Isolation of the viral DNA replication complex from adeno-associated virus type 1-infected cells.

Copyrighti 1977 AmericanSociety forMicrobiology Printedin U.S.A.

Isolation

of the Viral DNA

Replication

Complex

from

Adeno-Associated Virus

Type

1-Infected Cells

HIROSHIHANDAtANDHIROTOSHIMOJO*

InstituteofMedicalScience, University of Tokyo,4-6-1,Shirokanedai, Minato-ku, Tokyo 108,Japan

Received for publication 6 June 1977

The

replication

complex

active in

adeno-associated virus type

1

(AAV-1)

DNA

synthesis

in vitro was

solubilized,

with a

nonionic

detergent,

fromthenuclei of

human

embryonic

kidney

cells coinfectedwith AAV-1andan

early

temperative-sensitive mutant

(ts125)

of human adenovirus

type

5 at the

nonpermissive

temperature

(40.50C).

The

complex

sedimented with a mean size of 23S and

containedparental AAV-1 DNA. Most ofthe DNA

synthesized

withtheAAV-1

DNA

replication complex in vitrowasAAV-1

DNA,

asrevealedby DNA-DNA

hybridization

and

sedimentation

in a

neutral

sucrose

gradient. However, it

sedi-mentedinan alkaline sucrose

gradient

asmolecules smaller thanAAV-1 DNA

(14.4S).

The AAV-1 DNA

replication

complex

was notformed in cells infected

with AAV-1alone.

Adeno-associated

virus

(AAV)

is a defective

parvovirus

that is

capable

of

multiplying

only

in cells coinfected with

adenovirus

(1, 8, 15, 23).

The

analysis

of the AAVDNAhasbeen difficult

due to the simultaneous

replication

of

helper

adenovirus

(17).

Itwas

reported

that a

temper-ature-sensitive

(ts)

mutantofhuman adenovirus

type

31

(H31) (10,

13),

aswellasmutants

of

H5

and H12

(7, 24)

and avian

adenovirus

(9),

defec-tive in viralDNA

synthesis

atthenonpermissive

temperature,

assist the

growth

of

AAV.

Only

AAV DNA

replicates without the

replication

of

adenovirus DNA under these

conditions

(6, 13).

We

reported

that nuclei

isolated from

cells

coin-fected with AAV

type

1

(AAV-1)

and H31tsA13

atthe

nonpermissive

temperature

(400C)

were

active in AAV-1 DNA

synthesis

invitro

(5).

The

availability

of a

subcellular

system

capable

of

synthesizing

AAVDNA invitrowould facilitate the

analysis

of

AAV DNA

replication.

The

pres-ent

communication

describes the

isolation of the

AAV-1 DNA

replication

complex

from nuclei of

cells coinfected with AAV-1 and H5ts125 at

40°C,

as

well

asthe

analysis

of

DNA

synthesized

invitro.

It also shows that the

replication

com--plex

was not

formed in cells infected with

AAV-1alone.

MATERIALS AND

METHODS

Cells,

viruses,

andinfection. Secondary cultures

of humanembryonic kidney (HEK) cells and mono-layer cultures of KBcellswereused. The growth of AAV-1wasdescribedpreviously (6). A

temperature-tPresent address:Laboratoiy of ExperimentalPathology,

National Institute of Arthritis, Metabolism, and Digestive Diseases,Bethesda, MD 20014.

sensitive (ts) mutant ofH5,H5ts125,waskindly pro-vided by H. S. Ginsberg (4). H5ts125 was grown in KB cells and plaque-assayed in HEK cells at 330C

(permissive temperature). Confluent monolayers of HEK cells in 32-ounce (ca.960-ml) bottles (2 x 107 cells per bottle) were coinfected with AAV-1 and H5ts125 at 15fluorescent infectious units (2) and 30 PFU percell,respectively.Afteradsorptionfor2h at 370C, the cultures were incubated in maintenance medium (Eagle minimum essential medium [MEM]

with 1% fetal calfserum) at40.5°C (nonpermissive

temperature).

