Isolation and Partial Characterization of Different Defective DNA Molecules Derived from Polyoma Virus

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Isolation and

Partial Characterization

of

Different

Defective

DNA

Molecules Derived

from

Polyoma

Virus

MIKE FRIED

Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England

Received forpublication 19 November 1973

Supercoiled DNA molecules purifiedfrommousecellsinfected with

high-mul-tiplicity-passaged polyoma virus has a broader size distribution and sediments

moreslowly than DNA derived fromlow-multiplicity-passagedvirus. The shorter

DNA moleculesare predominately noninfectious. Virus populations containing

distinct size classes of defective virus DNAwere isolated by growing virusfrom

single cells infected by a defective and nondefective helper virus (infectious

center). This technique probably results in the cloning of defective virus

particles. By applying the infectious center method to DNA from various

fractions ofsucrosegradients it has beenpossibletoobtainshorter circular DNA

molecules ranging in size from 50 to 95% of the unit-length polyoma DNA

molecule. The shorter molecules inanyonepreparationarehomogeneous in size.

This class size is retained upon repeated passage of crude viral lysates at high

multiplicity. Thus far, all thepurified shorter DNA molecules testedappeartobe

noninfectious and largely resistant to cleavage by the RI restriction enzyme.

Someof the purified defective molecules have been found tointerfere with the

production of infectious virus upon co-infection with unit-length infectious

polyomaDNA.

Heterogeneity in the size of both polyoma and

SV-40 viral DNA extracted from viruspassedat

high-input multiplicities has been observed by

a number of workers (1, 7-9, 11-13).

Prepara-tions of this type contain a significant number

of shorter molecules that are predominately

noninfectious (1, 9, 11-13). If these shorter

moleculesrepresentpartially deleted virus

mol-ecules, they couldproveextremely useful in the

mapping and identification of viralgenes,

espe-cially those involved in neoplastic

transforma-tion. The following report describes the

isola-tion and partial characterization of different

polyoma virus preparations greatly enriched in

shorter supercoiled DNA molecules that range

in size from 50 to 95% of the unit-length

molecule.

MATERIALS AND METHODS

Virus stocks. The wild-type large plaque (PY)

strain and the temperature-sensitive mutant TS-A

havebeen described elsewhere (3). Virus stockswere

grown on mouse embryo cells, and the virus was

liberated from theinfected cells by freeze-thawingor

sonicationinthepresenceof thegrowthmedia(4),or

both.

Some of the properties of the high-multiplicity,

low-multiplicity, and starting virus (medium

multi-plicity) aresummarizedinTable 1.

The initial starting virus used to produce the

high-multiplicity virus had been passed withnogreat

care over a number ofyears. The virus was passed

undiluted foranumberofpassages,usually infecting5

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x 106 mouse embryo cells with 0.5 ml of lysate. High-multiplicity virus is denoted by DEL followed bythepassagenumber. For instance, the fifthpassage

TABLE 1. Characteristics of virus produced at differentmultiplicities

Characteristic

~~Low-

Medium-

High-Characteristic | multiplicityvirus multiplicityvirus multiplicity virus

PFU/HAU ... 106 105 As low as 103

PFU/physical particle... 1/20 1/200 Aslowas 1/20,000

DNAPFU/ggofDNA ... 106 105 As low as 103

Yield of PFU/2 x 106 cellsa (Single

cycle) ... 8 x 109 8 x10" As low as 8 x 106

YieldofDNA/5 x 106cellsa...5-8....58g 2-4gg As low as 0.5-1 tg

aInput multiplicity of 5 PFU/cell.

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of thewild-type virus (PY) is referred to as PY-DEL-5

and the sixth passageofTS-AasTS-A-DEL-6.

Low-multiplicity virus wasderived from the

medi-um-multiplicity virus by isolating single plaques from dishes containing less than four plaques per dish. The plaques were picked into 1 to 1.5 ml of growth medium

and freeze-thawed three times. Each plaque usually

contained between 104 and 106 PFU. Approximately

onehalf of the virus from the plaque was used to infect

5 x 106 cells. The virus from this infection was

harvested after5 to 7daysat 37Cor10 to14days at

31.5 C. "4C-labeled viral DNA purified from cells

infected with low-multiplicity virus at an input of 1

PFU/cellwasusedasthestandard unit-length marker

DNA insucrosegradients.

