Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

JOURNALOFVIROLOGY,JUlY1970,p.69-77

Copyright ©1970 American Society for Microbiology

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

Identification of the Simian Virus 40 Which

Repli-cates

When

Simian Virus 40-Transformed

Human

Cells Are Fused with Simian

Virus

40-Transformed Mouse Cells

or

Superinfected with Simian

Virus

40

Deoxyribonucleic

Acid

SAUL KIT, T. KURIMURA, McKAY BROWN, AND D. R. DUBBS Division of Biochemical Virology, Baylor College of Medicine, Houston, Texas 77025

Received for publication 5 March 1970

Simian virus 40 (SV40) was rescued fromheterokaryons of transformed mouse and transformed human cells. To determine whether the rescuedSV40wasprogeny of theSV40genomeresident in the transformedmousecells,the transformed human

cells,orboth, rescueexperimentswereperformedwith mouselinestransformed by plaque morphology mutants ofSV40. Thetransformedmouselines thatwereused

yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used didnot

release SV40spontaneouslyorafter fusion with CV-1cells.From each mouse-human

fusion mixture, onlytheSV40resident in the transformedmousecellswasrecovered.

Fusion mixtures ofCV-1 andtransformedmousecellsyieldedmuchmoreSV40 than

those from transformed human and transformed mouse cells. The rate ofSV40

formation was also greater from monkey-mouse than from human-mouse

hetero-karyons. Deoxyribonucleicacid(DNA) from SV40strains whichformfuzzy, large-clear, or small-clear plaques onCV-1 cells was also usedto infectmonkey (CV-1

and Vero), normal human, and transformed human cell lines. The rate ofvirion formation and the final SV40 yields were much higher from monkey than from

normalortransformedhuman cells.Onlyvirus with theplaquetypeof theinfecting

DNAwasfound inextractsfrom the infected cells. Two uncloned sublines of

trans-formed human cells [W18 Va2(P363) and W138 Val3A] releasedSV40

spontane-ously.Virusyieldswere notappreciably enhancedbyfusion with CV-1 cells. How-ever, clonal lines ofW18 Va2(P363) did notrelease SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of W138 Val3A cells did continue to shed SV40 spontaneously.

JensenandKoprowski (12)wereabletorescue simian virus 40 (SV40) from fusion mixtures of

SV40-transformed mouse (SV40-3T3-101) and SV40-transformed human or monkey cells but notfrom control cultures of fused SV40-3T3-101

cells alone. It was not established, however, whether the progeny SV40 was derived from the viral genome in the transformed mouse cell, the transformed humancell,orbothcells,orwhether therescued SV40was arecombinant (20). Swetly

et al. (25) showed that some normal and

SV40-69

transformed human lines relatively resistant to

infection by intact SV40 virions do produce

in-fectious SV40 wheninfected with SV40 deoxyribo-nucleic acid (DNA). Again, it was not shown whether virus progeny were derived from the

superinfectingSV40DNA, from the SV40 genome resident in the transformed human cells, orfrom both.

In the presentstudy, theorigin of the rescued SV40has been ascertainedby fusingtransformed human celllines withmousecellstransformedby

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plaque morphology mutants of SV40. The trans-formed mouse cell lines which were used yield fuzzy, small-clear, and large-clear plaque strains ofSV40 when fused with CV-1 cells (7).Theyields and plaque morphology of virus rescued from fusion ofmouseand human cells werecompared with those obtained from fusion of mouse and

monkey cells. In addition, replicationofSV40 was studiedin human transformed cells superinfected with SV40 DNA isolated from the plaque

mor-phology mutants.

MATERIALS AND METHODS

Cell lines. CV-1 (11) and Vero (29), established lines of African greenmonkeykidney cells, were sub-cultured at weekly intervals as previously described (16). The mKS-BUIOO cells are SV40-transformed mouse kidney cells which are deficient in thymidine

kinase activity (8). The mKS-U13 and mKS-U46 linesaremousekidneylinestransformed by ultraviolet

(UV)-irradiated SV40 clone 307L (6). Cell-free

ex-tractsofthetransformedmouse kidneylines didnot

contain detectableinfectiousSV40,butviruscould be rescued from eachofthetransformedmouse linesby fusingthemwithsusceptibleCV-1cellsin the presence

of UV-irradiated Sendai virus (UV-Sendai). Virus rescued from mKS-BUIOO and mKS-U13 cells pro-duces large-clear plaques on CV-1 monolayers. The SV40 rescued from mKS-U46 cellproducessmall-clear plaques. The 3T3(U4) cells are a 3T3 mouse line transformed by SV40 rescued from mKS-U4 cells. After fusion with CV-1 cells, only fuzzy type

infec-tious centers were formed. Another cell line, 3T3 (4-88)J-3,wasisolated from cultures transformed with

a mriixture ofSV40(mKS-U4) and SV40(mKS-U88).

