Derivation and characterization of POJ cells, transformed human fetal glial cells that retain their permissivity for JC virus.

0022-538X/87/030755-09$02.00/0

Derivation

and Characterization of

POJ

Cells, Transformed Human

Fetal

Glial Cells

That Retain Their

Permissivity for JC Virus

CHRISTIAN

MANDL,lt

DUARD L. WALKER,2 AND RICHARD J. FRISQUE1*

Departmentof Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania

16802,1

and Departmentof Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 537062

Received 25 August1986/Accepted 10November 1986

The study of the medically importantpolyomavirus JC virus is limited to only a few laboratories, primarily because thepermissive cell system most often used, primary human fetal glial cells, isdifficultto obtain and propagate. We have introduced mutations at the origin of DNA replication of JC virus and transformed glial cells with thereplication-defective genomes. Although normal glial cell cultures rapidly lose their permissivity for the virus after subculture, the transformed cells (designated POJ) had a greatly expanded life span and remained permissive for JC virus even after 30 passages in vitro. POJ cells constitutively express a functional Tprotein that complements the replication defect of lethal early-region mutations in JC virus. We expect that these cellswill greatly facilitate the study of this human virus.

JC virus (JCV), a human polyomavirus, infects most people early in life and in some immunocompromised

indi-viduals causes the fatal demyelinating disease progressive

multifocal lqukoencephalopathy. In spite of the medical

importance

If

JCV, many laboratories have been

discour-aged from studyingit because a convenient cell system has not been available. Primary human fetal glial (PHFG) cells, used in the initial isolation of JCV in 1971 by Padgett and co-workers (30), have been the cells usually used for the growth ofthe virus. These cells pose a number of serious problems for the virologist. (i) Fetal brain tissue is difficult to obtain insufficient quantitieson aregular basis. (ii) Cultures

of PHFGcells are a mixedpopulationof cell typescomposed

primarily ofastrocytes and spongioblasts. The latter cell is

presumedtobethe precursoroftheoligodendrocyte, the cell

in which JCV multipliesmostefficiently(28).(iii) The ratio of astrocytes to spongioblasts varies considerably between cultures prepared atdifferent times, and specialhandlingis required toobtain the preferred spongioblast-rich cultures.

Once "established" inculture, these cellsrapidlylose their

abilitytosupport JCVreplication. (iv)The lytic activity of JCV, evenin PHFGcells, isinefficient;thegrowth cycle is

prolonged, and cytopathic effects are difficult to recognize

(28). Typically, infectedcells are maintained in culture for 4 to5 weeks before virus is harvested.

From theverybeginning, major effortshave beendirected

at finding amore suitable cell system forpropagating JCV. Many kinds of cells, both

primary

and established lines,

havebeentestedwithlittle success(39a).Afewhumancells

(e.g., embryonic kidney, amnion, andurine-derived

epithe-lial cells) do support JCV replication tovarious degrees (1, 27,36);however,theyalso suffer fromrestrictedavailability

and difficulthandling.Inaddition, largenumbers of defective

virionsaredetected in viruspools prepared fromthese cells. The needfor a useful cell system for thegrowthof JCV in vitroprompted us to attempt to derive apermissive cell line

by transforming PHFG cells with

replication-defective

(Ori-) mutants ofJCV. This

approach

has been used

suc-cessfully for simian virus 40

(SV40)

(COS

cells

[11])

and

*Correspondingauthor.

tPresent address: Institute ofVirology, University ofVienna, Vienna, AustriaA-1180.

mousepolyomavirus (COP cells and Polyoma COS cells [2,

39]). In these instances, the objective was to obtain a

complementing cell system to propagate viral mutants and vectors. In our case, however, the primary goal was to derive apermanent cell line for propagating wild-type JCV.

RecentlyMajoretal. (21) transformedPHFG cellswith the sameSV40mutantsused togenerate COS cells (12).

Signif-icantly, these cells (designated SVG cells) support the

rep-lication ofJCV to approximately the same degree as the parent PHFG cells. While offering animportant alternative forthegrowth ofJCV,wefeel these cells have two serious drawbacks.First, fromthestudies ofMajor et al., it appears that the SV40 T antigen constitutively expressed in SVG

cells does not interact effectively with the JCV regulatory

machinery. A more serious obstacle to the practical use of

SVGcellsinvolvesthepossibility of recombination between

theintegrated SV40 sequences and input JCV DNA. Inthis paper we describe the first deletion and insertion

mutants of JCV constructed by site-specific mutagenesis techniques. Several ofthese mutants have beensequenced

and theirbiological functions investigated.Themutantshave allowedus toidentifysequencesrequiredfor the replication oftheJCV genome.

Wealso report theuseofOri-mutantsofJCVtoestablish transformed PHFG cell lines (designated POJ, for PHFG cellstransformedby Ori- JCV)that support thelytic growth of JCV. Becausethey constitutively express afunctional T

protein,POJ cellscomplementthereplication defect of JCV early-region mutants. Details of the derivation and

charac-terization ofthis cellline arepresented.

MATERIALS ANDMETHODS

Cells and virus. PHFG cells were obtained from 10- to

16-week-old abortusesasdescribed earlier(28). BothPHFG

and POJ cells were grown in Dulbecco modified

Eagle

(DME) medium

supplemented

with 10% fetal calf serum.

Duringthe DNA

replication

and virus

production

assays,the serum concentrations were reduced to 3 to 5% to prevent cells from

becoming

toodense and

sloughing

off the

plates.

JCV (Madl

strain)

was

propagated

in PHFG cells and

purifiedasdescribed

by

Padgett

etal.

(28).

Mutant construction and analysis. The

plasmid pMITC

(ABam)

containing

the

complete

genome ofJCV inserted 755

on November 10, 2019 by guest

http://jvi.asm.org/

M ITOC 9 1 Hindm(4498)

477' t pM TCABam) pBR322

co

l624 np

FIG. 1. PlasmidPMITC(ABam)usedto constructJCVmutants.

pMITC(ABam) contains the entire genome of prototype JCV

(Madi)inserted into the EcoRI site ofpBR322. pBR322 sequences between the unique Sall and Clal restriction sites have been

deleted, thereby eliminating the single Hindlll site in the vector.