Preparation ofthe AAV-1 DNA replication

complex. HEK cells were harvested at 18 h after

coinfection with AAV-1 and H5ts125, washed with phosphate-buffered saline (PBS), and suspended in

reticulocyte standard buffer (10 mM NaCl, 10 mM

Tris-hydrochloride, pH7.4, 1.5 mMMgCl2)at2x107

cells per ml inanice bath for30min. Theswollen cells were disrupted with five strokes in a tightly

fitting Douncehomogenizer,andthe nucleiwere col-lected by centrifugation at 2,500 rpm for 5min at

40C.Thenuclearpelletwaswashed withreticulocyte

standard buffer andsuspendedat 2 x 107nuclei per ml in TK buffer (50 mMTris-hydrochloride, pH 7.9,

25 mM KCl) containing 0.1 mM EDTA and 2 mM dithiothreitol (DTT). The nuclear suspension was lysed by addition of 0.5% dodecyl poly (P = 11.2)

oxyethylene ether(KAO-AtlasCo. Ltd., Tokyo) (27)

at0°Cfor30min. Then,thelysate wascentrifuged

at 4°Cin anSW27rotor at 25,000 rpm for 30 min. Thesupernatantwasdialyzed against TK buffer

con-taining0.1mMEDTA,20%glycerol,and2mM DTT

at 4°C for 3 h and used immediately or stored at -80°C. A protease inhibitor, phenylmethylsulfonyl

fluoride(Sigma),wasadded tosolutions at a concen-tration of1 mMforpreparationofthecomplex.

Assayforendogenous DNAsynthesisinvitro.

The reaction mixture (100

pi)

contained 50 mM

Tris-hydrochloride, pH 7.9, 2.5 mM MgC12, 2 mM DTT,

444

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100FM each of dATP, dCTP, and dGTP, 20

.uCi

of

[3H]TTP

(46Ci/mmol), andapreparation of the DNA

replicationcomplex containing about 35

gAg

ofprotein.

After incubation at370Cfor various times, the incor-poration of[3H]TTP into anacid-insoluble fraction

wasmeasuredasdescribed previously (5). The protein

content was determined by the method of Lowry et aL (12).

Partial purification of the replication

complex

by sucrose gradient centrifugation. The

dialyzed

soluble complex was layered onto a linear 15 to 30% (wt/vol) sucrose gradient in TK buffer containing 0.1 mMEDTA, 10% glycerol, and 2 mM DTT. The gra-dient wascentrifugedin anSW41 rotor at 30,000 rpm

for20hat

40C.

Aportion of each fraction was assayed for endogenous DNA polymerase activity. The re-mainder of each fraction was frozen rapidly and stored

at-800C.

Analysisof DNA synthesized in vitro. After the

reaction, the mixture was treated with Pronase (1 mg/ml, self-digested at 370C for 1 h) and 25 mM EDTA at 370Cfor 30min,followed by further

incu-bation for 60 min after addition of 0.1% Sarkosyl.

Then,DNAwasextractedwithphenol.The aqueous

phase containing newly synthesized DNA was

ana-lyzedbyaneutralsucrosegradient(5to20%[wt/vol]

in10mMTris-hydrochloride, pH7.5, 0.1 MNaCI,10

mMEDTA)at30,000 rpm for14hat40CinanSW41 rotor or by an alkaline sucrose gradient (5 to 20% [wt/vol] in0.5MNaCl,10mMEDTA,0.3NNaOH) at40,000 rpm for16hat40CinanSW41rotor.After

centrifugation,thegradientwasfractionated,and the

trichloroacetic acid-insoluble radioactivity in each

fractiontrappedon amembranefilter(Millipore)was

counted.DNA-DNAhybridizationwith AAV-1DNA,

H5DNA, and cellular DNAwas carried out as

de-scribed

previously

(22).

RESULTS

Conditions

for

solubilization

of the

rep-lication

complex.