Theclonalsubline, 3T3(4-88)J-3, gaveall small-clear

plaques when fused with CV-1 cells (7).Transformed

mousecell lines werepropagated as described earlier and subcultured twice weekly (8). HeLa(BU25), a

sublinederivedfrom HeLaS3,isdeficient inthymidine

kinase activity (15). Cells were grown in Eagle's

minimumessentialmedium(MEM;AutoPOW,Flow

Laboratories, Rockville, Md.) supplemented with 10% calfserumand subcultured atweekly intervals.

Primarycultures of humanembryonickidney(HEK)

were obtained from HEM Research, Inc. Rockville, Md., andweremaintainedas confluent cultureswith Eagle'sMEM plus 10% fetalcalfserum.

Fourlines ofSV40-transformed human cells were

employed in this study: W98 VaD, W98 VaH, W18 Va2, and W138 Va13A. The W98 Va line, derived from human skin, wastransformed by SV40 in 1963 (10). The sublines W98 VaD and W98 VaH of this culturewereisolatedfromapoolof cells, frozen after cultures becametransformedbutbefore they reached

thecrisisstate(21). W18Va2isanSV40-transformed culture derived from human buccal mucosa (21). Ampoules of the sublines W98 VaD (passage 124), W98VaH(passage 117), andW18Va2(passages 160 and 363), which werefrozen in 1966, were obtained from David Porter, UCLA School ofMedicine, Los

Angeles, Calif. The WI38 Val3A, a line of SV40-transformed humanembryonic lungcells(10, 12),was

obtained from Paul Kruse, Noble Foundation, Ardmore,Okla. Cultures of transformed human cells were grown in Eagle's MEM with 10% calf serum andsubcultured atweekly intervals.

W98 VaD,W138 Val3A, and W18 Va2 cells were cloned without a feeder layer (22) in R5a medium (16) containing 1% SV40 antiserum and 10 or 20% calf serum in 60-mmpetri dishes. Clones were picked from plates containing less than five colonies.

SV40-transformed cell lines used in this studywere

all T antigen-positive as determined by immuno-fluorescencetests.

Virus strains. SV40(mKS-U4), SV40(mKS-U46), SV40(mKS-U88), andSV40(mKS-U94) wererescued from mouse kidney cells transformed by UV-irradi-ated SV40. SV40(mKS-U4) produces fuzzy plaques

onCV-1 monolayers. Theother threestrains produce small-clear plaques. Parental SV40 clone 307L pro-duces large-clear plaques. Parental SV40 and SV40 strains rescued from transformed cell lines were

as-sayed by plaque titration on monolayer cultures of CV-1cells (16). Sendai virus was grown in the allantoic

cavityof 10- to 11-day-old embryonated eggs (6). Preparation and assay of infectious SV40 DNA.

Infectious SV40 DNA was extractedfromculturesof

CV-1 cells infected with parental SV40 clone 307L

orwith SV40strainsrescuedfromtransformedmouse

kidney cells, by using the p-aminosalicylate-phenol

method (19). SV40 DNA was separatedfrom cellular DNA by nitrocellulose chromatography (19). The infectivity of SV40 DNA was determined by plaque titration on monolayer culturesofCV-1 cells (17).

Testingcell-free extracts for SV40.Cell-freeextracts were preparedfrom5-to7-day-old transformed and normal monolayer cultures. The cells (107 to 3 X 10)

wereremoved from the glass andsuspended in2mlof culture medium. The cells weredisruptedbyfreezing

and thawing and by sonic oscillation at 10 kc for 3 minat4C.Sampleswereassayedfor SV40 on CV-1

monolayers.

Rescueof SV40 from transformed celllines.

Trans-formedhuman or mousecells (5 X 106) weremixed with CV-1 cells (107) in the presenceofUV-Sendaias

previously described (6). Samples of UV-Sendai-treated cell mixtures were planted in 60-mm plastic

dishes with 106freshlytrypsinizedCV-1cells in5mlof growth medium. Afterincubation overnighttopermit

thecells to adhere to the surface, the liquidmedium

was replaced with an agar overlay. A second agar

overlaycontainingneutralredwasadded8 to 10days

afterfusion. Infectiouscenters werecountedat 14 to 25 days (frequency of induction test; see Fig. 1 of reference6).