JCV DNAcontainsthree Hindlllsites: onesiteatnucleotide 4498

occurs within theuniquecoding regionof small-tprotein,asecond site at nucleotide4914 lies withinthe shared coding sequences of bothearly proteins,andthe third site at nucleotide 5112 is found in the control region ofJCV adjacentto the center of thepresumed origin of DNA replication. Closed bars represent pBR322

se-quences,andopen bars indicate viral sequences thatare translated

intothelargeandsmall Tproteins.Thenarrowlinerepresentsviral

sequences. Numbers refer to nucleotidepositions on thegenomic

mapofMadl(9).Numberingstarts within the25-np dyad symmetry thoughtto include theoriginofreplicationandproceedstowards the latecoding region.The entireplasmidis8,868np inlength,including 5,130 np of JCV DNA.

into theEcoRI siteofpBR322 (Fig. 1)wasusedto construct

deletionand insertion mutants ofJCV. Briefly, the plasmid

waslinearizedby partial digestionwith therestriction

endo-nucleaseHindlIl (20 ,ug ofDNAwas digestedwith 20 Uof

the enzyme for 20 min at 37°C). The HindIII recognition sequence occurs three times within the viral DNA: at nucleotide4498intheregion encodingthecarboxyterminus

of the small tprotein,at nucleotide 4914in theoverlapping

sequences specifying the amino termini of the large and

small T proteins, and at nucleotide 5112 adjacent to a

twofold symmetrical sequence assumed to be the center of

theJCVoriginof DNAreplication (9).Afterextraction with phenol and chloroform, the DNA was precipitated with

ethanol and suspendedin S1nucleasebuffer(280mMNaCl,

50 mM sodiumacetate, pH 5.2,1 mMZnCl2).Portions of the

sample weredigestedwith different amountsof Si nuclease (0.5 to 10U/Iug ofDNA)for20minat 20°C. This treatment

causedanincreasingnumberofnucleotides to beremovedat

both ends ofthe linear DNA. To create insertion mutants,

thelinearizedpMITC(ABam)DNAwassuspendedinbuffer

(50mMNaCl,10mMMgCl2,1mMdithiothreitol) containing all four deoxynucleotides (1 mM each) and ATP (0.1 mM). Five units of the large fragment of DNA polymerase I (Klenow reagent)wasadded,andthemixturewasincubated

at roomtemperaturefor 30min. Following S1 nucleaseand

Klenowtreatments,full-lengthlinearplasmidwasseparated

fromcircularforms andsmaller linearfragments by

fraction-ation on a 1% low-melting-point agarose gel at 4°C. After

purification, DNA was recircularized by ligation with T4

DNA ligase at aDNA concentration of less than 10 ngl,ul. Competent Escherichia coli DH-1 cells were transfected with this DNA andplatedontoLB agarplatescontaining 30

jig

ofampicillin per ml (23). Small-scale plasmid prepara-tions of individual ampicillin-resistant colonies (14) were digested with HindlIl, and mutants were identified by the absence ofoneof the three HindIll sites.

Following S1 nuclease treatment approximately 10% of the plasmids screened contained deleted HindIll sites.

Al-though allthree types ofmutants were obtained, mutations affecting the HindIII site atnucleotide 4498 occurred about 10 times more frequently than at the other two sites. A number of mutants were chosen for further study. To estimatethe sizes of the deletions in these mutants, restric-tion enzyme analysis was performed, and the sizes of the cleavage products from mutated and wild-type DNAs were comparedon agarose and polyacrylamide gels. DNAs con-taining relatively small deletions were sequenced by the Maxam and Gilbert technique (24). Each mutant in this serieswasdesignated by the letter S (forS1nuclease) anda number. These mutants are characterized in Table 1.

Mutations arising from treatment with Klenow reagent weredetected at nucleotides 4498 and 4914, but not 5112. As expected, the DNAs from these mutants contained an inser-tion(duplication) of the sequence 5'-AGCT-3'. This series of mutantswasdesignated by the letter K (for Klenow reagent) and anumber (Table 1).

This reportfocusesonthecharacterization anduseof the mutantsalteredattheHindlIl siteatposition 5112, particu-larly the mutants S-15 (35-nucleotide-pair [np] deletion), S-19 (11-np deletion), and S-27 (10-np deletion). In the complementation experiments outlined below, the mutant S-1, which has a 67-np deletion around nucleotide 4914 (affects large and small T proteins), will be discussed.

DNA transfection. Transfection of mammalian cells with viral DNA was done by either the modified calcium phos-phate (40) or the DEAE-dextran (34) procedure. The first transfectiontechniquewasused in thetransformation

exper-TABLE 1. Mutantsof JCV affected in theoriandearly codingsequences

Mutant Mutated Mutation Mutation T-Ag Transformation

regiona sizeb boundaryc expressiond of PHFG cells

K-1 t +4 4499-4502 + NTe

S-6 t -72 4467-4538 +

-S-21 t -26 4486-4511 +

-S-44 t -8 4498-4505 + NT

K-2 T/t +4 4915-4918 -

-S-i T/t -67 4871-4937 - NT

S-18 T/t -15 4908-4922 +/-

-S-36 T/t -13 4912-4924 NT NT

S-3 ori -59 5072-5130 +

S-8 ori -64 5072-5135 +

S-15 ori -35 5093-5127 +

S-19 ori -11 5108-5118 + +

S-27 ori -10 5107-5116 + +

aMutations wereintroducedat theHindIll sites atnucleotides4498, 4914,

and5112,which affectedsmall t,sharedlarge and small T,andorisequences,

respectively.

bSize in nucleotide pairs; +,insertion; -,deletion.

cBased on thenumberingscheme ofFrisqueet al. (9).

dTantigen (T-Ag) expression monitoredin PHFG cells 3 days

posttransfec-tion. A few cells transfected with S-18 DNA gave a weak, questionable nuclearfluorescence.

eNT,Nottested.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.62.300.69.241.2] [image:2.612.318.557.484.649.2]

T-binding I T - binding II

| S 17np | P17n

1

5n ~ A

5060 5070 5080 5090 5100 5110 5120 5130 10 20 29

5 -GAAAAACAAG GGAATTTCCC T;GCCTCCTA AAAAGCCTCC AC CCCTTACTACTT GAG TAAGCTTGA GGCGGAG GCGGCCTCGGCCT CCTGTATATA TAAAAAAAA

3-CTTTTTGTTC CCTTAAAGGG ACCGGAGGAT TTTTCGGAGG TGCGGGAATGATG CTC ATTGAAC CT CCGCCTC CGC CGAGCCGGAGGACATATAT ATTTTTTTT

HindM

S-27 10Onp _4 -27

S-I19 Ilnp S-l9

S- 15 '35np S -15

S-3 -59np -S-3

[image:3.612.65.541.65.206.2]

S- 8 64np S- 8

FIG. 2. Deletion mutations in the regulatoryregion of JCV. The regulatory region of JCV includes 393 np located between the coding regionsforearly and late proteins. This stretch of DNA includesa number of control elements including promoter and enhancer sequences andthe origin of DNAreplication. Only a portion of this region is depicted in the figure, and earlyand late coding information is to the left and right,respectively, of the sequences shown. The 25-np dyad symmetry (S) is assumed to be the center of the replication origin andspans nucleotides 5118 to 12. This symmetry differs from those ofSV40 and BKV at a single position. This nucleotide substitution disrupts the symmetry of the two human viralori signals (indicated by asterisks). Adjacent to this symmetry is theHindlllrecognition site (nucleotides 5112 to 5117) used in constructing the JCV origin-defective mutants. The arrows point to the cleavage positions. Also shown are two additionalsymmetries (17 np and 19 np) and a 17-np palindromic sequence. Two T-antigen-binding sitesare indicated by open bars and include severalcopies of the contact consensus sequence 5'-(G>T)(A>G)GGC-3' (6, 37).It is likely that the actual binding sites involve sequences extending to both sides ofthese pentanucleotide sequences. The sizes and borders of the deletions in five differentori mutants areindicated. In someinstances short repeats at the deletion boundaries make positioning of the borders ambiguous. To remain consistent, borders were always drawn as far to the left (i.e., towards the early coding region)as possible. The AT-rich region containing the TATA box of the early viralpromoter was not affected in any of the deletion mutants.

iments; the second method was used in the DNA replication assays.