The

rate

of DNA synthesis

in HEK cells

coinfected with AAV-1

and

H5ts125

began

to increaseat 6 h

postinfection

(p.i.) and

reached its maximumat16 h

p.i.

(Fig.

1). Nuclei

were isolated from cells at 18 h

p.i.

and

solubilized

with various kinds

of

detergents.

Only

alowlevel of

endogenous

DNA

polymerase

activity

wasfound in the

supernatant,

when

the

nuclei

weresolubilized

by Nonidet P-40, Triton

X-100,

Brij

58,

or

Sarkosyl.

The

highest

level

of

activity

wasfound when the nucleiwere

solubi-lized

by dodecyl

polyoxyethylene

ether.The

ac-tivity

of the

solubilized

complex

was also

de-pendent

on the concentration of

NaCl

during

solubilization.

The

complex

solubilized at

var-ious

concentrations

of NaCl was

dialyzed and

assayed

for the

endogenous

DNA-synthesizing

activity

in vitro

(Fig. 2).

Theresult showedthat 0.5M

NaCl

was

optimal

for solubilization of

the

complex

with the

detergent.

The

extract similarly

prepared from the nuclei

of

HEK

cells infected

with H5ts125

alone

x

4 8 12 14 16 18 20 22

Hoursafter coinfection with AAVI and H5ts 125

FIG. 1. RateofDNAsynthesisinHEK cells after

coinfectionwith AAV-Iand

HMts125.

Confluent

mon-olayersofHEKcells in 2-ounce(ca.

60-mI)

bottles(2

x

log

cells per bottle) were coinfected withAAV-1

andH5ts125 at 15

fluorescent

infectious

units and

30 PFU per cell,

respectively,

and incubated at

40.50C.

At intervals of2 h, the cells were labeled with

f3H]thymidine

(2

auCi/ml)

for30min, and the

radioactivity

incorporated

into the acid-insoluble

fractionwascounted.

0

x E

0

0)

0

I-0.

c

(I

0 0.1 0.2 0.3 0.5 0.7 1.0 Concentration ofNaCI(mole)

FIG. 2. Effectof

NaCl

concentrationonthe

solu-bilization ofthe

complex

with the

detergent.

The

nuclei isolated

from

the HEK

cells

coinfected

with

AAV-1 and

H5ts125

were

lysed

with 0.5%

dodecyl

polyoxyethylene

ether

containing

the indicated

con-centrationsof NaCl.The

lysate

was

centrifuged.

The

supernatantwasdialyzedand

assayed

for

DNA

po-lymerase

invitro. Theamount

of

PHJTTP

incorpo-rated into anacid-insoluble

fraction

wasmeasured

(0).

The extractprepared

from

the nuclei

of

HEK cells infectedwithH5ts125 alonewas

similarly

as-sayed

(0).

showed

noorvery

little

DNA-synthesizing

ac-tivity

(Fig.

2).

Conditions for DNA

synthesis

in

vitro

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446

with the

replication complex. The

pH

opti-mum for endogenous DNA

polymerase activity

was

broad, ranging from pH

7.5 to8.5

(Fig. 3A).

Although ATP and

KCI

were

not

essential for

the

activity, the addition of

1

mM ATP

and

25

mM

KCI

gave maximal stimulation

(Fig.

3B and

D).

Mg2"

was

essential for the

activity (Fig. 3C),

and the

optimal

Mg2e

concentration

was

2.5

mM. The

high NaCl concentration (0.5 M)

showed the

inhibitory

effect on the activity (Fig.

3E).

All four

deoxyribonucleoside

triphosphates

were

required for maximal

activity (Table

1).

An

appreciable activity detected in the absence

of one to three

deoxyribonucleoside

triphos-phates may be due

to

the

remains of these

x

E

C)

0

0~

Q.

I

A

01 l

6 7 8 9

pH

substrates

in

the

extract

(3, 21, 26). RNase

in-hibited the reaction

slightly. Actinomycin

D

and

sodium

pyrophosphate inhibited the polymerase

activity

markedly.