Alternatively, 2-ml samples of the UV-Sendai-treated mixtures, equivalent to 4 X 106 CV-1 and 2 X 106 transformed cells, were planted in 8-oz

pre-scription bottles (ca. 240 ml). Eighteen milliliters of growth medium was added, and the cells were in-cubated at 37 C for 7 days. The cells were scraped fromtheglass, centrifuged, and resuspended in2ml of

theoriginalgrowth medium. The cellsweredisrupted bysonicoscillationfor 3minat10 kc inaRaytheon Sonic Oscillator. SampleswerethentitratedonCV-1 indicator cells (see Fig. 1 of reference 6). In some

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IDENTIFICATION OF SIMIAN VIRUS 40

experiments, transformed mouse cells (5 X 106) were fused with an equal number oftransformed human

cells and plantedin prescription bottles as described above.

Infectious center assay. To determine the capacity of transformed human cells to form infectious centers, 0.1-ml cell suspensions containing 106, 105, or 104 transformed cells were plated on 5-day-old CV-1

monolayers in plastic petri dishes. Agar medium (0.2ml) was added to fix theplated cells to the mono-layers. Thenan overlay of 5 ml of agar medium was

added. A second agaroverlaycontainingneutral red was added 8 days later, and infectious centers were

scoredfrom day12today23 (8,16).

Replication of SV40 in cell Unes infected with SV40 DNA. Confluent monolayer cultures containing 1.5 to 3.1 X 106 cells in 2-oz prescription bottles (ca.

60 ml) or 10 X 106cells in 8-oz prescription bottles (ca. 240 ml) were used. Fresh growth medium was

added to thecultures 1 day prior to theinfectionwith SV40 DNA. Forinfection, themediumwasremoved,

and the monolayers were washed with phosphate-buffered saline lacking Mg2+ or Ca2+ (PBS-). Then 0.25 ml of diethylaminoethyl (DEAE)-dextran (1

mg/mlinPBS-) was added. After incubating for 30

minatroomtemperature withintermittent agitation,

the DEAE-dextranwasremoved andinfectiousSV40 DNAwasaddedattheinput multiplicitiesindicated in

Fig.I to 3.Aftera30-minadsorptionperiod,thecells werewashed with PBS-.Five- and 20-ml amounts of growth mediumwere then added to the 2- and 8-oz

bottles (ca.60and 240ml),respectively.Thecultures wereincubatedforvarious periods oftimeat37C and then stored at -20 C. Afterthawing, the cellswere

removed from theglassanddisrupted by sonic oscilla-tion.Samplesweretitratedforviralinfectivityon CV-1

indicatorcells.

RESULTS

Detectionof SV40incell-freeextractsof

trans-formed humancells.Atthestartofthisstudy,

im-munofluorescence tests were performed which

verifiedthat thetransformedhumancellswereall

positive for the SV40Tantigen.Routinetestsof

cell-free extracts were also carried out to deter-mine whether the transformed human lineswere

free from infectious virus. Extracts fromsublines

W98 VaD and W98 VaH did not exhibit

de-tectable virus. Contrary to expectation (12, 20, 21), however, it was observed that a subline of W18 Va2 cells was shedding virus. Relatively

large amounts of SV40 (more than 104PFU per 107cells) wereconsistently detected in extracts of W18 Va2cells frozen atpassage 363. The SV40 recovered from W18 Va2(P363) cells formed

ragged plaquesonCV-1 cells and thusdiffered in

plaque morphology from SV40 strains in use in

our laboratory. However, it resembled inplaque

morphology the SV40 strain LP-4, isolated from Hilleman strain Rh 911 at the Wistar

Insti-tute (R. Carp, personal communication). Small

107

106

Icv-1

Vero

(I)I

-J

w

0

zsOD 104 xil W98VaD

L W98VaH

0-U)

103/

WI38Val3A

"__EmKS (BUIOO)

10

0 10 20 30405060 70 8090 100

HOURS AFTER INFECTIONWITHSV40(Clone307L)DNA

FIG. 1. Replication of SV40 after infection of

monkey, human, and mouse cell lines with SV40 (clone 307L) DNA.Theinputmultiplicities (PFU/cell)

of SV40 DNA were as follows: CV-1, 1.2; Vero,

0.7; W98 VaD, 4.6; W98 VaH, 1.3; WI38 Va13A,

1.2; and mKS-BUJOO, 1.7.

amounts ofSV40 wereoften, but notalways,

re-covered fromextracts of WI38 Va13A cells.For

example, approximately 200 PFU of SV40 per

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duced large-clear plaques on CV-1 cells,

resem-bling SV40clone 307L.

Approximately onein 102 W18Va2(P363) and one in 105 to 106 W138 Val3A cells initiated plaque formation when plated on CV-1

mono-layers. Treatment of mixtures of CV-1 and W18

Cl) J

(D

0

CL.