Assayof virus and infectious DNA. The plaque

assay usedto titer infectious virus and the

haemag-glutination assay used to titerphysical particles have

been described previously (4). Infectious viral DNA

wasassayed on mouse embryo cells by the

DEAE-dex-tranmethod ofThorne et al. (9).

Infectious center plaques. The cloning of

individ-ual defective virus particles from

high-multiplicity-passaged viruswas attempted by growing virus from

single cells infected by adefective and a nondefective

particle. Mouse embryo cells were infected at a

multiplicity of less than, or equal to, 1 PFU/cell, but

at amultiplicity of greater than one particle per cell.

The inputmultiplicity dependedonthe defectiveness

ofthe stock asestimated from the

PFU/hemaggluti-nation unit (HAU) ratio. Usually a variety of input

multiplicities wereusedforeach viruspreparation.

After virus absorption the cells werewashed three

timeswith medium and then incubatedfor2 to 3 hin

the presenceofreceptor-destroying enzyme and

anti-polyoma antiserum to reduce and inactivate the

unabsorbed virus (4). The infected cells were then

trypsinized and reseeded at concentrations ranging

from50to5,000cells with2 x 106uninfectedmouse

embryocells per 50-mm petri dish. After2to 3h when

the cells had settled, the media were removed and

agarwas addedasforanormal plaqueassay.

When the infectiouscenterplaques developed they

were picked into 1 to 1.5 ml of medium with a

wide-bore pipette. The picked plaques were then

freeze-thawedthree timestorelease the virus from the

plug of agar. Thevirus wasthen grownup on mouse

embryo cells at high multiplicity. The supercoiled

DNA derivedfromvirusproduced bythese infectious

center plaques was then sedimented in neutral

su-crosegradients.

Viral DNA extraction and purification of

su-percoiled molecules. Infected mouse embryo cells

wereincubatedforeither4daysat37Cor7to8days

at 32C(usuallyinthepresenceof2MCiof [3H

Ithymi-dine and 1 ug of cold thymidine of medium per

milliliter) before viral DNA was extracted by the

selective method of Hirt (5). The Hirt supernatant

fluidwasextracted withphenol,and the nucleicacid

was precipitated by the addition of 2 volumes of

ethanol at -20C. The supercoiled and

nonsuper-coiled DNA were separated by using an ethidium

bromide-cesium chlorideequilibrium gradient (6).

Neutral sucrose gradients. Linear 5to20% (wt/

vol) sucrose density gradients containing 0.14 M

NaCl, 0.01 MTris-hydrochloride (pH 8.5), and 0.004

M EDTA were used for sedimentation analysis of

purified supercoiled DNA. [3H]thymidine-labeled

DNAsamples with or without [14C]thymidine-labeled

marker DNA involumes of 0.2 to 0.3 ml were layered

on topofgradients that were centrifuged in anSW40

rotor at 28,000 rpm for 17 h at 19 C.The tubes were

pierced from the bottom, and either 5 or 10 drop

fractions werecollected on 12-inch (2.542 cm)

What-man no. 1 filter paper. There were 64 to 67 10-drop

fractionsor128 to 1345-drop fractions. The DNA was

acid precipitated and the radioactivity determined.

Thesizeof slowersedimenting peaks, expressed as a

percentage of the unit-length marker DNA, was

calculated from the equationofVinograd (2).

Cleavage withRIendonuclease. The R,

endonu-clease was a gift ofW. R. Folk. The digestion was

carried out in a buffer containing 0.05 M

Tris-chlo-ride, 0.05 M NaCl, and0.01M MgCl2at 37C for 30

min. Theenzyme wasin20-fold excess at a ratio of 1

Lliter ofenzyme to 1 ,g of DNA. The reaction was

stopped by the addition of EDTA to a final

concentra-tionof 0.03 M.

RESULTS

Isolation of TS-A-shorter DNA molecules.

It wasthought that a TS-A viral DNA deleted

in genes coding for capsid proteins could be

rescued by a temperature shift of mouse cells transformed and maintained atthe

nonpermis-sive temperature (10). Therefore, the isolation

of shorter DNA molecules from TS-A was

at-tempted.