Indirectimmunofluorescence assay. Atvarious times after DNAtransfectionorvirusinfection, cells growing on 12-mm coverslipswerefixed for90 s in a 1:1mixture of acetone and

methanol. To detect T antigen, serum from a hamster

bearingaJCV-orSV40-inducedtumor wasadded to cells for 30 minat37°C. Afterthecells were rinsedwith

phosphate-buffered saline (PBS), fluorescein-conjugated goat

anti-hamsterimmunoglobulin G (IgG) serum was added. Thirty

minutes later the cover slips were rinsed with PBS and distilled water and mounted in buffered Gelvutol (10 g of polyvinyl alcohol in40 ml of PBSand 20 ml ofglycerol)on

microscope slides. The same procedure was followed for

capsid antigenexceptthatrabbitanti-capsidserum wasused as the first antibody and fluorescein-conjugated goat anti-rabbit IgGserum wasthe second antibody.

Viral DNA replication assay. Plasmid DNA containing a mutant orwild-type viralgenomewas digestedwithEcoRI andHhaItoyieldonelargefragment, representingtheentire

viral genome, and several small plasmid DNA fragments.

The linear viral DNA was separated fromthe smaller

frag-ments on a 1% agarose gel and isolated by electroelution. Viral DNA was recircularized during an overnight

incuba-tionwith T4 DNA ligaseat 14°C withlow DNA

concentra-tions (1to10 ng/,l). Aportion oftheself-ligated DNAwas

analyzed on agarose gels to determine the efficiency of

ligation.DNApreparations usedtotransfectcellscontained

atleast 50% circular forms. Cells were plated onto 35-mm

tissue culturedishestheday before transfection atdensities

that would yield nearly confluent monolayers after one

doubling. Transfections were

performed

by

incubating

the

cells for 75 min at

37°C

with 0.5 ml of DME

containing

DEAE-dextran(250

,ug/ml)

and 100 ng ofcircularized DNA.

Viral DNA was extracted by the method of Hirt

(13)

at severaltimepoints,

ranging

from0to21

days

posttransfec-tion. Aportion ofeachextract was

digested

with

DpnI

and

EcoRIby the scheme of Peden et al. (31).DpnIcleaves DNA

of bacterial origin (input DNA) but does not cut DNA

replicated ineucaryotic cells due toits sensitivity to

differ-ences in DNA methylation patterns in the two cell types.

EcoRI cuts JCV DNA once independent of methylation. Thus, inthese experiments,input DNA was cut into several smallerfragments while newly synthesized viral DNA was

linearizedby the singleEcoRI cleavage. Digestion products were fractionated on 1% agarose gels and transferred to

nitrocellulose filters bythe methodofSouthern(35). Follow-ing hybridization at68°C to a

32P-nick-translated

(32) JCV

DNA probe, viral DNA on the filter was detected by

autoradiography.

HAtiters.Thehemagglutination(HA) testwasconducted

asdescribed by Padgettand Walker (29). Briefly, doubling dilutions ofthe virus preparations were made in PBS. An

equal volume of 0.5% human type 0 erythrocytes from

donors lacking antibodies to JCVwas added, and the mix-ture wasincubatedfor3 hat4°C. The HAtiterwastakenas thereciprocal ofthehigher dilution ofthe virus suspension showing complete

agglutination

ofthe erythrocytes.

ViralDNAintegration patterns. Confluent monolayers of

cells from several 100-mm tissue culture plates were col-lected and incubated with pronase in the presence of0.5% sodium dodecyl sulfate for 4 h at 45°C.

High-molecular-weightDNAwaspurified byseveralextractions withphenol

and chloroform, followedby

precipitation

withethanol. All

manipulations were done as gently as

possible

to avoid

shearing the chromosomal DNA. RNA was removed

by

RNase treatment and ethanol

precipitation.

Cellular DNA (10

jig)

wasdigestedwith restriction enzymesthat cleaved

viral DNA sequences onlyalimited numberof times

(0

to3

cuts).This DNAwasloadedonto1or0.8% agarose

gels and,

after

electrophoresis,

transferred tonitrocellulose filters

by

the Southern

technique

(35). Viral DNA was visualized

by

autoradiography after

hybridization

to viral

probes

labeled with 32P by nicktranslation(32).

on November 10, 2019 by guest

http://jvi.asm.org/

S-19 97

M~ A 0C. 7 2 x 7 2

_m

FIG. 3. Replicative activityofJCV mutant DNAs in PHFG cells. PHFG cells were transfected with 0.1 ,ug of wild-type (w.t.) or

mutant (S-15, S-19, and S-27) JCV DNA. Each DNA had been separated from vector sequences and recircularized by ligation.

Viral DNA was extracted from the cells at 0, 7, and 21 days posttransfectionanddigestedwiththe restrictionenzymesDpnIand EcoRI.DNArepresenting 1/4of eachdigested sample (1/60of the

21-day wild-type DNA sample) was electrophoresed on a 1%

agarosegelandsubjectedto Southern blotanalysis (35).MlandM2

were wild-type inputDNAcleaved withDpnI plusEcoRI(1 ng of DNA) and EcoRI alone (0.3 ng of DNA), respectively. Newly synthesizedviral DNAmigratedasasingle5.1-kbfragment (sizeof

M2)andwasvisualizedby autoradiographyafterhybridizationtoa

nick-translated(32) pMITC(ABam) probe. Filmwasexposedfor5

dayswithoutan intensifyingscreen.