0

U)

10l

10

W 18 Va 2-9

U)

-J

-J

-i

w

o

0

NL.

0-0

Uf)

0 1020 3040 50 60 708090 100

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HOURS AFTER INFECTIONWITHSV40(Clone307L)DNA

FIG. 2.Replication of SV40 after infectionofmonkey

andhuman cell lines with SV40 (clone 307L) DNA. The inputmultiplicities (PFU/cell) of SV40 DNA were asfollows: CV-1, 1.7; HeLa(BU25), 1.6; W18 Va2

clones 1, 2, and9, 2.6.

107 cells were found in cell-free extracts ofcells testedafteronlythreepassagesinourlaboratory. Even after cloning, 8 of 12 clonal lines of W138 Val3A still continuedto shedSV40sporadically.

The SV40 isolated from W138 Val3A cells

pro-10

cv-HEK * W98VaD

pW98 VaH

- _6,

-A I~~~~

0 102030405060708090 100

HOURSAFTER INFECTION WITH SV40 (mKS-U4)DNA FIG.3.Replication ofSV40after infection ofmonkey andhuman celllineswith SV40 (mKS-U4) DNA. The

input multiplicities (PFU/cell) ofSV40 DNA were as follows: CV-J, 0.2; humanembryonic kidney (HEK), 0.5; W98 VaD,0.4;and W98VaH,0.2.

*

cv-102F

/

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IDENTIFICATION OF SIMIAN VIRUS 40 Va2(P363) cells, orCV-1 and W138 Va13A cells,

with UV-Sendai prior to plating with freshly trypsinized CV-1 cells (frequency of induction

test) didnot significantly increase the number of cellsproducing SV40 infectiouscenters.

Eleven clones were isolated from W18

Va2(P363) cells. Viruswasnotdetectedincell-free

extracts from any of these lines. Moreover, cell-free extracts of another subline of W18 Va2, frozenatpassage 160, also failedtoyield SV40 in

numeroustests.

Afteritwas discovered thattwo of the human transformed cell lines were spontaneously

shed-ding SV40, rigorous testing ofothertransformed

linesinuse inourlaboratory wasinstituted.

Ex-tracts oftransformed cell lines, after assay on

CV-1 monolayers, wereusedforthreesuccessive blindpassagesinCV-1 monolayers. Extracts from each blindpassage weretestedfor virus onCV-1 indicator cells. Virus was not detected by this method inextractsfrom TSV-5(hamster), mKS-A (mouse), W98 VaD,orW98VaHcells. Viruswas

detected inextracts from W138 Va13A, and the virus yield increased by a factor of 103 during

threeblindpassagesinCV-1 cells.

Attempts torecoverSV40 after fusion of

trans-formedhumanlineswith CV-1cells.To determine

whetherSV40 could be rescued from transformed human cell lines thatdidnotshed virus

spontane-ously, mixtures of transformed human and

sus-ceptible CV-1 cellsweretreatedwith UV-Sendai (6). The fusion mixtures were grown in 8-oz

prescription bottles (ca. 240 ml) for 7 days, harvested, and sonic-treated. Extractsweretested onCV-1 indicator cells forinfectious SV40. The

frequency ofinductiontest(6)wasalsoperformed

to determine whether heterokaryons of

trans-formed and susceptible cells formed infectious

centers onCV-1 monolayers. SV40 was not

re-coveredinanyof theexperimentswith W98 VaD orW98 VaH, norfrom any of 10clonal lines of

W18 Va2. FiveoftheW18 Va2 clonal lineswere

also self-fusedby UV-Sendaitreatment and then cultivated for 7 days. No infectious virus was

recovered.

Rescue of SV40 after fusion of transformed humancellsandmousekidneycellstransformedby plaquemorphologymutantsofSV40. Ithas previ-ously been shown that fusion of transformed

mouse and human cells yields SV40 (12). How-ever, it was not possible to discernwhether the rescuedSV40wastheprogenyof theresidentviral genome of the mousecell, of the human cell, of

bothcells,or arecombinant.Inthepresentstudy, transformed human cells were fused with mouse

kidney lines transformed by plaque morphology

mutants ofSV40. Five transformed mouse lines wereused: (i) 3T3(U4)whichproduces only large

fuzzy plaqueSV40;

(ii) 3T3(4-88)J-3

which

yields

only the small-clear plaquetype; (iii) mKS-U46 which

yields

the small-clear plaque type;

(iv)

mKS-U13 and (v) mKS-BU100 which both yield

large-clearwild-typeSV40 afterfusionwith CV-1

cells (6,7).

