativedensity

gradient

(Fig. 1) was

ana-FRACTION NUMBlER

FIG. 1. Preparative isopycnic centrifugation of

newly synthesized DNA in RV-infected cells.

RV-infectedRT cells (multiplicity of infection, 5PFU/

cell) werelabeled with [3H]Tdr (5 pACilml) at12 h

p.i. foraperiod of1h. 3H-labeled DNAfromtheHirt

supernatant(purifiedasdescribed)wassubmittedto

anequilibrium density gradient centrifugation in 6

mlofCsCl (1.72 g/cm3) at35,000 rpmfor48 h at

20°C in aSpincorotor65. Fractions werecollected

fromthe tube by bottompuncture. Portions(10 d)

fromeachfraction werespottedonfilters,andtheir

radioactivitywas counted. Portionswere also taken

tomeasuretherefractiveindex. Arrows indicate the

positions ofcellularDNA and RV DNA added as

markers inaparallel gradient.

lyzed by velocity sedimentation in a 5 to 20%

neutral sucrose gradient. The pattern of sedi-mentation illustrated in Fig. 2 shows twopeaks ofradioactivity that were not found in mock-infected cells. The major peak (M)accounts for 80% of theradioactivity, and the minor (D) for 20%. To study these two species of DNA, frac-tions M and D were separately pooled and puri-fied by an additional sucrose centrifugation.

Hydroxyapatite chromatography. Elution pattems fromhydroxyapatite chromatography of RV DNA and of DNA species M (DNA-M) andD(DNA-D) were studied. Under the exper-imental conditions used (see Materials and Methods), native dsDNA was eluted from

hy-droxyapatitewith 0.55 M phosphate buffer, pH 7.85, and denatured DNA with 0.15 M phos-phate buffer. These characteristics were illus-trated in our experiments with rat cellular DNA (Fig. 3a, b). By contrast, RV 3H-labeled DNA was eluted with 0.3 M phosphate, which wasunexpected for ssDNA (Fig. 3c). Consider-ing that by increasConsider-ing concentrations of phos-phate buffer the elution from hydroxyapatite

mainly depends on thesecondary structure of the DNA (1), this result strongly suggests that RVDNA has aspecialstructure. A short

palin-dromic region could account for the value ob-tained for the molarity of elution. Since minute virus of miceDNA has been found to contain a

small duplex region (2 to 5%) ofthe viral ge-nome (G. J. Bourguignon, P. J. Tattersall, and D. C. Ward, Abstr. Int. Congr. Virol., 3rd, p. 181, 1975), we also studied the behavior of this DNA in hydroxyapatite chromatography and observedanelutionpattern similartothat ob-tained with RV DNA (data not shown). More-over, apalindromicsequence hasalso been

sug-gestedfor the terminal segments of

adeno-asso-ciated virus DNA strands on the basis of the results of Koczot et al. (7) and Gerry et al. (3).

The DNA species M and D were elutedfrom hydroxyapatite with 0.55 M phosphate,

indicat-ing a double-stranded structure, whereas no radioactivity was found at either 0.15 or 0.3 M phosphate (Fig. 4a and c). Alkaline-denatured DNAofspecies MandDhavesimilarpatterns

ofelution fromhydroxyapatiteasshowninFig. 4 (b and d). Both DNA species, M and D, are resolved into two different peaks. One peak

elutes at 0.3 Mphosphate, and the otherpeak

at 0.55 M phosphate (about 30and 50%of the totaleluted radioactivityforspeciesMandD, respectively). Similar results were obtained with DNA that was heat denatured in Tris-EDTA buffer at 100°C for 3 min and then quickly cooled. It isimportantto stressthat0.3 Mphosphateisthetypicalmolarityof elution of

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RV DNA RV ssDNA and that 0.55 M phosphate is the

J

ffi D M molarity of elution of dsDNA. These results

J1

4

suggest that3H-labeled DNA of both species M

.~,15_IF and D is doublestranded. A denaturation

treat-ment converts a fraction of both species to ssDNA that is eluted at 0.3 Mphosphate as RV

0fi

l DNA, whereas the remaining molecules are

elutedatthemolarity correspondingtodsDNA.