Upon repeated passage of TS-A virus at

high-input multiplicity, it was observed that the yield of particles as well as the yield of

plaque-forming units decreased markedly. At

the sixth high-multiplicity passage the

PGU-HAU ratio was reduced to 2 x 103, which was

50-fold less than the starting virus inoculum and 500-fold less than low-multiplicity-pro-duced virus. This is equivalent to about 1

PFU/10,000 physical particles (usually about 50% ofthese particles do not contain any viral DNA). The major part ofthesupercoiledDNA extractedfromTS-A-DEL-6-infectedcells sedi-mented more slowly than low-multiplicity marker unit-length DNA in neutral sucrose

gradients.Acomparisonofthe widths of thetwo

sedimenting bands (Fig. 1) indicates that the

TS-A-DEL-6 DNA is moreheterogenous in size

than the low-multiplicity marker DNA.

To see ifcomplementation to form a plaque

between different defective virus particles

pres-ent in the TS-A-DEL-6virus population could

be detected, 43 infectious center plaques were

isolated after infection with TS-A-DEL-6 virus at a multiplicity of 0.01

PFU/cell

(approxi-mately 100physical

particles/cell).

All43

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E

CL

Fraction Number

FIG. 1. Neutral sucrose gradient sedimentation

analvsis of TS-A-DEL-6 DNA. Symbols: 0,

3H-labeled TS-A-DEL-6 DNA; 0, 14C-labeled

unit-length marker DNA.Inthis and thefollowing figures

only data from the lowerhalf of thesucrosegradients

arepresented.

tious center plaques when replaqued showed

linear plaque formation with dilution. This

suggests that these plaques were not produced

by complementation of two or more defective

virusparticles. All theplaquestested contained

between 104and 106 PFU.

To isolate shorter DNA molecules, seven

infectiouscenterplaques derived froman

infec-tion ofTS-A-DEL-6 atan input multiplicityof

0.001 PFU/cell (approximately 10physical

par-ticles/cell) weregrownupasdescribed in

Mate-rial and Methods. Theresulting virus wasused

to infecta large numberofmouseembryocells

fromwhich supercoiled DNAwasextracted and

purified.

The DNA from each of theseven plaqueswas

sedimented ina neutralsucrose gradientinthe

presenceofamarker fromlow-multiplicity viral

DNA. The width of each oftheseven

DNA-sedi-menting bands indicated that they were more

homogeneous in size than the DNA from the

uncloned TS-A-DEL-6 DNA. DNA from three

ofthe plaques sedimented coincident with the

marker, whereas two sedimented with

charac-teristics ofmolecules 5 to 10% shorter and two

approximately 15% shorter.

The infectivity of the fractionated DNA of

one oftheplaques (TS-A-DEL-6-2) containing

15% shorter molecules was assayed (Fig. 2). It was found that the peak of infectivity sedi-mented faster than the bulk of theDNA, and in 4 thesame region asthe low-multiplicitymarker

DNA. In the fractions containing most of the

15% shorter DNA, only background infectivity

was present. Eighty-seven plaques resulting

from infection with DNA from thelight side of the infectivity peakin theregionofthe bulk of the DNA were picked. All of these plaques contained 101 to 106 PFU and, on replaquing, produced alinear dose response withdilution. 2 o. Clonal isolation of PY shorter DNA

molecule. Because the shorter molecules de-rived from TS-A appeared to carry little or no

infectivity, complementation between

wild-type shorter DNA molecules and the various temperature-sensitive mutants, including TS-A, might be feasible. Therefore, the

isola-tion ofshorter DNApopulationswasattempted

from wild-type virus. After the second passage at high multiplicity of wild-type virus,

infec-tious center plaques were isolated and grown

up. Two out of three plaques tested showed a

u

c x

9

x

9

FRACTION NUMBER

analvsisof DNA from infectiouscenterplaque TS-A-DEL-6-2. (A) DNA infectivity of various gradient

fractions of TS-A-DEL-6-2 DNA. Symbols: 0,

3H-labeled TS-A-DEL-6-2 DNA; A, infectious DNA

plaque-forming units. (B) Sedimentation behaviorof

TS-A-DEL-6-2 DNAinrelationtounit-length marker DNA. Symbols: *, 3H-labeled TS-A-DEL-6-2 DNA;

0, "4C-labeledunit-length marker DNA.