The results of these experiments (Tables 1-4) indicate three significant facts. (i) The SV40 re-covered from fusion mixtures of transformed mouse cells and transformed human cells was always of the same plaque morphology as the

SV40 resident in thetransformed mousecell (see

footnotes of Tables 1, 2, and 4). Thus, in all experimentsshowninTable 1, inwhich 3T3(U4) was used in the fusion, only SV40 ofthe fuzzy

plaque morphology was rescued. In contrast,

when

3T3(4-88)J-3

wasfused with CV-1orhuman transformed cells

(Table 2),

only SV40 of the

small-clear plaque morphology was rescued.

When mKS-U13 or mKS-BU100 was used (Table 4), wild-type SV40 (large-clear plaque

type) wasrecovered. (ii) Virus yieldswere much lowerwhen transformed mouse cells were fused with transformed human cells than when fused with CV-1 cells. (iii) Inmanyinstances, fusionof

transformedmousecells with transformedhuman

cells

failedtoyield SV40 (Tables 2 and 3). Kinetics of SV40 formation after fusion of trans-formed mouse cells with either CV-1 or human transformed cells. Thepreceding experiments dem-onstratethat virusyieldsareconsiderablygreater 7 days after incubation of fusion mixtures of

transformed mouseand CV-1 than after cultiva-tionoffusion mixturesoftransformed mouse and

transformed humanlines. UV-Sendai-treated mix-turesofCV-1 andtransformedcellscontainCV-1 cellswhich havenotundergone fusion.Thus, in 7

days theunfused CV-1 cells may undergo

sec-ondary infection by SV40 released from

hetero-karyons. Hence, the

yield

ofSV40 is amplified.

However, anamplificationof SV40yieldprobably does not occur when fusion mixtures of trans-formed mouse and transformed human cells are

employed, because the transformed human cells are resistant to superinfection by intact SV40

virions. Thus, it was

important

to determine whether both therateandthe total

yield

ofSV40 weregreater from CV-1 and mouseheterokaryons

than from transformed human andmouse

hetero-karyons. We used mKS-U13 and mKS-BU100, which release large-clear plaque types of SV40h afterfusion with CV-1 cells, to answer this

ques-tion.Fusion mixtures oftransformed mouse

cells

and eitherCV-1 or transformed human cells were incubated for various times. Sonic extracts were thenpreparedandassayed onCV-1 indicator cells (Table 4). SV40 was detected in fusion mixtures 73

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TABLE 1. Rescueoffuzzy plaque type SV40 after fusion of3T3(U4) cells withCV-J cells orwith transformed human cellsa

SV40yield (PFU/culture) after fusion of 3T3(U4) cells with

CV-1 W98VaD W98 VaH W18 Va2-1 W18 Va2-2 W18 Va2-4 W18Va2-6

WP

1V62 a 8.8 X 103 0.5 X 10' 0.5 X 10

b 1.1 X 10' 0.5 X 10' 0.5 X 10

c 1.3 X 104 0.5 X 101 0.8 X 10 d 1.4 X 10' 5.0 X 101

e 1.2 X 104 2.4 X 10b f 7.2 X 104 2.3 X102b

g 1.2 X 106 0.3 X 101 0. 7 X 101 3.0 X 101

h 2.4 X 105 0.3 X 10k

1.5 X 106 6.0 X 10'

aHeterokaryonswereincubated for 7 daysin prescription bottles (8 oz, ca. 240 ml). Extracts were

titrated on CV-1 indicator cells. All plaques formed on CV-1 cells byrescued viruswerefuzzy.

bW98 VaDclone 1A was used.

TABLE 2. Rescue of small-clear plaque type SV40

afterfusion of3T3(4-88) J-3 cells with CV-1 cellsorwith transformed humancellsa

SV40yield (PFU/culture)after fusion of 3T3(4-88)J-3 cells with Expt

CV-1

W98

VaD

W98VaH

(P1V)2

a 1.5 X 104 5.1 X 102

b 1.1 X 104 0 0.1 X 10'

c 1.2X 104 0 0

d 8.0 X 102 3.0 X 101

e 1.1 X 103 Ob

f 1.4 X 104 Ob

g 3.0 X 10' 3.0 X 101

aHeterokaryons were incubated for 7 days in

prescription bottles (8 oz, ca. 240 ml). Extracts were titrated on CV-1 indicator cells. Plaques

formed on CV-1 indicator cells were small and

clear.

bW98 VaDclone IA wasused.

ofmKS-U13andCV-1 cells

by

24 hrafter fusion.