10-_

lThe

experimental conditions of denaturation

used are assumed to prevent intermolecular renaturation of DNA (low ionic strength, low DNA concentration, and room temperatures).

s-

lWe

willshowinthe

following

experiments

that the DNA which is eluted at 0.55 M phosphate afterdenaturationderivesfromintramolecular

"snap-back" renaturation.

I6y

\

Hybridization.

To examine the viral

specific-bo,tom

ity

of3H-labeled DNA extracted from

RV-in-nwn,mFractinr

fected rat cells, species M and D were heat denatured in 0.01x SSC and reannealed in 0.3 FIG. 2. Sedimentation in a neutral sucrose gra- MNaCl at 65°C with increasing quantities of dientofaHirtsupernatant DNAfrom RV-infected or unlabeled RV DNA. After 48 h the DNA re-mock-infectedcells. 3H-labeled DNA was layered on

the top of a 5 to20%sucrose gradient in 1 MNaCI

matring

ss was degraded by

Sp

nuclease, and

and Tris-EDTA buffer and centrifuged at20°Cfor trichloroacetic acid-precipitable radioactivity 150 min at 54,000 rpm in a Spinco SW56 rotor. wasdetermined (see Materials and Methods). If

Fractions were collectedfrom thebottomof the tube we assumethatspeciesM and Darecompletely

directly on glass fiber filters, and the radioactivity of homologous to RV DNA, which contains only

dried filters was counted. The arrow indicates the the"viral strand," saturatingconcentrations of

sedimentation position ofRV DNA addedasmarker unlabeled RV DNA would be expected to

dis-(24S under the conditions used). Symbols:

*,

3H- place all viral strands from species M and D

labeled DNA collectedfrom the major peak, corre-

(i.e.,

50%

ofthe

radioactivity,

assuming that

sponding to the horizontal bar ofFig. 1; 0, 3H-

3H-labeled

DNA is equally labeled on both labeledDNA of a Hirt supernatant

firom

mock-in- 3

fected cells labeled for 1 h andextracted asdescribed strands). The 3H-labeled DNA extracted from

for infectedcells.FractionscorrespondingtoDNA-M RV-infected cells could thus be considered as

and -D werecollected separatelyfrom a similargra- 100% virus specific. As already indicated, a

dient. fraction of the alkaline-denaturedDNA species

a b c

E

06

X,

2030, ,

Q15 Q30 0.55 Q.15 030 0.55 0.15 0.30 Q55

[image:4.508.53.241.60.275.2]

Phosphate buffer)Moes/Liter)

FIG. 3. Hydroxyapatitechromatographyofpurified3H-labeledcellular and RV DNA. 3H-labeled DNA in

0.01 Mphosphate buffer, pH 7.85, was adsorbed at room temperature to 0 25 g ofhydroxyapatite.Stepwise

elution wasperformed at56Cbyincreasing molarities ofphosphate buffer, pH 7.85 (from01 to0.7 M), as

described inMaterials and Methods, and the radioactivity of thetrichloroacetic acid-precipitable material of

each eluate was counted. (a) NativecellularDNA; (b)alkali-denaturedcellular DNA; (c) RV DNA.

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90 GUNTHER AND MAY

Phosphatebuffer(Moles/Liter)

FIG. 4. Hydroxyapatite chromatography of

3H-la-beled DNA-Mand-D, by the method described in

Fig. 3. (a) Native DNA-M; (b) alkali-denatured

DNA-M; (c) native DNA-D; (d) alkali-denatured

DNA-D.