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sharpsingle peakofDNAabout5 to 10%shorter

than the marker, whereas the DNA from the A

third plaque co-sedimented with the marker.

An attempt was made to enhance for even

shorter populations by growing up virus from

the DNA fromthelight sideofhigh-multiplicity X DNA peaks. Supercoiled DNA, purified from -PY-DEL-5 sedimented through a neutral

su-crosegradient, showedabroaddistribution and

sedimented moreslowly than the marker DNA (Fig. 3). DNA from the peak fraction and a

number of fractions from the light side of the

peak of the PY-DEL-5DNA were usedtoinfect B

mouse cells. The viruses produced from these 3

DNA infections were used to infect a large

number of mouse cells from which the

[image:4.494.265.455.63.330.2]

super-coiled DNAwas purified andanalyzed. i 2

Figure 4shows the sedimentation characteris- 2

tics of DNA derived from these various frac- T

tions. It can be seen that the DNA obtained 5

from the peak fraction 14 is similar in its size

distribution to that ofthe parental PY-DEL-5

gradient (Fig.

3A). With the DNA derivedfrom ,c

Il 0

the

lighter

fractions,

two

peaks

of DNA were 20 30

resolved; one co-sedimented with the marker Fraction Number

DNA, whereas the other sedimented more FIG. 3. Neutral sucrose gradient sedimentation

slowly depending on its origin in the parental

analvsis

of PY-DEL-5 DNA. (A) Sedimentation of

graien.Iwafundtha th aoun

ofsloerPY-DEL-5

DNA. The DNA of fractions 14-22 was

gradient. It was found that the amount of slower used to makevirus stocks that were furtheranalyzed

sedimentingDNA decreases muchmorequickly (see Fig. 4). (B) Sedimentation behavior of PY-DEL-5

than the unit-length DNA upon infection with in relation to unit-length marker DNA. Symbols: 0,

increasing dilutions of the virus derived from 3H-labeled PY-DEL-5 DNA; 0,

"4C-labeled

unit-thevarious fractions.Thusitwouldappearthat length marker DNA.

14, 15 6

k.

J: X~

3,

'0

2-1Ao 0 2200 1 251 5 0 20 25

/1

3. 1711.

15. 130D~~~~~~~~3/3

Fractlion Number

FIG. 4. Neutral sucrose gradient sedimentation analysis of supercoiled DNA isolated from mouse cells

infected with virus derived from the DNA of gradient fractions 14-22 (Fig. 3A). Symbols: *, 3H-labeled DNA

derived from gradient fractions 14-22; 0, "4C-labeledunit-length marker DNA.

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the shorter molecules are defective and are

dependent on the unit-length molecules for their growth.

The viruses derived from fractions 21 and22

(Fig. 4) were used to produce infectious center

plaques. Fourteen infectious center plaques

were grown up and theirDNAwasanalyzed. In

all cases most ofthe DNAco-sedimented with

the marker. In most of the preparations a

shoulder or another peak of DNA was distin-guishable. Table2lists theapproximate size of the slower sedimenting DNA relative to the unit-length molecule. Figures 8A and 9A show representative gradients of preparations

con-tainingmolecules75and60% oftheunit-length molecule, respectively.

Infectivity of the shorter molecule. It was

already found that the infectious DNA derived

from TS-A-DEL-6-2 (Fig. 2) sedimented inthe region ofthe unit-length molecule rather than with the bulk of the DNA. It is ofinterest to

know if all the DNA sequences present in the unit-length molecules of polyoma virus are

essential for the production of new infectious virus. Thus it was decided to look at the infectivity ofother isolated shorter DNA mole-cule populations.