Greater SV40 yields were recovered after 41 or 48hr. SV40was notdetected41 hr afterfusion in

mixturesofmKS-BU100 and W98 VaD-lA cells,

but smallamountsofvirusweredetectedat48 hr in fusion mixtures of mKS-U13 and W98 VaH. After 72 hr, SV40 was found in all fusion

mix-tures, but virusyieldswere

considerably

higherin mixtures containing CV-1 cells thanin mixtures

containing transformed human cells. Virus re-covered from fusion mixtures ofmKS-BU100 or

mKS-U13 with transformed human cells

pro-,duced

large-clear

plaques

onCV-1indicatorcells.

Superinfection

of transformed cell lines with

SV40DNA.

Replication

ofSV40didnotoccurin

TABLE 3. Effects offusion of SV40-transformed mouse kidney cells with CV-1 or with

transformed human cells on the rescue of SV40a

SV40yield (PFU/culture)after fusion with SV40-transformed mouse

line

CV-1 W98

|W98

C1 VaD VaH

mKS-U46 (sc)b 7.6 X 104 Oc mKS-U13 (lc) 8.9 X 105 Oc

mKS-U13 (lc) 1.0 X 10' 0 0

mKS-U13 (lc) 8.2 X 104 0 0

mKS-U13 (lc) 4.6 X 103 0 0

aHeterokaryons were cultivated for 7 days in prescription bottles (8 oz, ca.240 ml), harvested,

and extracts were assayed on CV-1 indicator cells.

bmKS-U46 and mKS-U13 yield small-clear (sc) andlarge-clear (lc) plaquetypeSV40,

respec-tively, after fusion with CV-1 cells.

cW98 VaD cloneIAwasused.

W98 VaD or W98 VaHcells aftersuperinfection withSV40 virions(unpublisheddata),in confirma-tion offindings bySwetlyet al. (25). Therefore,

SV40replicationwas studiedintransformed

hu-man and mouse lines superinfected with SV40 DNA. In each case, the plaque morphology of progenySV40,the yield of SV40, and thekinetics

of SV40replicationwere compared withnormal monkeyorhumanlinesinfected with SV40 DNA. Infectious DNA fromfive SV40 strainswasused.

InFig. 1 and2,DNA fromparental SV40 clone 307L (large plaque type) was used. In Fig. 3, DNA from SV40(mKS-U4; fuzzy plaque type)

was used. In other experiments, DNA from

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IDENTIFICATION OF SIMIAN VIRUS 40

TABLE 4. Recovery oflarge-clear plaque type SV40 at various times afterfusion of U13 or mKS-BUI0O with either CV-1 ortransformedhuman cells

r_ SV40yield(PFU/culture)b after

Tnoe fusingtransformedmousecells with

Transformed ;

mouseline C - - ______V

*~. cx--i W98

H _1 VaD-1Ac W98VaH

hr

I mKS-BUI0O 41 1.5 X104 0 50 1.7 X105 0 72 >2.0 X105 1.5 X101

2 mKS-U13 24 0.5 X101 0

48 4.4 X-103 2.0-X 101 72 2.5 X 104 2.2 X 103 aSV40 recovered in both experimentsfrom CV-1 orfrom transformed human cellswas of the large-clear plaque mor-phology.

bCulturesinexperiment1 wereplantedat3 X 106cellsin prescription bottles (4 oz, ca. 120 ml) containing 10 ml of medium. Cultures in experiment 2 wereplanted at 3 X 106 cells in prescription bottles (8 oz, ca.240ml) containing 20 ml of medium.

cW98VaD-lA isaclonal lineof W98 VaD.

SV40(mKS-U46), SV40(mKS-U88), and

SV40(mKS-U94; small-clear-plaque types) was

used.

Theexperiments inFig. 1 to3indicate that, in

CV-1 cells infected with SV40 DNA, virus

in-creased rapidly 24 to 72 hr postinfection (PI), reaching titers of5 X 106to107PFU per106 cells. Thekinetics ofSV40

replication

inVerocellswere

similar, but

slightly

lower

yields

were obtained.

SV40 replication did not occur in mKS-BU100 cellssuperinfected withSV40 DNA. (It should be notedthattheinfectivity observed2hr PI of cells

with SV40 DNA

probably

represents residual DNA-DEAE-dextran inthesonic extractsofthe cells.)

When transformed human lines were superin-fected with SV40 DNA, the eclipse

period

was

longer, and thevirusyields obtained 48, 72, or96 hr after infection were

considerably

lower than withCV-1 orVero cells.

Replication

ofSV40 in

cultures of human

embryonic

kidney or

HeLa(BU25)

infected with SV40DNA was

simi-lartothatin cultures of transformed human cells

superinfectedwithSV40DNA.