RESULTS

JCV mutants with deletions around nucleotide 5112 are

replication defective. A dyad symmetry is located near the replication origin of SV40; nearly identical symmetries are

found inthecorresponding regionsof the JCV and BK virus (BKV)genomes(9, 38). The three viralsequences differata

single position (nucleotide 5124 in JCV). This base change

disturbs the otherwise perfect symmetry in the JCV and

BKV sequences and accounts for the absence of a BglI

restriction site inthese two DNAs. Gluzmanand co-workers

(12) tookadvantageof theBglI sitepresentwithin the SV40 origin region to construct the Ori- mutants of SV40 that

were used to derive the COS cell line (11). The most

convenient restriction site nearest to the center of the

presumedJCVoriginsequences(the symmetryextendsfrom

nucleotide 5118 to 12) is the Hindlll recognition sequence

encompassing nucleotides 5112 to 5117. A diagram of this region, (Fig. 2) indicates the boundaries of the deletions of

thefive mutations obtained atthis HindlIl site. Itshould be

notedthatS-27retainedthecomplete 25-nptwofold

symme-try, while mutants S-19 and S-15 were missing the

5'-terminal nucleotideand the 5' halfof the symmetry,

respec-tively. To test thereplicativeabilityof thesemutants,0.1 ,ug ofthe S-15, S-19, and S-27 DNAs(plasmid sequences were

removed and the viral DNAs were recircularized) were

transfectedintoPHFG cellsbyamodification of the

DEAE-dextran method (34). Low-molecular-weight DNA was

ex-tracted fromthecells atseveraltime points following

trans-fection andsubjectedto theDpnI replicationassay (31) (see

Materialsand Methods). Newly synthesizedDNAcould be

detected as early as 3 days after wild-typeJCV DNA was

added to the cultures (Fig. 3). The amount of replicated

DNA increased steadilyuntilthe final time pointat 21days

posttransfection. Viral DNA synthesis was not detected at

anytimein cellstransfected with the mutantDNAs. It was

concluded thatmutantsS-15,S-19,and S-27werereplication

negative (Ori-).

T-antigen expression and transforming activity of JCV

mutants. A major reason for constructing Ori- mutants of JCV was to see whether they could be used to transform PHFG cells intopermanent cell lines that retained theability

to support the growth of JCV. To determine whether this approach wasfeasible, itwasfirstnecessaryto testwhether the mutants induced Tantigen, since thisprotein isrequired

for stable transformation. In addition to the five mutants altered in the origin region, several mutants affectedat the other twoHindlIl sitesweretested. PHFG cellswereseeded onto coverslips in 16-mm wells and transfected with 0.5plg

of intact plasmid by the modified calcium phosphate tech-nique (40). Three days later cells were stained for Tantigen byanindirect immunofluorescence assay (Table 1). All Ori-and small-t mutants induced T antigen in theglial cells, while the T/t mutantsfailed to do so.

Once we had determined that some of the mutantswere capable of inducing T antigen in PHFG cells, our efforts were directed to establishing permissive transformed cell lines. PHFG cellswere seeded onto 60-mm dishes (5 x 105 cells perdish) and transfected the following day with 2 ,ug of intact mutant plasmid DNA (Table 1). The medium (DME

plus10% fetal calf serum) was changed every 4days, andas cultures reached confluence, cells were trypsinized and transferred to new dishes at a ratio of 1 to 2. At 8 weeks posttransfection (passage 4), patches of T-antigen-expressing cells were observed in cultures infected with mutants S-19 and S-27. By 10 weeks (passage 6), cells in thesetwoculturesbegantodemonstrateanaccelerated rate ofgrowth andanaltered shape (cells became elongated and weremuchlargerthan uninfectedglial cells). More than 50% of the cells in each culture were producing T antigen. Cells transfected with the other mutants remained indistinguish-able from those in mock-infected cultures; cells did not contain viralproteins and their growth rate declined steadily until passage 8, when cell division ceased. On the basis of these observations, we concluded that the cells transfected with S-19 and S-27 DNA were transformed (Table 1). We named these transformants POJ-19 andPOJ-27, for PHFG cells transformed by origin-defective JCVmutants S-19 and S-27. Furthercharacterizationindicated that these cells had apartiallytransformed phenotype.By passage 8, essentially

100% of the POJ-19 andPOJ-27 cells expressed T antigen. Thecells hadatendencytopileupin small clusters and were able to grow in medium supplemented with low (1%) con-centrations ofserum. Although POJ cells grew faster than their normal counterparts,they still grew slowly relative to

fullytransformed cells. Furthermore, the cells grew poorly if subcultured at low cell densities. We found that POJ cells canbetrypsinizedandpassedat aratio of 1 to 4about once aweek. As aresultof their sensitivity to growth at low cell

densities, wehavehaddifficultycloning POJ-19 and POJ-27

cells;

cloning efficienciesonplasticwith medium containing 10% fetal calfserum range from 0.1 to 1.0%. However, we succeeded in obtaining a number of clones of POJ-19 and POJ-27 cells that have been called POJ-1 and POJ-2,

respec-tively. Thegrowth properties and appearance of the cloned cell lineswere indistinguishablefrom those of the uncloned POJ cells. After approximately 70 cell generations, both cloned anduncloned POJ cells entered crisis; the growth rate decreased dramatically and the cells enlarged, became

rounded,anddetachedfromthe culture dishes. Attempts to nurse these cells through this period continue; however, becauselarge numbersof cells from earlier passages can be

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.94.281.69.210.2]

0 3 7 14 21

r---I I---~-'-~-~I

a N OJ N' N7 7

° I O 0 0 0

MiCL L - a -,:,CL -m aLL

M 20_a t 0 1 0 m 0 = 0 I: 2 C a- a- a EL aI a a a~ a

0 3 7 14 21

L r-

r-b

77 7-

r-

2-IN N (M N N U C} C\lN

0 0 0 0 0 0 0 0 0 0

MI M2 a a a a a a a a a a

4,

*

4WO ~ ...

[image:5.612.133.489.69.239.2]

..>

FIG. 4. Replication of wild-type JCV DNA in POJ and PHFG cells. Viral DNA was extracted fromPOJandPHFG cells, 0, 3, 7, 14, and 21days after the cells were transfected with 0.1 ,ug of wild-type JCV DNA. DNA (1/4 of the POJ-2 and POJ-27 samples, 1/12 of the PHFG sample) wasdigested withDpnIplusEcoRIand analyzed by the technique ofSouthern(35). Amounts of the PHFG samples were reduced threefold to account for the difference in cell number at the time of transfection. An additional threefold reduction was made in all 21-day samplestoavoidpossible overexposure of these bands. Shown arecomparisons of the accumulation of replicated viral DNA in POJ-2 and PHFG cells (a) andin POJ-2 and POJ-27 cells (b). Input JCV DNA cleaved with DpnI andEcoRI (M1)orEcoRIalone(M2) served as markers. Mlrepresents 3 ngand 1 ng of DNA in panels a and b, respectively; M2 represents 1 ng and 0.3 ng of DNA in panels a and b, respectively. Films were exposed for 6 days (a) or 7 days (b).

frozenfor future use, their entry into crisis is not considered aseriousproblem.