The 75% DNA molecules derived from virus

of the plaque designated 21-2 were purified through twosucrose gradients. The final DNA

preparation (Fig. 5) contained 3 x 102PFU/ug of DNA. This is equivalent to a 104-fold

de-TABLE 2. Sizes of shorter DNA moleculesofvirus

derivedfromfraction21and22

Approximate size Infectious center slower

plaques

~~~~of

slower

plaques sedimentingDNAa

Fraction21

21-1 75%

21-2 75%

21-3 50%

21-4 50%

21-5 75%

21-6 None

21-7 None

Fraction22

22-1 60%

22-2 None

22-3 75%

22-4 None

22-5 90%

22-6 75%

22-7 65%

aThe sizes of the slower sedimenting DNA,

ex-pressed as a percentage of the unit-length marker

DNA, was calculatedfrom the equation of Vinograd

(2).

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1 09 20 30 40 Fraction Number

analvsis of 75% DNA derived from virus from

infec-tious center plaque21-2. Thepeakcontinuing the 75%

DNA wasisolated from two successive neutral sucrose

gradients before being sedimented with the marker

DNA. Symbols: *, 3H-labeled 75% 21-2 DNA; 0,

"4C-labeledunit-length marker DNA.

crease in terms of plaque-forming units per

moleculesofDNAincomparison with low-mul-tiplicity viral DNA. (Table 1). Sixplaquesthat

arose fromthe 21-2 75% DNAwere pickedand

replaqued. Individualplaquesweregrown up at

low multiplicity. Supercoiled DNA obtained fromthe virus fromtheseplaqueswasanalyzed

in sucrose gradients and each plaque that

de-rived DNA showed a single peak that co-sedi-mented with the marker DNA, asillustratedin

Fig. 6. This resultsuggeststhe small amount of

infectivity present in the 75% molecules of

plaques 21-2 is probably due to contamination with unit-length molecules.

The infectivity found in 90 and 50% mole-cules, isolated aftertreatment with the

restric-tionenzyme

RI

(seebelow),wasless than15and

125

PFU/4g

of DNA, respectively. All the shorter moleculesso faranalyzed are defective inthe production ofnewinfectious virus.

A B

0 1 \ R

I~~ ~~~~2§i

;0-F-ct..N-mb.,F{b um

FIG. 6. Neutral sucrose gradient sedimentation analvsis of DNA derived from virus from two plaques

thataroseafterinfection with purified 75% 21-2 DNA

(Fig. 5).Symbols0, 3H-labeled DNA from 75% 21-2

plaques; 0, "4C-labeledunit-lengthmarker DNA.

I

10-1

6

I

IL

u

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J.VIROL.

Restriction enzyme

RI

sensitivity of the

shorter molecule. Circular polyoma virus DNA

is cleaved to linear molecules at one unique

region by

RI

restriction enzyme (D. Robberson

and M.Fried, manuscriptinpreparation).Viral

DNA derivedfromplaques grown at low

multi-plicity was found to be greater than 95%

sensi-tive tocleavageto linear moleculeswith the R,

restriction enzyme (Fig. 7).

A number ofpreparations containing both a

peak of unit-length molecules and a peak of

shorter moleculesweretested for their

sensitiv-ity to cleavage with

R,.

So far none of the

shorter molecules (14 isolates) were found, to

any great degree, to be sensitive to cleavage

with the R, enzyme. Unit-length molecules in

the same preparations were cleaved to linear

molecules (Fig. 8 and 9). If detectable amounts

ofthe shortermoleculesweresensitiveto cleav-age with

RI,

a disappearance of the shorter supercoiled molecules should have occurred and

apeak of DNA sedimentingatthe region where

the shorter linear molecules sediment would

have been observed.

Treatment with

RI

revealed that resistant supercoiled molecules about 85 to 95% of the unit-length molecules are present in varying

amountsin most preparationsproduced athigh

multiplicity. These R,-resistant molecules are

particularlyobvious in preparations containing DNA less than 80% where they appear as a

shoulderor a separate peak (Fig.8 and 9). Treatment with

RI

was used to purify the shorter molecules. The preparations were

treated with

RI,

and the resistant shorter

super-coiled moleculeswereseparatedfromthe sensi-tive linear molecules by equilibrium density centrifugation. Velocity sedimentation was

analvsis of low-multiplicity DNA before and after

treatment with restriction enzyme RI. (A) Before

treatment withRI. (B)AftertreatmentwithRI. The marker DNAwasaddedaftertheR,reactionhadbeen

terminated.Symbols: *, 3H-labeledlow-multiplicity

DNA; 0, 14Cunit-length marker DNA.

A

F,..ti.. Number

analvsis of DNA from infectious center plaque 22-3

(Table 2) before and after treatment with restriction

enzyme R,. (A) Before treatment with RI. (B) After treatmentwithRI.The markerDNA was added after

the RI reaction had been terminated. Symbols: 0,

3H-labeled 22-3 DNA; 0, "4C-labeled unit-length

marker DNA.

6 A 6

5