The plaque morphology of progeny SV40

re-covered from transformed human cells

superin-fected with SV40 DNA was also studied. Only

the large-clear plaque type virus was recovered from W98 VaD, W98 VaH, W18 Va2-1, W18 Va2-2, and W18 Va2-9 superinfected with DNA

from SV40 clone 307L (large-clear plaquetype).

This is ofparticularinterestsince theparentalline from which the clonal lines W18 Va2-1, W18

Va2-2,and W18 Va2-9 wereisolated (W18 Va2) spontaneously sheds SV40 with a ragged plaque morphology. When W98 VaD or W98 VaH cells were superinfected with DNA from SV40(mKS-U4), which forms fuzzy plaques on CV-1 cells, only SV40 with the fuzzy plaque morphology was produced. In additional experiments, the DNA from SV40(mKS-U46), SV40(mKS-U88), and SV40(mKS-U94) was used to superinfect W98 VaD cells. These three virus strains form small-clear plaques on CV-1 cells (6). Extracts of W98 VaD cells superinfected with SV40 DNA from these strains formed only small-clear plaques on CV-1 cells.

DISCUSSION

In confirmation ofthe experiments of Jensen and Koprowski (12), we found that SV40 was produced by fusion mixtures of transformed mouse and transformed human cells. However,

onlytheSV40residentin the transformed mouse cells was recovered. Either the SV40 genome resident in the transformed human cells was not activated, or defective particles were produced which were not detected in the plaque assay on

CV-1 cells.Similarly,inthesuperinfection

experi-ments, only the replication ofthe superinfecting DNA wasdetected. The fate of the SV40 genome

resident in the transformed human cells is un-resolved;itmayhavebeenreleased from integra-tionincells superinfected

by

SV40 DNA. Either replicationwasminimal, however,ortheparticles produced were noninfectious for CV-1 cells.

Thepreceding experimentssuggestthat factors

essential for SV40 replication are not present in

mousecells andmaybesuppliednotonlybyCV-1 but also bysome normal or transformed human

cells. The experiments in which transformed

hu-mancellswereinfected with DNAsignify thatthe

postulated replication factors mayfunction with nonintegratedSV40DNA.Experiments bySwetly

et al. (25) also indicate that theessential factors

in monkey cells may act upon nonintegrated

SV40 DNA. Swetly et al.

(25)

infected mouse

cells with SV40 DNA and fused the mousecells 24 hr laterwithmonkey cells. Replicationof the SV40 DNA occurred in the monkey-mouse

heterokaryonsbutnotinunfusedmousecells

in-fectedwith SV40 DNA alone.

Of the transformed human lines studied, the W98 VaD, W98 VaH, W18 Va2(P160), and the clonal lines of W18 Va2(P363) did not produce

infectious SV40spontaneouslyorafterfusionwith CV-1 cells.Since the factors essential for

replica-tion ofnonintegratedSV40 DNA were

probably

available, this also suggests that the SV40 ge-nomesresident in these transformed human cells 75

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[image:7.487.39.230.96.225.2]

were defective (6, 14) or that they were not re-leased fromintegration.

A small number of cells in the cultures of parental W18 Va2(P363), parental W138 VaI3A, and eight clonal lines of W138 Val3A spontane-ously produced virus. Studies are in progress to

determine whether secondary and tertiary clones ofW138 Val3A cellswillcontinue to release virus spontaneously (1, 9). In the case of the Wi8 Va2(P363) cells, the amount of virus produced was sometimes as great as that recovered from

fusionmixtures of CV-1 and certain transformed mouse cells. Moreover, the number of infectious centers formed by W18 Va2(P363) cells was as great as thatobtained by plating fusion mixtures

of CV-1 and transformed mouse cells on CV-1 monolayers. Therefore, this subline was unsuit-able forthe analysisof rescue fromheterokaryons of transformed mouse and human cells and for the SV40 DNA infection experiments.

The observation that W18 Va2(P363) and W138 Val3Acells shed virus is explicable if these cell lines contained a complete complement of SV40genetic informationand the factorsessential

for SV40replication.Increasing theavailability of

the essential replication factors by formation of

heterokaryons with CV-1 cells did not increase

thepercentageof W18Va2(P363)orW138 Val 3A cells which released virus. Moreover, in fusion

mixtures of CV-1 and transformed mouse cells, the percentage of transformed mouse cells in

heterokaryons greatly exceeded the percentage

whichproducedSV40.The lasttwofindingscould

meanthat additional regulatory mechanisms

op-erate to preserve the integrated state. At any given time, only a certain percentage of the cells may be competent to release the SV40 genome fromintegration.

It is common for mouse lines transformed by

nondefectiveSV40 toexhibit thefollowing prop-erties

(6,

8, 12, 14, 21, 26-28): (i) theydo not pro-duce virus spontaneously;

(ii)

however, they do

produce SV40 after fusion with monkey cells.