M and D undergoes intramolecular renatur-ation. The denaturingconditions used for hy-bridization experimentswerechosen toreduce this intramolecular renaturation by reducing themolecular weight. The DNAspeciesMand D were converted to ssDNA of a molecular

weight of4 x 105 (25%of thegenomelength) by

boilingDNA 15minin0.01x SSC. Denatured

DNAspeciesMand D showedanS1 resistance

of 9% in the absence ofreannealingtreatment. After reannealing, 3H-labeled DNA species Mshowedan

S,

resistanceof 92.2 + 7.5%(mean value + standard deviation forseven independ-entdeterminations). When increasing quanti-ties ofunlabeled RV DNA were added, this percentage first decreased and finally leveled

offat47%with 200ngof unlabeled DNA.This

valueremainedessentially unchangedwith

ad-dition ofincreasingamountsof RV DNAupto

880ng(Fig. 5).WhenexcessheterologousDNA was added to the incubation medium, the

amount ofreannealing of 3H-labeled DNA-M

was about 80% (Fig. 5). Thismean value was

obtained with unlabeledDNAfrom RT(rat)or

CV1 (monkey) cells and from SV40. To take intoaccountthisnonspecific hybridization, the experimental value ofrenaturationofDNA-M obtained with excess unlabeled RV DNA was

multiplied byacorrectingfactor of100/80,and we thus obtained a corrected value of 60%.

Thus,40%ofthelabeled DNA-Mwasdisplaced

by the unlabeled RV DNA, corresponding to

80% homology for this species. This value is underestimated ifwe consider that 9% of the

DNA is resistant to an

S,

nuclease digestion

without reannealingtreatmentandisprobably counted with the DNA remaining double stranded after renaturation. Similar results

wereobtained with 3H-labeled DNAspeciesD,

except that 3H-labeled DNA-D reannealed to theextentofonly75.6 + 14.7%(meanvalue +

standard deviation obtained with seven

inde-pendent determinations). Purified 3H-labeled DNA-Dpreparationswerealwaysinlower

con-centrations than 3H-labeled DNA-M

prepa-tions.Thus, reassociation conditionswere

prob-ablynotoptimal, since the standarddeviation

wasrelatively high. When unlabeledRVDNA was added in excess to DNA-D, better

condi-Unlabelled DNA added(ngl

FIG. 5. Variations of the percentage of

renatur-ationof3H1-labeledDNA-M as a functionof

increas-ing quantities of unlabeled RV DNA. 3H-labeled

DNA-M labeled from 12 to 15 hp.i. was denatured in

0.01x SSC fori5min at 100°C and then reannealed

in 100 ,ul of 0.3 MNaCI for 48 h at 65°C in the

presence of varying quantities of unlabeled DNA.

Each assay wasperformed with 10 ng ofDNA-M. At

the end of thereannealing period two equal portions,

designated A and B, were taken; the radioactivity

was counted directly in the trichloroacetic

acid-pre-cipitable material ofportion A, and the radioactivity

ofportion B was counted in thetrichloroacetic

acid-precipitable material after anS1nuclease digestion.

All results are expressed as the percentage of the

countsper minute remaining indsDNAafter theS,

nuclease digestion (portion B) as compared to the

total counts per minute(portion A). Unlabeled DNAs

added: *, RVDNA; A\,RTDNA;*,CV1 DNA; 0,

SV40 DNA.

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tions of concentration wereobtained. The per-centageof renaturation ofDNA-D inthe pres-enceof 100 and 200 ng ofunlabeled RVDNA was 46 and45%, respectively. These valuesare similar to the corresponding values obtained with DNA-M. Both species appear to possess the same extent of virusspecificity(over80%).

Sucrose sedimentation. Species M and D of

3H-labeledDNAwerestudiedseparatelyin su-crose gradients (Fig. 6). Sedimentation coeffi-cients were 14.5S for DNA-M and 18.3S for DNA-D ascompared to the 16Ssedimentation coefficient of SV40 form II DNA added as a marker (Fig. 6a, d).Since the species M andD have been observed as linear molecules with

the electron microscope (M. Gunther, H. Bu-jard, and G. Hayward, manuscript in prepara-tion), it waspossible tocalculate themolecular weight of each species. To obtain an accurate evaluation of the molecular weight of DNA-M, we compared, in the same gradient, the sedi-mentationproperties of DNA-Mandthelinear form ofSV40 DNA obtained after Hpa II endo-nuclease digestion (data not shown). DNA-M sedimentedslightlymoreslowly: the difference in sedimentation coefficients was0.4S, which

corresponded to a molecular weight of 3.15 x 106for DNA-M, with a molecular weight of 3.4 x 10c for SV40 DNA. The ratio (molecular

weightofDNA-D/molecular weight of DNA-M)