POJ cells support the lytic growth ofJCV.The next step in our studies was to ask whether POJ cells had retained the

ability oftheparental glial cells to support the replication of

JCVDNAandthepropagationof JCV. To address the first part of this question, POJ-2 cells and PHFG cells were

transfected with0.1 ,ugof circularJCVDNA, and at several

time points low-molecular-weight DNA was extracted from

the cellsandsubjectedtotheDpnI replicationassay (Fig. 4). Thecultures ofprimarycells weregenerally more dense than

those ofthelarger POJ cellsatthe startofeach experiment,

and therefore the total numberof cells per dishwas

deter-minedpriorto eachtransfection. These values were used to

adjust the amount of sample applied to the agarose gels

(indicated in each figure legend) so that band intensities

could be considered torepresent the DNA froman equiva-lent number of cells. Extractions at day 0 represented the amountof viralDNAstill associatedwith the cells at the end

of the transfection procedure. Results indicated that a greater amountofDNA wasrecoveredfromthePOJ-2cells

thanfrom the PHFG cells. Thisfindingwasreproducible and

may reflect the larger surface area of the POJ cells or

possible differences in the efficiency ofDNAuptakein the two celltypes. The amount of residual input DNAquickly decreasedin the PHFGcells,but wasstilldetected inPOJ-2 cells aslate as 14days posttransfection. Fragments ofinput

DNA generatedby DpnI cleavage became more diffuse on the Southern blots with time,

indicating

slow but steady

degradation

of

input

DNA

by

the cell. The

persistence

of

DNA inPOJ-2 cells may be partlyexplained by the

possi-bility

that more DNA enteredeach POJ cell

initially. Newly

synthesizedDNA(uncutby

DpnI,

linearized

by

EcoRI)was

first

detected

in each cell type as early as 3 days posttransfection. By 7

days

the band

representing

DNA

replicated

in POJ-2 cells was stronger than its counterpart

from PHFG cells. At 14

days

the band intensities were

roughly equal for the POJ-2 and PHFG

samples.

At about this timeadecreasewasobserved in the cell number of both

DNA- andmock-transfected POJ cultures, and this decline was very apparent by 3 weeks. The data show that the amount ofDNA synthesized by 21 days had continued to

increaseinbothcell types; however, by this time the amount of DNAextracted from thePHFG cellshad begun to surpass thatobtained fromPOJ-2 cells.

ThePOJ-2 cells used in this experiment had a relatively

highpassagenumber(ca. passage 35)and were within a few

generations of crisis. We suspected that POJ cells from earlier passagesmighthave a betterchance of surviving the entire course of the replication experiment and that such cells would presumably accumulate greater quantities of

replicatedviral DNA at the latertime points. To pursuethis possibility,POJ-2 cells(ca.passage35)andunclonedPOJ-27 cells(passage12)weretransfectedwithwild-type JCVDNA and testedfortheirabilityto supportviral DNA replication (Fig. 4b). Results similar tothose obtainedfor the

POJ-2-PHFGcellcomparisons (Fig. 4a)wereobtained; JCVDNA

replicatedmost efficientlyinPOJ-2 cells onlyatearly times posttransfection. In this experiment the amount of viral DNAinPOJ-2 cellsdecreasedslightly between

days

14and

21, again reflecting a significant loss of cells from these

cultures. POJ-27 cells, however, maintained a healthy ap-pearance throughout the experiment, and cell numbers

re-mainedconstant.The amountofreplicatedDNAdetected in POJ-27 cells inweeks2and 3oftheexperimentfarexceeded

thatfoundin POJ-2 cells. ViralDNAaccumulatedto an even greater extent in POJ-19 cells (ca. two- to threefold more than in POJ-27 cells; data not shown). These studies indi-cated that the uncloned POJ cells

supported

JCV DNA

replication better than

permissive

PHFGcells.

After

establishing

that POJ cells

supported

JCV DNA

replication, experiments were

designed

todemonstrate that

infectious virions were

produced by

these cells. Confluent cultures of

POJ-19,

POJ-27,

and PHFG cells were estab-lished in 24-well

(16-mm-diameter)

cluster

plates

containing 12-mm cover slips. Each well was inoculated with 100 HA

units ofprototype MadlJCV. Virus

production

was moni-tored by immunofluorescent

staining

of infected cells to

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.59.299.88.250.2]

TABLE 2. JCV multiplication inPOJ-19, POJ-27,and PHFG cells

CellsDays after Meanno.ofnuclei

Cells

Dayioatio

containingcapsid HAunitsb

inoculation atgn-S_{antigena ± SD}

PHFG 7 87 11 93

14 607 ± 125 587

21 715 ± 73 1,696

28 1,045 + 99 2,240

POJ-19 7 649 ± 118 267

14 434 ±42 747

21 333 ± 61 427

28 341 ± 20 427

POJ-27 7 92± 27 120

14 132 ± 16 373

21 158 ± 51 320

28 254± 41 533

aMeannumber(±standarddeviation) ofantigen-containingnucleionhalf

ofa12-mm coverslip. Basedonelevencountsfrom threeexperiments. bTotalyield;meanofthreeexperiments.

detect viral capsid antigens and measuring HA

activity

in lysatesoftheinfected cells. Atweekly intervals coverslips

were removed for infected-cell counts and cells were

har-vested for virus assays (Table 2). Virus from the 28-day

harvest of one experiment was passed through four

addi-tional serial passages ofPOJ-19 and POJ-27 cells in 25-cm2 flasks. The final virus yield was a 10,000-fold increase in

POJ-19and a5,000-foldincrease in POJ-27overthe

original

virusinoculum,demonstratingthatthe cellsproduced com-pleteinfectious virus.

POJcellssupport DNAreplication ofJCVcarryingalethal early-region mutation. The primary reasonfor

deriving

POJ cellswasto obtaina permanentcellline that could be used topropagatewild-type JCV. However, if these cells

consti-tutively

expressed

afunctionalTprotein, theywouldalsobe usefulfor complementingthe growthofJCV mutants

carry-ingalethalearly-region mutation. Toinvestigate this possi-bility, we made useofthe JCV mutantS-1, which failed to

induce detectableTantigen productioninPHFG cells(Table

1). The DpnI assay was used to determine the replicative ability ofS-1 DNAinPHFG and POJ-2cells. As expected, S-i DNA didnot replicate in PHFG cells; however, newly synthesized viralDNAwasdetected inPOJ-2 cells, indicat-ingthatafunctionalTprotein was presentin thetransformed

cells(Fig. 5). Althoughreplicated viral DNAwasdetectedin the POJ-2cells, the amount of DNA was significantly lower than that following transfection with wild-type JCV DNA.

Substituting POJ-19 or POJ-27

celis

for the POJ-2 cells in

these experiments did not raise the levels of replicated S-1 DNA observed atthelatertime points (data not shown).

Integration patterns of JCV DNA in cloned POJ cells. The presence ofS-19 and S-27 DNA in POJ-1 and POJ-2 cells,

respectively, was examined by the approach of Botchan et al. (3) and Ketner and Kelly (17). Results from these

experiments revealed complex integration patterns of JCV DNAin the cellular genomes of these two cell lines. When restriction enzymes were used that did not cleave the parental

DNAs

(e.g.,Bcll andEcoRV), each band resolved onthe blotrepresented at least oneindependent integration event.BothPOJ-1andPOJ-2 cells had viral DNA integrated

intothe cellular DNA at four or more sites (Fig. 6a). DNA

fragments comigrating with the supercoiled and nicked cir-cular forms of the parental plasmids were not detected,

suggesting thatfree

episomal

viral DNA was absent in the cells. At least three ofthe

integrated

sequences in POJ-1 cellsandoneinPOJ-2 cellswereshorter than the

transform-ing

DNA.To

investigate

thenatureoftheinsertions

further,

cellular DNA was

digested

with restriction enzymes that

cleavedthe

parental

DNAsonce

(Fig.