None ofthetransformed human lines behaved in this way. Either the transformed human lines produced virus spontaneously or they failed to produce virus spontaneously or even after fusion with monkey cells.

A somatic hybrid of CV-1 and SV40-trans-formed mouse cells (mKS-BUIOO) did not produce virus spontaneously (18). Fusion with additional CV-1 cells did, however, lead to re-covery of infectious SV40. The monkey-mouse hybrid cells contain few monkey chromosomes but essentially the entire complement of

inKS-BU100chromosomes. This suggests that chromo-somal determinants required for SV40 recovery

werelacking.Itremainstobeseenwhether clonal

lines of transformed human cellscanbe obtained which do not spontaneously release virus but which can be activated to do so by fusion with CV-1 cells. Transformed human cell lines that release virusonly after fusion with CV-1 cells will beuseful for studies of the "helper" function of either superinfecting SV40 DNA or the SV40 genome resident in transformedmousecells.

The yields of SV40 in rescue experiments and after infection with SV40 DNAwere lower from transformed human lines or from HeLa(BU25) and HEK cells than from monkey cells. This suggeststhat thedesignation"semipermissive"or

"semirestrictive" would be appropriate for the humancell lines used in this study. In contrast, primary and established lines of African green monkey kidney cells are highly permissive for nondefective SV40 virions (4, 5, 16, 24, 27), but normalandtransformed murine cell linesarevery nonpermissive (19, 25). Nontransformed human cell lines vary in their susceptibility to SV40 virions (1). Ingeneral, replication isabortive,

al-though afew cells inaninfectedhuman culture do producevirus (1, 3-5, 10, 23). Failure of human cell linesto replicate intact SV40 virions can be

attributed, at least in part,to a blockin penetra-tion and uncoating of virions, because normal and transformed human cell lines do replicate superinfecting SV40 DNA (25; Fig. 1-3).

How-ever, the concentrations of essential replication

factors may have been suboptimal in thehuman cell linesstudied here.

Further experiments are needed to elucidate themechanismsunderlyingsemipermissiveness or

semirestriction. With respecttothesuperinfection experiments described inthis study, itwas recog-nized that CV-1 cells grow more slowly than transformed human celllines. Aneffortwasmade

to standardize the metabolic conditions of the host cells, but it ispossiblethatwe chose

condi-tionsthatwere

optimal

forSV40 DNA

replication

in CV-1 but not in human cells. The fact that

SV40 virions releasedafterone

growth

cyclemay

superinfect CV-1, but not human cells,

compli-cates interpretation ofthe experiments. Also, the percentage of transformed human cells which initiate SV40 replication after addition of SV40

DNAisnotknown.Theinductionof Tantigenor

synthesis of early SV40-messenger ribonucleic acid (RNA) cannot be used as a measure of initiation of infection since all transformed cells contain T antigen and early SV40-messenger RNA. Preliminary experiments indicate that about 1

%l

ofHeLa(BU25) cells and about4% of

CV-1 cells synthesize Tantigen46to 48 hr after

SV40DNA infection. Aaronsonand Todaro (2)

found less than0.01 and1.5% of humandiploid

fibroblasts T antigen-positive after SV40 virion

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IDENTIFICATION OF SIMIAN VIRUS 40

andSV40 DNA infections, respectively. Carp and Sokol (5) found

5%7c

of African green monkey

kidney, but only 0.11%7 ofW138cells, T antigen-positiveat 44 hr after infection with SV40 DNA.

On theother hand, about 80to100%, of African green monkey kidney or CV-1 cells were T

antigen-positive after infection by SV40 virions

(5, 14). Preliminary experiments suggest that,

after SV40 DNA infection, 10 to50 times fewer infectious centers are formedby the transformed

human cell lines,W98 VaD and W98 VaH, than by CV-1 cells (S. Kit and D. R. Dubbs, unpub-lishedexperiments). Experimentstodetermine the penetration and degradation of infecting DNA

and the burst sizeperinfected cellare being

car-riedout.

ACKNOWLEDGMENTS

Thisinvestigationwassupported bygrants from the National

Science Foundation (GB 8469), the American Cancer Society (E291F), the Robert A. Welch Foundation (Q-163), and by Public Health Service grants CA-06656-07, l-K6-AI-2352, and

5-K3-CA-25,797 from the National Cancer Institute and the

NationalInstitute ofAllergyandInfectious Diseases.

We thank S. Tokuno, Marjorie Johnson, Carolyn Smith, Judith Rotbein,and Jannie Corbin for able technical assistance.

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