This

observation suggested

the involvement of RNA

synthesis in DNA

syn-thesis. The lack

of inhibition

by

a-amanitin

in-dicated that RNA

polymerase

II

(11) was

not

involved in DNA

synthesis

in this system.

Under

optimal conditions,

DNA

synthesis

increased

linearly for 40 min. Maximum synthesis was

obtained by incubation

at

370C

for 100 min

(Fig.

4).

Partial purification of

the

replication

complex by

sucrose

gradient

centrifuga-tion.

The

soluble

replication complex

was

par-tially purified

by sedimentation in a neutral

2 3 4 5 10

ATP(mM) MgCI2(mM)

3

E

o 2

0.

25 50 75 100 0.5 0.1 0.2 0.3

KCI(mM) NaCI(M)

FIG. 3. Conditionsfor DNApolymerasein vitro. The crude DNA replicationcomplex was solubilized and assayed for DNApolymeraseunder thefollowingconditions: (A) at various pHvalues; (B,C, D and E) at various concentrationsofATP,

MgC42,

KCI, andNaCi,respectively.

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TABLE 1. Requirements for DNAsynthesiswith the viral DNA replicationcomplex

Reaction mixture Percentage of max-imalactivity

Complete

... 100

Deletions. -DTT ... 102

-dATP ... 73

-dCTP ... 31

-dGTP ... 38

-dATP, dCTP,dGTP ... .... 28

Additions. RNase (100ug/ml) ... ... 25

RNase (4

jig/ml)

... ... 41

Actinomycin D (5

jg/ml)

... 19

a-Amanitin (10

pg/ml)

... .... 99

a-Amanitin (0.5

pg/ml)

... 103

a-Amanitin (0.01

jig/ml)

... 98

Sodium pyrophosphate (10 mM) . 6

aDNA-synthesizing

activity of the complex was measured under theoptimal conditionsdescribed in the text with thedeletionsoradditionslisted above.

Theactivitywasexpressed by incorporationof

[3H]-TTP into the acid-insoluble fraction relative to that

inthecompletesystem.

4

3

0

E

0

'-' I

0 20 40 60 80 100 120

[image:4.500.46.241.79.258.2]

Incubation time(min)

FIG. 4. TimecourseofDNA synthesisin vitro. The

crude DNAreplication complexwassolubilizedand

assayed forDNApolymerase.Atthe timeindicated, the acid-insolubleradioactivitywasmeasured.

sucrose gradient. The complex, detected by

DNA-synthesizing activity, sedimentedas a

sin-gle broad peak withameansize of 23S(Fig. 5).

Theapical three fractions (fractions 12, 13, and

14) were pooledas the partially purified

repli-cation complex. The replication complex

ex-tracted from cells coinfected and labeled with

3

co,

X 2

E

0

I-5 10 15 20

Fraction number

FIG. 5. Sedimentation of the soluble complex by sucrosegradients. The crude DNAreplication

com-plex wasextracted, dialyzed, andcentrifuged in a

neutralsucrosegradient.Eachfractionwasassayed

forDNA-synthesizing activityasdescribed

(0).

The

coinfectedcells werelabeledat18h p.i. with

R3H]-thymidine (2 aCi/ml) for30minand treated in the

same manner. The nuclear extractwas centrifuged

similarly, and the acid-insoluble radioactivity in

eachfractionwascounted

(0).

Simian virus40DNA

components I(20S)and II(16S)wereusedasmarkers.

[3H]thymidine

sedimented similarly

(Fig. 5).

The

results indicate that the

complex

of

the

same

size is active both

in

vivo

and in vitro.

Nature

of

DNA

sequences

synthesized

in

vitro. The results of DNA-DNA

hybridization

revealed that the DNA

synthesized

in vitro with

the

replication

complex

consisted

mainly

of

AAV-1 DNA

(Table

2).

Very

little,

if

any,

H5

DNA

or

cellular DNA

was

synthesized.

The

results show that the

complex

is the AAV-1

DNA

replication complex.

Sucrose

density gradient

analysis

of

DNA

synthesized

in

vitro.