Fractionnumber

FIG. 6. Sucrose sedimentations of 3H-labeled DNA-M and -D. Neutral sucrose sedimentations were

per-formedasdescribedinthe legend ofFig. 2. Alkaline sucrose gradients (pH 13) were centrifuged at 20°C for

180minat54,000 rpm in a SpincoSW56 rotor. The 3H-labeled DNA-M sedimented in (a) neutral, (b) neutral

(after an alkaline denaturation), and (c) alkaline 5 to 20% sucrose gradients. The 3H-labeled DNA-D

sedimentedin(d) neutral, (e) neutral (after an alkaline denaturation), and (f)alkaline 5 to20%sucrose

gradients. In gradients b, c, e, and f, RV"4C-labeledDNA was added as a marker.SV40

"4C-labeled

DNA

form IIwasadded as a marker in parallel gradients. 16S arrows in neutral sucrose gradients represent the

sedimentation position of SV40 form II, and in alkaline sucrose gradients arrows16S and 18S represent the

positionsof the circular and linear strands derived fromSV40 DNA formI.

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92 GUNTHER AND MAY

wascalculated from the experimental

sedimen-tation coefficients 18.38 and 14.5S by using Studier's formula (18). This ratiowas 2

(calcu-lated value, 1.94). Thus, the DNA of species M

appearstocorrespondtothemonomeric ds form

of RV DNA, and the DNA of species D to a

dimer of species M.

Both DNA-M and -D were alkaline

dena-tured and studied separately in alkaline and neutral sucrose gradients (Fig. 6b, c, e, f). An

alkaline sucrose gradient of DNA-D (Fig. 6f)

showedtwopeaks whose sedimentation coeffi-cientscorrespondedto20.38 and 16S. The16S peak co-migrated with RV 14C-labeled DNA

markerand the20.3Ssedimentation coefficient found for the secondpeakwasclosetothe

coeffi-cientexpected (21S)for thecorresponding sin-gle-stranded dimer. Thus an alkaline sucrose

gradient of DNA-Dgaveessentiallytwospecies

of ssDNA inamolecularweight ratio of 2

(cal-culated value, 1.86). An alkaline sucrose

gra-dientof DNA-M showed thesametwopeaks of sedimentation(Fig. 6c), whichwillbe discussed indetail later. Inaneutralsucrosegradient of

denatured DNA-M, two peaks were obtained

withcoefficients of24Sand 14.5S (Fig. 6b). As

shown later, the 24S peak, which co-migrates with RV DNA, corresponds to the single-stranded monomer form, and the 14.5S to a

double-stranded snap-back DNA of monomer

length. Under the same sedimentation

condi-tions, DNA-Dgaveamajor peakat14.5Sanda

shoulder ofradioactivity at 24S, i.e., the posi-tion of RV ssDNA (Fig. 6e). Radioactivitywas notfoundatthe position for thesingle-stranded dimer (whose expected sedimentation

coeffi-cient would beranging about5OS). Thus, inan

alkaline sucrose gradient (Fig. 6c, f),the

sedi-mentation patterns of denatured DNA from

both DNA-M and -D showedtwo bands

corre-sponding to the single-stranded monomer and

thesingle-stranded dimer;inaneutralsucrose

gradient (Fig. 6b and e) the sedimentation

pat-ternsofdenaturedDNAfrom both DNA-M and

-Dshowedtwobandscorrespondingtothe

sin-gle-stranded monomer and to an additional

componentsedimentingat14.5S.