~~~~~~~~~~~~~~~5

7_!~41

~~~~~~~~~~~~~~~~~4

;

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

~~~~~~~~~~~~~~~~~~~~i

u 333

2

-2

10 20

S B 6

51

5

x6

4 4

3

13

0

12 /2

1

10

3 10 20 30

Fraction Number

analvsis ofDNA from infectious centerplaque 22-1

(Table 2) before andaftertreatmentwithrestriction

enzymeRI. (A) Before treatment with RI. (B)After

treatmentwithRI. ThemarkerDNAwasaddedafter

the RI reaction had been terminated. Symbols: 0,

3H-labeled 22-1 DNA; 0, "4C-labeled unit-length

marker DNA.

used as a further purification step for those

molecules of80% orless.

944 FRIED

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Interference. Experiments were performed

to determine the effect of the various shorter molecules on the growth of

low-multiplicity-produced wild-type DNA. Mouse cells were

infected with different preparations ofpurified shorter DNA

plus

wild-type low-multiplicity DNA. Theyields of virusproduced at 31.5and

38.5 C of the dual infections were compared

with the yield producedby thewild-typeDNA alone. Table 3 lists the results of such an

experiment. Whereas co-infection with circular supercoiled SV-40 DNA, or linear DNA from uninfected cells, produced littleor noinhibition

ofthe productionofwild-typevirus,someofthe preparations ofshorter DNAproducedasmuch

as a 10-fold inhibition. Those shorter molecules derivedfromTS-Avirus areequallyeffective in inhibitingatthe permissive andnonpermissive

temperature. Thisinhibitory effect will present

a problem in the use ofgenetic

complementa-tiontests toidentify viralgenes present insome

ofthe shorterDNA molecule preparations.

DISCUSSION

It has been possible by the techniques de-scribed above to produce different viral DNA

preparations that are greatly enriched in

indi-vidual distinct classes ofsupercoiled, defective DNA molecules, ranging insize from 50 to95%

oftheunit-lengthpolyoma DNA molecule. The rationaleofthe infectiouscentermethodwas to

clone defective virus particles by growing the

progeny ofindividual cells infected byonlyone

defective particle in thepresence of a

nondefec-TABLE 3. Interference of different DNAs with PY

DNAa

YieldofPFU/ YieldofPFU/ DNA cultureat cultureat

31.5C 38.5C

PY+85%TS-A-DEL-6-2 1.3 x 106 2.5 x 105

PY + 85%TS-A-DEL-6-5 2.5 x 106 5.5 x 105

PY+ 95%PY-DEL-2-3 6.0 x 106 9.5 x 105

PY+75%22-3 3.1 x 106 5.5 x 105

PY+SV-40 1.3 x107 2.5 x 106

PY+uninfectedmouse 1.6x 107 3.5 x 106

cell linear DNA

PY 1.5 x107 3.0 x 106

aAtotal of 8 x 105 mouse embryocellswere infected with 0.25Agofunit-length (PY) DNA in the presence

orabsenceof 0.05mgofeach DNA to be tested. After

infection thecells were trysinized and 4 x 105 cells

wereplatedinto each oftwo 35-mm petri dishes with

medium containing receptor-destroying enzyme (4).