Most of the in

vitro-synthesized

DNA

sedimented

at

the

posi-tion

coinciding

with the

marker

AAV-1

DNA

(14.48)

when

analyzed by

sedimentation in

a

neutral

sucrose

gradient (Fig.

6).

DNA

synthe-sized

ina 5-min reaction sedimentedassmaller

components

inan

alkaline

sucrose

gradient (Fig.

7A).

However,

prolonged

incubation didnot

re-sult

in

the

elongation

of the

smaller molecules

(Fig.

7B

and

C). No decrease in the size of

3H-labeled

AAV-1 DNAwas

detected

when AAV-1DNAwas

incubated

with the

replication

com-plex under the

same

conditions, suggesting

that

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TABLE 2. DNA-DNA hybridization of DNA synthesized in vitro

DNAimmobilized(%)

InputDNA(cpm)~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - Blankfilter

Input DNA (cpm) AAV-1 DNA H5 DNA Cellular DNA

(cpm)

(cpm) (cpm) (cpm)

DNAsynthesizedinvitroa (8,400 2,856(34)b 391(4.7) 428(5.1) 60

cpm)

AAV-1 DNAc(5,800cpm) 2,088(36) 93(1.6) 87

(1.5)

58

H31 DNAC (5,200cpm) 104(2.0) 2,236 (43) 88(1.7) 53

HEK DNAc(8,800 cpm) 185(2.1) 167(1.9) 1,848 (21) 64

aDNAsynthesizedin vitrowasextractedasdescribed in Materials andMethods,

dialyzed against

O.JX

SSC

(0.15MNaCl plus0.015 M sodiumcitrate)and 10 mMEDTA,and annealed with AAV-1DNA,H5DNA,and humanembryolivercell DNA immobilizedonmembranefilters. DNA-DNAhybridizationwascarried outas

describedpreviously (22).

bFigures inparenthesesshow percentages of DNAhybridizedtoinputDNA.

cAscontrols,labeledDNAfrompurified

visions

ofAAV-1, H5,and fromgrowingHEKcellswereincluded

in thetest.

15

x

E

5

5 10 15 20 25

Fraction number

FIG. 6. Analysis of DNA synthesized in vitro in neutralsucrosegradients.DNAsynthesizedinvitro

with thecomplexat37°C for60 minwassedimented

in aneutralsucrosegradient. Theposition of 3P-labeled AA V-I DNA(amarker) isshown byan arrow.

the formation ofsmaller-sized DNAduring the

in vitro reaction wasnot due to the action of

DNasein thecomplex (Fig. 7D).

InvolvementofparentalAAV-1 DNAin

thereplication complex.Confluent

monolay-ersof HEK cellswerecoinfectedwithH5tsl25

and[3H]thymidine-labeled AAV-1. At18 hafter

coinfection,thecomplexwasextracted and

sed-imentedina sucrose gradient. The radioactive

peak coinciding with the position of the AAV-1

DNAreplication complexwasdetected(Fig. 8).

About 1% of theinput AAV-1 DNA was found

in the

replication complex.

When HEK

cells

were infected with3H-labeled AAV-1

alone,

no

radioactivepeakwasdetected.

DISCUSSION

The

AAV-1 DNA

replication complex

was

solubilized and

partially

purified from the nuclei

of HEK cells coinfected with AAV-1 and

H5ts125

at

40.5°C,

to

establish

an

in

vitro

system

for AAV DNA

replication. The complex, with

a mean

size of 23S, synthesized exclusively

AAV-1

DNA

as

identified

by DNA-DNA

hybridiza-tion and sedimentahybridiza-tion in

a

neutral

sucrose

gra-dient.

However,

sedimentation in

an

alkaline

sucrose

gradient showed that the products

con-sisted

mainly of shorter chains than

AAV-1

DNA. The

results suggest

that the

replication

complex

lacks

some

factors,

such

as

DNA

ligase,

which

are present in the intact isolated

nuclei

and

necessary

for the formation of

complete

AAV-1 DNA molecules, since AAV-1 DNA of

the

mature

size

was

synthesized

in vitro with

isolated nuclei

(5).