The fractions(A, B, B', C, and C') indicated

inFig. 6werepooled and examinedon

hydroxy-apatite and in sucrose gradients. The DNA

frompool Awasessentially single stranded,as

indicatedby its molarity ofelution(0.3M phos-phate)inhydroxyapatitechromatography (Fig. 7a) and resedimented asthe RV DNA in

neu-tral or alkaline sucrose gradients (data not

shown). The DNA from pools B and B' was

elutedwith 0.55Mphosphatefrom hydroxyapa-tite (Fig. 7b); i.e., it behaved as dsDNA and

sedimentedat20.3Sinanalkalinesucrose

gra-~4C

,,, 40 E

3CL

I30

20

0₁₀

- 10

D

0.15 030 0.55 0.15 0.30 0.55 Phosphate buffer (Moles/Liter)

FIG. 7. Profile of elution from hydroxyapatite of

3H-labeled DNA from pools A and B corresponding

to horizontal bars inFig. 6. The method isas

de-scribedinthe legend ofFig. 3. (a) PoolA; (b) pool B.

dient (Fig. 8a). Thus the DNA from pools B and B'appearedtobe doublestranded; this DNA in neutral sucrosebehaved as adouble-stranded

monomer and in alkaline sucrose as a

single-stranded dimer. DNA from pools C and C', i.e.,

an"alkaline"single-stranded dimer, renatured

when neutralized, and then it was eluted at

0.55 M phosphate from hydroxyapatite (data

notshown) and sedimented at 14.5S in a

neu-tralsucrosegradient, asexpectedfora

double-stranded monomer (Fig. 8b). These results

show that the rapidly renatured DNA arose

fromanalkaline single-stranded dimer. To

ac-countfor theseobservations,we supposethat in

such a single-stranded dimer, both strands,

viral andcomplementary, arelinked byan al-kali-stable covalent link, giving rise, when neutralized, to a hairpin structure. Molecules whichsnapbacktothis hairpinstructurehave the characteristic monomer length. On the

basis of thisobservation, we proposestructures

for themonomeric and dimeric species (see Dis-cussion).

DISCUSSION

In this work,we have shown thepresenceof

two species of DNA in RV-infected rat cells afterlabeling with[3H]Tdr. The DNAobtained by an SDS-pronase selective extraction from

RV-infectedratcells andcentrifuged inaCsCl

gradient formed a band slightly heavier than

that of theratcellular DNA (Fig. 1).The DNA in this band, when sedimented in a neutral

sucrosegradient,wasresolved intotwodistinct

peaks, M and D (Fig. 2). Both species were

shownto bedoublestranded by their molarity ofelution (0.55 M phosphate buffer) during hy-droxyapatite chromatography (Fig. 4) andtobe

atleast 80%virusspecific by displacement hy-bridization experiments with saturating

con-centrations ofunlabeled RV DNA. Analysis of

a b

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10 20 30 40 50 60 70 10 20 30

Fractionnumber

FIG. 8. Sedimentationof3H-labeledDNAfrompoolsBand C(correspondingtohorizontal bars inFig.6).

(a)Alkalinesucrosegradient of3H-labeled DNAfrom poolB (a); RV "4C-labeled DNA was added as a

marker(0);(b) neutral sucrosegradientof3H-labeledDNAfrom pool C (0); "4C-labeledDNA-Mwasadded

as amarker(0).

thetwo speciesbysucrosegradient

centrifuga-tion with various DNA markers (SV40 DNA form II, SV40 DNA linear form III, and RV DNA) showed that species M of 3H-labeled

DNAcorresponded to the linear duplex form of RVDNA,with amolecularweightofabout3 x 106, whereas species Dcorrespondedtothe lin-eardimeric duplexof species M.

The resultsfrom sucrosesedimentation anal-ysis (Fig. 6and 8), whencombinedwith those

obtainedbyhydroxyapatiteexperiments (Fig.4

and7), haveshown that a fraction of both spe-cies snaps back to a hairpin monomer after

denaturation. This propertyleadsus to propose

thefollowingstructuresfor the DNAspeciesM

and D(seeFig. 9 and 10).

Monomeric DNA preparations probably con-sistof a mixture of two types (1and2) of

double-stranded molecules (Fig. 9). These structures are consistent withthe behavior ofdenatured

monomericdsDNA. Themonomergives riseto twokindsofsingle-strandedmolecules in

alka-line sucrosegradients:one(of monomer length) comes from structure (1) and the other (of di-merlength) comes from structure(2); the latter snaps back when neutralized into a hairpin monomer.