One half ofthe dishes were incubated at 38.5 C and

theother halfat 31.5 C. After 68 h at 38.5 C or 144 h

at 31.5C, the virus was harvested. The yield of

plaque-formingunits per culture is shown.

tive helpervirus particle. The observation that

30 to60% of theinfectious center derived virus

isolates contained no detectable shorter

mole-cules suggests that cells that produced shorter

molecules were infected by only one (or just a

few) defective particles. The success of this

method in the cloning of individual particlesis

supported by thefollowing observations. (i)The

shorter molecules inany oneisolateare

homoge-neous in sizeand this size molecule isretained

upon repeated passage of crude viral lysatesat

high input multiplicity. (ii) Some of the

puri-fied shorter molecules have been shown to be

homogenous with respect to their nucleic acid

sequences by heteroduplex studies (Robberson

and Fried, submitted for publication) and by the production of molar equivalents of

frag-mentsaftertreatment with multicut restriction

enzymes (Griffin and Fried, manuscript in

preparation). (iii) The shorter molecules are

representative of the size distribution of the

DNA population ofthe virus used to produce the infectious centers (Fig. 4 and Table 2).

The finding that shorter molecules retain their sizewhen grown with different isolatesof

unit-length infectious DNA molecules (Fried, unpublished data) suggests that the shorter moleculesareactually derivedfromtheoriginal population as opposed to being continuously generated by particular unit-length molecules. The infectiouscentermethod requiresonly that the defective particlebeing isolatedcan coexist

with the infectious unit-length molecule inthe formation of a plaque and thus is nonselective

to thisextent.

In most high-multiplicity-passaged virus

stocks, 85 to 95% length DNA makes up the

majority of the non-unitlengthDNAmolecules

(see Fig. 1 and 3). This may explain the

presenceof85 to95%molecules, as a second size

class, in some of the defective DNA isolates. This second size class was usually observed in

isolates containing defective DNA of less than

80% unit length (Fig. 8 and 9). The defective

DNAmoleculesso far studied have been

nonin-fectious and have depended, forcontinued

pas-sage, on complementation with unit-length

in-fectious viral DNA. The shorter DNA is thus

itself maintained by passage of virus stocks at

high multiplicity. This passage has probably

led, insome cases, to the generation of the 85 to

95% shorter molecules discussed above. The

prevalence ofthe 85 to 95% molecules may be

explained by the less efficient encapsidation

into astable particle of smaller than 85% DNA,

thus conferring a selective advantage to these

newly generated larger defective molecules. It

cannot be ruled out, however, that the second

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class of defective DNA is due to infection by

morethanonedefective particleofthe cell that

producedthe infectious center.

So far all the shorter molecules tested have been found to be predominately noninfectious

andresistant tocleavage with the

RI

restriction

enzyme. Thereasonforthisresistance to

cleav-age by

RI

is unclear at this time, but may be revealed when the structure ofthese molecules is determined. The loss ofthe R, cleavage site

might indicate that this region is not very

essential for viral growthor thatthe defectives

aregeneratedbya common mechanism. The finding that some of the shorter mole-cules interfere with the production of infectious virus is not too surprising. On continued high-multiplicity passage both the yields of

infec-tious virus and virus particles decrease

mark-edly. Continued high-multiplicity passagealso results in the production of predominately

shorter than unit-length supercoiled DNA.

Per-haps thistypeof interferenceplayssomerolein

vivo. Defective viruses, whether due to an

addition of host sequences or repetitions of

specific viral sequences, may have a selective advantage and replicate faster and at the

ex-pense ofthe infectious virus.Suchamechanism

would lead to the slowing down of the virus infection within a high-multiplicity virus area,

suchas alesion, and might allowmoretime for

the host defense mechanism, like the immune

response, to act before the virus infection has

damagedtoomany cells.

Thetransforming abilityofdifferent purified defective DNA is now under investigation. By

this means it maybe possibleto identify those

portions of the viral genome necessary for

neo-plastic transformation, as well as other viral functions.

Some of the classes of molecules isolated contain only a portion of the viral genome

(Robberson and Fried, manuscript in

prepara-tion). Thus, the isolation procedures described above present a way of obtaining large and probably homogeneous quantitiesofarestricted portion of this small viral genome that can be

replicated in vivo.The biological, biochemical,

and genetic studies ofthese deleted molecules

should lead to a better understanding of the properties of polyoma virus.

ACKNOWLEDGMENTS

Iamindebted to Moira Griffiths for her excellent technical assistance. I would also like to thank Bill Folk and Don Robberson for their helpful discussions and advice, and Beverly Griffin and Elsebet Lund for their valiant effort in helping in the preparation of this manuscript.

LITERATURE CITED

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