6b).

BstXIand

BamHI

cutwithin the

early

coding region

of

JCV,

whileBstEIIcuts

within the late coding sequences. Scal cleaves the

pBR322

vectoronly. The large number of bands observed on

blots

representing

the BstXI- and

BamHI-digested

samples

sug-gested

that most of the viral inserts retained these two

restriction sites. It is

possible

that several of these inserts contained anintact copy ofthe JCV

early

region

and

might

therefore

specify

functionalT

proteins.

The BstEII and ScaI

digests were less

complex,

suggesting

that several of the

integrated viral copies were

missing

these restriction sites. Onthe basis of these results with

single-cutting

enzymes, it appears that the

integration

of viral DNA in POJ cells

frequentlyinvolved sequencesfromthelatecoding

region

of the virus orfrom the

plasmid

vector.

Digestion

of S-19 and S-27

plasmid

DNAs with PvuII yielded four fragments, one ofwhich contained the entire

JCVearly codingregion (2.5 kilobase [kb] fragment).

Cleav-age of POJ-1 and POJ-2 cellular DNA with this restriction

enzyme gave a strong 2.5-kb band (data not shown),

sup-porting

the

assumption

thatseveralintact

copies

of the entire

early

region

are present in the POJ-1 and POJ-2 cellular genomes.

A second cloned cell line derived independently from POJ-27 cells

(designated

POJ-2c)gaveJCV DNA

integration

profiles

that were identical to those seen in POJ-2

cells,

indicating that both cell lines arosefrom the same

transfor-mationevent(datanotshown). Thisresult may indicatethat

the original transformation was a rare occurrence and that the whole population of uncloned transformed cellswas the progeny of only a few individual transformants. Our diffi-culty in deriving the POJ cell lines would support this suggestion. Alternatively, the precursor cells ofthe cloned

0

0

o

MMMM

a f

3

0

o T

a- a0

7

N 0

I

o I:

a-

a-i4

0 -) IL

o fr

a- a

2

r~~

C0

-~

U-o lZ

a a

46I

FIG. 5. Replication activity of a JCV early-region mutant in POJ-2 andPHFGcells Thereplicative activityofS-iDNA inPOJ-2 andPHFG cellswasinvestigatedasdescribed in thelegendtoFig. 4exceptthatno further reduction was made in the amountof the 21-day samples. Additional bands in thedigest of the7-day POJ-2 extract resulted fromincomplete Dpnldigestion of the DNA. The amountsandidentities of the

Ml

andM2markersare asgivenin the

legend toFig. 3.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.327.546.467.642.2]

-{cl EcoRV

Ml M22M3 i1 2 2'M4 Ml NM2

*BstX BOmH BstEI1 Sco

Ml M2M3 2 2 2 21 Ml

a

formSn

form

8.9kb

--7.1kb Om

b

89kb - o 7.1kb

-5.1 kb

_..1mI

_a

_aa

p

40 _ of _

_1

*

_-U

_

U.

on

U

5.1kb-X _{18kb- t}

Af

4

FIG. 6. Integration ofviral DNA in the cellular genomes of POJ-1 and POJ-2 cells. Cellular DNA (10pg) was digested with various restriction enzymes, electrophoresed on 1% agarose gels, and transferred to nitrocellulose filters (35). JCV DNA minus pBR322 vector sequences wasradioactivelylabeled by nicktranslation (32) and used as a virus-specific hybridization probe. The following DNAs were used assize markers: linearizedpMITC(ABam), theparental plasmid(M1;8.9 kb),pMITC(ABam)cleaved withHindIII(M2; 7.1, 1.8, 0.4, and 0.2 kb), linearized JCVDNA (M3; 5.1 kb), and uncut form I and formIIpMITC(ABam)(M4; 8.9 kb). Theamounts of marker DNAs loaded on the gelrangedfrom5 x 10-6 to 2 x 10-5 pug(1 x 10 ,ug of DNA is equivalent to the amount of viral DNA in 10 ,ug of cellular DNA if 1 copyofthe genome is present in each cell). Thefilms were exposed for 2 days with an intensifying screen. (a) Digestion of POJ-1 (lanes 1) and POJ-2(lanes 2) cellular DNAs withBcIIandEcoRV,which do not cut S-19 and S-27 DNAs. (b)Digestion of POJ-1 (lanes 1) and POJ-2 (lanes 2) cellular DNAs with BstXI,BamHI,BstEII,andScaI, each of which cleaves S-19 and S-27DNAs once.

POJ cell lines may have had a selective growth advantage over other transformants in the culture. Because of this advantage, such cells may have become predominant in the uncloned population prior to cloning or may have had a

higherplating efficiency during the cloning operation.

DISCUSSION

The studies presented here describe our attempts to

es-tablish a permissive cell line for the propagation of the human polyomavirus JCV. Although our approach paral-leled that taken by Gluzman (11) in deriving COS cells, its success wasdifficult to predict because of several problems

inherent to the JCV system. To derive a JCV COS cell

equivalent, three requirements had to be met:

origin-defectivemutantshadtobe constructed,PHFG cells had to be transformed by the mutant DNAs, and the cells had to

retain their permissivity for JCV. Although the JCV ori sequenceshad notbeen defined precisely,weexpected that

thefirststepwouldbe straightforward. Problems associated

with steps two and three were considered more serious

obstacles to the establishment ofa useful cell line. These

problems includedthehighly inefficient transforming activity ofthevirus (10, 15)and theinability of normal PHFG cells,

once passaged a few times in culture, to support JCV

replication (B. L. Padgett and D. L. Walker, unpublished observations).

Basedonits closehomology with the SV40DNA

replica-tionorigin, we assumed that the 25-np dyad symmetry found atnucleotides 5118 to 12 in the JCV genome formed partof

thereplication origin ofJCV(8, 26). A

HindIlI

cleavagesite

isfoundnearthis symmetry, and becauseofthe

infrequent

occurrenceof thisrecognition sequence in the JCV genome

(threetimes),

HindIlI

wasused in the

site-specific

mutagen-esisprocedure. Mutationswereobtainedatall three HindIII sites. Thoseoccurringatnucleotides 4498

(within

thesmall-t coding sequences)and 4914(withinsharedcodingsequences

forlarge and smallt) will prove useful in future studies aimed atinvestigatingtheJCVearly proteins. Recent experiments

indicatethat the JCV Tprotein contributes to the restricted lytic and transforming activities of the virus in vitro (4). The

small-tmutantsconstructed in this study will prove useful in

determining the role ofthe small-t protein in the biological

activity of this virus.Themutationsatnucleotide 4498 affect the termination codon of small-t translation or the donor splice site of small-t transcript processing. Most of these mutant DNAs induce T antigen expression in PHFG and BHK-21 cells; the lattercells also become morphologically transformed.