A large proportion (about 50%) of dimeric moleculesspontaneously renatured, after dena-turation, to hairpin monomeric molecules. Di-meric molecules must have a structure such that viral and complementary strands are

5'

V

3 '

(1)

3'C

59

5'V~) (2)

[image:8.508.45.452.59.285.2]

3'

C

FIG. 9. Proposed structure for the

double-strandedmonomericspecies(DNA-M)which

proba-blyconsistsoftwotypes (Iand2).Onestrand in each

duplexin anRV viral strand (v), and the other is the

complementary strand (c). In type 2, v and c are

covalently linked,forming a hairpinmolecule.

5'

V C

3'

39 C V

5'

FIG. 10.Proposed structure for the

double-stranded dimeric molecules (species D). Each linear

strand in the duplex is composed of one RV viral

strand and one complementary strand, which are

covalently linked end to end.

93

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94 GUNTHER AND MAY

linked by an alkali-stable covalent link, as rep-resented in the scheme in Fig. 10.

This is inaccordance with the fact that radio-activity was not detected in neutral sucrose gradients in the region theoretically

corre-spondingto asingle-strandeddimer. After neu-tralization single-stranded dimers were always found in the region of monomeric dsDNA. A fraction of denatured dimeric DNA did not snap

back and behaved as single-stranded mono-meric molecules in neutral or alkaline sucrose

gradients. Thus, it is likely that a fraction of

dimeric moleculesmusthaveanickat(ornear)

the middleofthestrand(s).

There is another possible structure for the

dimer. Dimeric molecules might arise by the joiningbyhydrogen bondsoftwohairpin mono-mers, occurring either ininfected cells or dur-ingthe DNAextraction-purificationprocedure.

Suchastructureneedsaterminalredundancy

if the joining occurs between a viral and a

complementary strandandapalindromic struc-ture at the ends of the strands ifthejoining

occursbetween twoviral-ortwocomplementary strands. The existence of the latter structure cannotberuledout,althoughitseems

unlikely

if we assumethat dimericmoleculesplayarole inthe RV DNAreplication.

In conclusion, two new species of virus-spe-cificdsDNAwerefoundinRV-infectedratcells: onespecies(the more

abundant)

ismonomeric (a one-unitlength);theother isdimeric (a

dou-ble-unit length). Both species contain

cova-lently linkedviraland

complementary

strands (Fig. 9 and 10), which

strongly

suggests that

theyare synthesizedby a

self-priming

mecha-nism. A similar hypothesis was

suggested by

Tattersalletal., whoreportedthe existence ofa

hairpinmonomerform of intracellular minute virusof mice DNA(20).

Recently,

Strausetal. (17)identifiedreplicative intermediatesincells

infected with adeno-associated virus type

2,

whichconsist of

covalently

linked

plus

and mi-nus DNAstrands.These authors have

proposed

areplicationscheme that involvesa

self-prim-ingmechanism. Itappearslikelythat the

repli-cation ofdifferent parvovirus DNAisa unidi-rectional and self-priming process. Further studies are necessary to obtain a detailed scheme of RV DNA

replication.

We are pres-ently studying the relationship between the replicative intermediates andtheirrole inthe synthesis of progeny ssDNA.RV DNA

replica-tionappearstoinclude differentsteps:

conver-sion of parental ssDNA to a double-stranded form then synthesis of some other double-stranded replicative intermediates

(of

mono-meric and dimeric length), one

replicative

in-termediate species acting as a template to prog-eny ssDNA. If every step requires a

self-prim-ing process, RV DNA must have a hairpin du-plex structure at both ends of the viral strand.

The behavior of RV DNA on hydroxyapatite (Fig. 3c) and preliminary results obtained with

S1 nuclease digestion suggest that RV DNA contains a short duplex region(s), which is in accordance with recent results from L. Salzman (Virology, in press). Further studies on RV DNAconcerning the structure of the ends of the molecules and the ability of RV DNA to be utilized as a primer template, as has been shown with minute virus of mice DNA (Bour-guignon etal.,Abstr. Int. Congr. Virol., 3rd, p. 181, 1975), arerequired.