The

analysis

of the

parental

AAV-1 DNA in

an

alkaline

sucrose

gradient

would also

provide

an

explanation

for this

ob-servation. No AAV DNA

replication complex

was

formed

incells infected

with

AAValone. It has

been

reported

that AAV

virions contain

single-stranded DNA,

which is either a

plus

or

minus

linear

strand

(14, 18). After single

infec-tion

with

AAV,

the

effective

adsorption and

penetration

of

the

AAV genome

into the nucleus

was

observed

(20;

H.

Handa, unpublished data).

The

conversion of

single-stranded

DNA to

dou-ble-stranded

DNA

(replicative

form)

must be

necessary

for the

AAV

replication cycle,

as in

the

replication

of other

parvoviruses (16, 25).

The above

results

suggest that the

replicative

form is not

formed

in

cells after

infection

with

AAV alone. Further studies

are necessary to

448

HANDA AND SHIMOJO

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x E

o-

0.5-I

.q

5min

41

6

x E

I 31

21

[image:6.500.96.392.60.402.2]

15min

FIG. 7. AnalysisofDNA synthesized in vitro in alkaline sucrosegradients. DNA synthesized in vitro

with thecomplexat37Cfor(A) 5, (B) 15,and(C)60 minwassedimented inalkalinesucrosegradients. (D)

3H-labeledAAV-1 DNA was sedimentedsimilarly afterpreincubation with the AAV-1 DNA replication complexat370Cfor60min. Theposition of32P-labeledAAV-1 DNA(a marker)is shownbyanarrow.

elucidate the defective step of the AAV DNA

10 replication.

We have suggested that a helper factor(s)

induced by infection with adenovirus is closely

related to the formation ofadenovirus-specific

T antigen (H. Handa, K. Shiroki, and H. Shi-mojo, submitted

for

publication).

Further

puri-x E.' 8 fication of the AAV DNA replicationcomplex

5 and the establishment of a complete system

I, t .would provide amethodto investigatethe

na-ture of factors involved in the AAV DNA

repli-f\: \ cation.

ACKNOWLEDGMENT

We aregratefultoK.Shirokiforcooperation and toK. Oda forcritical reviewof themanuscript.

1 5 10 15 20

FIG. 8. Involvement ofparentalAAV-1 DNA in thereplication complex. HEKcells werecoinfected

with

13H]thymidine-labeled

AA V-1(0.1cpm/ fluores-centinfectious unit)andH5ts125at40.50C.Symbols: AAV-1 DNA replication complex extracted

sedi-mented in aneutral sucrosegradient

(0);

extract

preparedfromthe cellsinfectedwith

13Hlthymidine-labeledAAV-I alone

(-4*);

and 3H-labeledAAV-1DNAextractedfrom purifiedvirions

(---0).

The

position ofthe AAV-1 DNAreplication complex is

shownbyan arrow.

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This work wassupported bygrants from theMinistryof Education, Science and Culture, Japan, and the Princess TakamatsuFund for Cancer Research.

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3. Chang,L. M. S.1973. Lowmolecularweight deoxyribo-nucleic acidpolymerase from calf thymus chromatin. I.Preparationof homogeneousenzyme. J.Biol. Chem. 248:3789-3795.

4. Ensinger,N.R., andH.S.Ginsberg. 1972.Selection and preliminarycharacterization of temperature-sensi-tivemutantsof type 5adenovirus.J. Virol.10:328-339. 5. Handa, H., andILShimojo.1977.ViralDNAsynthesis in vitro with nuclei isolated from adeno-associated virus type 1-infected cells.Virology77:424-428.

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8. Hoggan,M.D., N. R. Blacklow, andW. P. Rowe. 1966.Studiesofsmall DNA viruses found invarious adenoviruspreparations: physical, biological,and im-munological characteristics. Proc. Natl. Acad. Sci. U.S.A.55:1467-1471.

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