Inthe present studyourprimary interest was in mutants that had deleted the HindIII site at nucleotide 5112. This

cleavage site occurs within a 19-np dyad symmetry lying adjacenttothe25-npsymmetry presumed to be the center of the JCV origin of DNA replication. Three DNAS (S-15,

S-19, and S-27) mutated at this site were tested in aDpnI

assay and were found to be replication defective. The

deletion inmutantS-27wasconfined to the19-np symmetry,

indicatingthatmutations affectingsequences next to the ori center and between

T-antigen-binding

sites I and II were sufficienttointerfere with JCV DNAreplication.Recentlyit has been shown that insertions and deletions in the

corre-sponding region of the SV40 genome abolish viral DNA

replication by disrupting the spacer function of these se-quences(5, 20).

The two origin-defective JCV mutants with the smallest

deletions (S-19 and S-27) transformed PHFG cells to

yield

thecell lines called POJ-19 and POJ-27. Thesecellssupport JCV DNA

replication

and virus

production

atlevels compa-rable to those observed with the traditional cell system, PHFG cells. It was also noted that the JCVlytic cyclewas faster in the POJ-19 cells than in POJ-27 or PHFG cells.

Although the

primary

cells

rapidly

lose their

ability

to support the

lytic cycle

of JCV

(prior

to theirsenescence at passage8), POJ cells remain

permissive

nearly

until the time

of crisis(passages35to

40).

Similar resultswere

reported by

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.612.100.522.70.241.2]

Major and co-workers with their SVG cell line (21). While SVG cells do support JCV replication, the SV40 T antigen producedin SVG cells apparently fails to interact effectively with the JCV ori sequences. This is surprising, since this

protein does bind to the JCV origin region (7) and recent results demonstrate that plasmids containing JCV ori se-quences replicate in COS cells, presumably through their

interaction with SV40 T antigen (19; K. Lynch, W. F.

Chuke, and R. J. Frisque, unpublished results). Moreover,

studies with hybrid polyomavirus genomes containing SV40 and JCV DNA sequences indicate that SV40 T protein interacts with the JCV regulatory sequences to yield viable

viruses (4). An additionalproblem withthe SVG cell line is

the possibilityofrecombination occurringbetween the

inte-grated SV40genome and the input JCV DNA. It has been shown thatrecombinantviruses doarisewhenSV40 mutants are propagated in COS cells (11, 16, 18, 33); the extensive homology between JCV and SV40 may also favor these recombination events. Our results with JCV-SV40 hybrid viruses constructed in vitro indicate that, should recombi-nants arise inSVG cells, some will be viable and may have aselectivegrowthadvantage over the input JCV. Because of the immunological cross-reactivity of many of the JCV and

SV40 proteins, it might not be possible to identify such

recombinantswithout closeexamination of the viral DNA by

molecularbiological methods.

POJ cells represent the first permissive cell system for

JCV that constitutively produces a wild-type JCV large-T protein. In combination with COS and SVG cells, POJ cells

form ausefulcollection of cell lines forinvestigating

DNA-protein interactions of the closely related viruses JCV and SV40. This is especially relevant now, since recent studies indicatethat the JCV Tproteininteracts lessefficientlythan

its SV40 and BKV counterparts with homologous and

het-erologous polyomavirus regulatory sequences (4).

As with COS cells, the POJ cell lines will be used to propagate viruses carrying lethal early-region mutations.

Althoughthepresentstudies demonstratedtheability of POJ

cells to support DNA replication of the JCV T-antigen

mutant S-1, the replication efficiency of the DNA was

significantly lower than that ofwild-type JCV DNA. Itwill be necessary to test additional early JCV mutants in these

cellstodeterminewhetherthis resultis aconsequence of the

cell system or the particular mutant. The first possibility seemsunlikely; immunoprecipitation andSi nuclease

anal-ysesindicatethatauthentic-sized JCV T antigen is produced and the proper mRNA start sites are utilized in POJ cells

(22). This information, togetherwith the viralDNA integra-tion data presented in this paper, suggests that POJ cells express significant levels of an unaltered T protein, and

thereforethe cell system is probably notresponsible for the

reduced replication of S-1 DNA. It is possible that this mutant produces a nonfunctional T polypeptide that is not detected in transfected cells by immunofluorescentstaining.

Suchapolypeptide might be capable of interfering with viral DNA replication by forming inactive oligomers with wild-type Tprotein monomers.

Southern blot analysis of POJ-1 and POJ-2 cells revealed that integration of viral sequences into the host genomes occurred at a minimum of four to five unique sites. Similar

complex

patterns

have been reported to occur in

JCV-transformed hamster cell lines and tumors (22, 25, 41). In

parallel studies,theSV40 transformantsfrequently exhibited

simpler integrationpatterns. It is possible that JCV

transfor-mation requiresmultipleintegration events; if this is found to

be a prerequisite, it might explain the poor transforming

activity of JCV, since theprobabilityofmultiple integation

eventswould likely be low.

The JCV mutants and POJ cell lines described in this report should allow us to pursue new avenues of study for this poorly understood yet medically important polyoma-virus. We believe that POJ cells will greatly facilitate the study of this virus and that theavailability of these cells will encourageothers to undertake JCV research.

ACKNOWLEDGMENTS

We think Fran Reilly forthe preparation ofthismanuscript. This workwas supported by grants from the American Cancer Society (MV-168), by Public Health Servicegrants from the Na-tional Cancer Institute (CA-38789) and the National Institute of Allergy and Infectious Diseases (AI-11217), and by the Tracy RuhrupMemorial Fund.

LITERATURE CITED

1. Beckman, A. M., K. V. Shah, and B. L.Padgett. 1982. Propa-gation and primary isolationof papovavirus JCinepithelial cells derived fromhumanurine.Infect. Immun.38:774-777. 2. Binetruy, B., G.Rautmann, G. Meneguzzi, R. Breathnach, and

F.Cuzin.1982.Bovinepapilloma virus 1-pBR322 and polyoma-pBR322 recombinants as eukaryotic vectors, p. 87-92. In Y. Gluzman (ed.), Eukaryotic viral vectors. Cold Spring Harbor Laboratory, ColdSpring Harbor,N.Y.

3. Botchan, M., W. Topp, and J. Sambrook. 1976. The arrange-mentof simian virus40 sequences in the DNAof transformed cells. Cell 9:269-287.

4. Chuke,W.-F., D. L. Walker, L. B. Peitzman, and R. J.Frisque. 1986.Construction and characterization ofhybridpolyomavirus genomes.J. Virol. 60:960-971.

5. Deb, S., A. L. DeLucia, C.-P. Baur, A. Koff, and P.Tegtmeyer. 1986. Domain structure of the simian virus 40 core origin of replication. Mol.Cell. Biol.6:1663-1670.

6. DeLucia, A. J., B. A. Lewton, R.Tjian, and P. Tegtmeyer.1983. Topography of simian virus 40 A protein-DNA complexes: arrangementofpentanucleotide interaction sitesattheorigin of replication. J.Virol. 46:143-150.