ACKNOWLEDGMENTS

We wish to thank G. Hayward for his helpful discus-sions, and F. Koczot, N. Salzman, and P. Sheldrick for a critical reading of the manuscript. We also thank J. Rose forthe preprint ofreference 17 (Straus et al.), L. Salzman for thepreprint of her article inVirology, P. Nardeux for a gift of Hpa II endonuclease-cleaved SV40 DNA, and P. Tattersall for a gift of14C-labeled minute virus of mice DNA. We acknowledge M. 0. Blondel for her excellent technical assistance.

LITERATURE CITED

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3. Gerry,H.W.,T.J. Kelly,and K. I. Berns. 1973. Ar-rangementofnucleotidesequences in adeno-associ-atedvirusDNA.J. Mol.Biol.79:207-225.

4. Hallauer, C., G. Kronauer, and G. Siegi.1971. Parvovi-ruses as contaminant of permanenthuman cell lines. I.Virusisolationfrom 1960-1970.Arch.Gesamte Vi-rusforsch.35:80-90.

5. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369.

6. Kilham,L., and L. J. Olivier. 1959. A latent virus of ratsisolatedin tissueculture. Virology 7:428-437. 7. Koczot,F.J., B.J. Carter,C. F.Garon, and J. A. Rose.

1973. Self complementary of terminal sequences within plus andminusstrandsofadeno-associated virusDNA. Proc.Natl. Acad. Sci.U.S.A. 70:215-219. 8. May,P., andE.May. 1970.The DNA of Kilham rat

virus. J.Gen. Virol. 6:437-439.

9. Rhode,S. L., III. 1973. Replication process ofthe parvo-virus H-1. I. Kinetics in aparasynchronous cell sys-tem.J.Virol. 11:856-861.

10. Rhode, S. L., III. 1974. Replication process ofthe parvo-virusH-1. II. Isolation and characterization of H-1 replicative form DNA. J. Virol. 13:400-410. 11. Robinson, D. M., and F. M. Hetrick. 1969.

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12. Rose,J. 1974. Parvovirus reproduction, p. 1-51. In H. Fraenkel-Conrat and R. Wagner (ed.), Comprehen-sivevirology,vol.3.PlenumPublishing Corp., New York.

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14. Salzman, L. A., and W. White.1973.In vivo conversion of the single-stranded DNA of the Kilhamratvirus to adouble-strandedform. J. Virol.11:299-305. 15. Salzman,L.A.,W. L.White, and T. Kakefuda 1971.

Linear, single-stranded deoxyribonucleic acid iso-lated from Kilhamratvirus.J.Virol. 7:830-835. 16. Siegl,G., andM.Gautschi.1973.The multiplicationof

parvovirus Lu HI inasynchronizedculturesystem.I.

Optimum conditions ofvirusreplication.Arch. Ges-amteVirusforsch. 40:105-118.

17. Straus, S. E., E. D. Sebring, andJ. A. Rose. 1976. Concatemers of alternating plus andminus strands are intermediates in AAV DNA synthesis. Proc.

Natl. Acad. Sci.U.S.A. 73:742-746.

18. Studier, F. W. 1965. Sedimentation studies of the size

andshape of DNA. J. Mol. Biol. 11:373-390. 19. Tapiero, H.,M. N. Monier, D. Shaool, and J. Harel.

1974.Distributionof repetitioussequencesinchick

nuclear DNA. Nucleic Acids Res.1:309-322. 20. Tattersall, P.,L. V.Crawford, and A. J. Shatkin. 1973.

Replication of the parvovirusMVM.H.Isolation and

characterization of intermediatesinthe replication of the viral deoxyribonucleic acid. J. Virol. 12:1446-1456.

21. Tenant,R.W., and R. E.Hand.1970.Requirement of cellularsynthesisfor Kilham ratvirus replication. Virology 42:1054-1063.

22. Vogt, V.M. 1973. Purification and further properties of single-strand-specificnuclease from Aspergillus ory-zae.Eur. J. Biochem. 33:192-200.

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