7. Frisque, R. J. 1983.Regulatorysequences andvirus-cell inter-actions of JC virus. Prog.Clin. Biol.Res. 105:41-59.

8. Frisque, R. J. 1983. Nucleotide sequenceof theregion encom-passing the JC virus origin of DNA replication. J. Virol. 46:170-176.

9. Frisque, R.J., G. L. Bream, and M. T. Cannella. 1984. Human polyomavirus JC virusgenome. J.Virol. 51:458-469.

10. Frisque, R.J., D. B. Rifkin, and D. L. Walker. 1980. Transfor-mation of primary hamster brain cells with JC virus and its DNA.J. Virol. 35:265-269.

11. Gluzman, Y. 1981. SV40transformed cells support the replica-tionof early SV40mutants. Cell23:175-182.

12. Gluzman, Y., R. J. Frisque, and J. Sambrook. 1980. Origin-defectivemutants ofSV40. Cold SpringHarbor Symp.Quant. Biol. 44:293-299.

13. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mousecell cultures.J. Mol. Biol. 26:365-369. 14. Holmes, D. S., and M.Quigley. 1981. A rapid boiling method for

the preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

15. Howley, P. M., F.Rentier-Delrue, C. A. Heilman, M.-F. Law, K. Chowdhury, M. A. Israel, and K. K. Takemoto. 1980. Cloned human polyomavirus JC DNA can transform human amnion cells.J. Virol.36:878-882.

16. Jasin, M., J. deVilliers,F. Weber,and W.Schaffner.1985. High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes. Cell 43:695-703.

17. Ketner, G., and T. Kelly, Jr. 1976. Integrated simian virus 40 sequences intransformedcell DNA: analysis using restriction endonucleases. Proc. Natl. Acad. Sci. USA 73:1101-1106. 18. Lanford, R. E., and J. S. Butel.1984. Construction and

on November 10, 2019 by guest

http://jvi.asm.org/

terizationofanSV40mutant defectiveinnucleartransport of T antigen. Cell 37:801-813.

19. Li, J. J.,and T. J. Kelly. 1985. Simianvirus 40 DNA replication in vitro: specificity of initiationand evidence forbidirectional replication. Mol. Cell. Biol. 5:1238-1246.

20. Li, J. J., K. W. C.Peden, R. A. F. Dixon, and T. Kelly. 1986. Functional organization of the simian virus 40 origin of DNA replication. Mol. Cell. Biol. 6:1117-1128.

21. Major, E. O., A. E. Miller, P. Mourrain, R. G. Traub, E. deWidt, and J. Sever. 1985. Establishment ofaline ofhuman fetal glial cellsthat supportsJC virus multiplication.Proc. Natl. Acad.Sci. USA82:1257-1261.

22. Mandl, C., and R. J. Frisque. 1986. Characterization ofcells transformed by thehuman polyomavirus JCV. J. Gen. Virol. 67:1733-1739.

23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: alaboratory manual, p. 86-96, 249-253. Cold Spring HarborLaboratory, ColdSpringHarbor, N.Y.

24. Maxam, A. M., and W. Gilbert. 1980. Sequencingend-labeled DNAwith base-specific chemical cleavages.MethodsEnzymol. 65:499-560.

25. Miller, N. R., W. London, B. L. Padgett, D. L. Walker, and W. C. Wallen. 1983. Thedetection ofJC viral genome in owl monkeytumors. Prog.Clin. Biol. Res.105:271-288.

26. Miyamura, T., H. Jikuya, E. Soeda, and K. Yoshiike. 1983. Genomic structure of human polyoma virus JC: nucleotide sequence of theregioncontaining replication originand small-T-antigengene. J. Virol. 45:73-79.

27. Miyamura, T., K. Yoshiike, and K. K. Takemoto. 1980. Char-acterization of JC papovavirus adapted to growth in human embryonic kidneycells. J. Virol.35:498-504.

28. Padgett, B. L., C. M.Rogers, and D. L. Walker. 1977.JC virus, ahuman polyomavirus associated with progressive multifocal leukoencephalopathy: additional biological characteristics and antigenic relationships. Infect. Immun. 15:656-662.

29. Padgett, B.L., and D. L. Walker.1973.Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J. Infect. Dis. 127:467-470.

30. Padgett, B. L., D. L. Walker, G. M. ZuRhein, R. J.Eckroade, and B. H. Dessel. 1971. Cultivation ofpapova-like virusfrom human brain withprogressive multifocalleucoencephalopathy. Lanceti:1257-1260.

31. Peden,K. W. C.,J.M.Pipas,S. Pearson-White, and D. Nathans. 1980. Isolation of mutantsofananimal virus inbacteria.Science 209:1392-1396.

32. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labelingdeoxyribonucleic acidtohigh specific activityin vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251.

33. Shaul, Y., 0. Laub, M. D. Walker, and W. J. Rutter. 1985. Homologous recombination between a defective virus and a chromosomalsequence in mammalian cells. Proc. Natl.Acad. Sci.USA 82:3781-3784.

34. Sompayrac,L.M., and K. J. Danna.1981.Efficient infection of monkey cells with DNA of simian virus 40. Proc. Natl. Acad. Sci. USA 78:7575-7578.

35. Southern,E. M. 1975. Detection ofspecific sequencesamong DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

36. Takemoto, K. K., P. M. Howley, and T. Miyamura. 1979. JC humanpapovavirus replicationin human amnion cells. J. Virol. 30:384-389.

37. Tjian, R.1978.Protein-DNAinteractionsatthe originof simian virus40DNA replication. Cold SpringHarbor Symp. Quant. Biol. 43:655-662.

38. Tooze, J. (ed.).1981. Molecularbiology oftumorviruses,part2, 2nd ed.: DNA tumorviruses. ColdSpring Harbor Laboratory, ColdSpringHarbor, N.Y.

39. Tyndall, C., G. La Mantia, C. M. Thacker, J. Favaloro, and R. Kamen. 1981. Aregion of the polyoma virus genomebetween the replication origin and late protein coding sequences is requiredin cisfor both earlygene expression and viralDNA replication. Nucleic Acids Res. 9:6231-6250.

39a.Walker, D. L., and R. J. Frisque. 1986. Biology and molecular biology of JC virus, p. 327-377. In N.P. Salzman (ed.), The Papovaviridae: the polyomaviruses. Plenum PublishingCorp., NewYork.

40. Wigler, M., A. Pellicer, S. Silverstein, and R. Axel. 1978. Biochemical transfer ofsingle-copy eucaryoticgenesusing total cellularDNAasdonor. Cell 14:725-731.

41. Wold, W. S.M., M. Green,J.K. Mackey,J. D.Martin,B.L. Padgett,and D. L. Walker. 1980. Integration pattern of human JC virussequencesintwoclones ofacell line established from a JC virus-induced hamster brain tumor. J. Virol. 33:1225-1228.

on November 10, 2019 by guest

http://jvi.asm.org/