Copyright @1970 AmericanSocietyforMicrobiology
Temperature-Sensitive Simian Virus
40
Mutant
Defective
in
a
Late Function
SAUL KIT, S. TOKUNO, K. NAKAJIMA, D. TRKULA, AND D. R. DUBBS Divisionof Biochemical Virology, Baylor College of Medicine, Houston,Texas77025
Received forpublication 7 May 1970
Atemperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been
iso-lated from nitrosoguanidine-treated and SV40-infected Africangreenmonkey
kid-ney(CV-1) cultures. Replicationofvirusatthenonpermissivetemperature(38.7 C)
was 3,000-fold less thanatthepermissivetemperature (33.5 C). Plaque formation
by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred
normally at 33.5 C but wasgrossly inhibited at 38.7 C. The time at which virus
replicationwasblockedat38.7 Cwasdeterminedby temperature-shiftexperiments.
Inshift-up experiments, cultures infected for various timesat33.5 Cwereshiftedto
38.7C. In shift-down experiments, cultures infected for various times at 38.7 C
wereshifted to33.5 C. All cultureswereharvestedat96hrpostinfection (PI). No
virusgrowth occurred when the shift-up occurred before 40 hr PI. Maximum virus
yields wereobtained at96 hrPI when the shift-down occurredat66hr, but only
about15% of the maximum yieldwasobtained when theshift-downoccurredat76
hrPI. These results indicate that SV40tsTNG-1 containsaconditionallethal
muta-tion in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen,
viral capsid antigens, and viral DNA, and induced thymidine kinase activity at
either 33.5 or 38.7C.The properties of the SV40 DNA synthesized in
mutant-in-fected CV-1 cellsat33.5 or 38.7 Cwereverysimilarto those ofSV40 DNA made
in parental virus-infected cells, as determined by nitrocellulose column
chroma-tography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by
velocity centrifugation in neutralsucrosegradients. Mutant SV40tsTNG-1 enhanced
cellular DNA synthesis in primary cultures ofmousekidneycellsat33.5and 38.7 C
and also transformed mousekidney cultures at 36.5 C. SV40tsTNG-1 was
recov-ered from clonal lines of transformed cells afterfusion with susceptible CV-1 cells
and incubation ofheterokaryonsat 33.5C, but not at38.7C.
A directwayofrelating viralgenefunctionsto
biochemical changes in infected cells is to study
temperature-sensitive mutants ofa virus. A
tem-perature-sensitivemutantisonethatproduces an
altered gene product (protein) that is functional
atonetemperaturebutnotatanother.By asking
whetheragiven biochemical changeoccursunder
conditions where the mutated viralgeneproduct
isnotfunctional, itcanbeestablished whether the
viralgeneproductisrequired for the biochemical
changetooccur.
By using temperature-sensitive mutants, three
or four polyoma virus genes have so far been
identified (2, 5-7, 22). Plaque morphology
mu-tants ofsimian virus 40 (SV40) have also been
described, someof whichreplicateverypoorlyat
theelevatedtemperatureof 40to41 C(14, 18, 19,
22, 23). However, useof the latter SV40mutants
hasbeen somewhat limited because of the
dele-teriouseffects ofsupranormaltemperaturesupon
thehost cells.
In thisreport, wedescribe theproperties ofan
SV40 mutant, tsTNG-1, which replicates nor-mally at 33.5 C, but not at 38.7 C. The mutant
was isolated from SV40-infected African green
monkey kidney (CV-1) cultures that had been
treated with nitrosoguanidine. The data to be
presented demonstrate that viral replication is
markedly inhibited at 38.7 C, but that induction
of early SV40 functions, replication of SV40
deoxyribonucleic acid (DNA), and formation of
viralcapsid (V) antigens do take place.
MATERIALS AND METHODS
Cell lines.CV-1,anestablishedlineof Africangreen monkey kidney cells, was grown in "R5a" medium containing 10% calf serum and 0.5% lactalbumin hydrolysate (13), or in Eagle's minimal essential 286
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SV40 TEMPERATURE-SENSITIVE MUTANT
medium (MEM;AutoPOW, FlowLaboratories, Inc., Rockville, Md.) supplemented with 10% calfserum.
CV-1 cells were subcultured at weekly intervals. Primary mouse kidney cell cultures were prepared from 10- to 14-day-old Swiss mice (Texas Inbred Mouse Co., Houston, Texas). Five-day-old cultures containing3 X 106cellspercultureweretransformed by infecting them with SV40tsTNG-1 at an input
multiplicity of 61 plaque-forming units (PFU)/cell (4). Transformed cells were routinely grown in monolayer cultures at 36.5 C and were subcultured twice weekly.Thetransformed cellswerealsocapable ofgrowthat38.7 C forthree to fourgenerations.The transformed cell linewasclonedatpassages6and17
in medium containing 1% SV40 antisera.
Trans-formed cell lines contained SV40 T antigen, as
de-termined by immunofluorescence (IF), but did not
contain detectable infectious virus. Cell-free extracts were prepared from cultures of transformed cells
which were incubated at 36.5 C for 4 days after
subculturing and thenwereincubated at 33.5 C for6
days. No SV40 was detected after plating the cell extracts onCV-1 monolayers ateither 33.5or36.5 C.
Virus.SV40wasgrownand assayed in monolayer cultures ofCV-1 cells(13).ParentalSV40clone307L, whichproduces largeclearplaquesonCV-1 cells,was
assayedat36.5 Cor33.5 C. MutantSV40tsTNG-1was assayedat33.5 C andproducessmall indistinctplaques
at this temperature. Sendai virus wasgrown in the
allantoiccavityof 10-to11-day-oldembryonatedeggs
(3).
Virus growth curves. Seven-day-old monolayer cultures of CV-1 cells in 2-oz prescription bottles
(ca. 60 ml) were infected at a multiplicity of five
PFUper cellina 0.2-ml volume for 2 hr at 36.5 C.
Thecultures were washed three times with
isotonic-glucose-salinesolution (GKN).Then 0.5 ml ofSV40 antiserum (diluted1 partto 9 partsofmedium) was added, andthe cultureswereincubated for 30minat
37C. (The SV40 antiserum was prepared in horses againstSV40strain Rh911; FlowLaboratories, Inc., Rockville, Md.) After washing cultures three times
with GKN toremovetheantiserum, growthmedium
(5.0 ml) wasadded and the cultures wereincubated ateither33.5or38.7C. At the timesindicatedinFig. 1,twocultureswereremovedfrom the incubator and
frozen. Cells and supernatant fluid were harvested
fromeach culture. The cellswere disrupted bysonic
treatment, and the virusyieldswereassayed onCV-1
monolayers. Parental SV40 clone 307L and mutant
SV40tsTNG-1 wereassayedat33.5 C.
For thetemperature-shift experiments,cultureswere
treated ina similar manner, exceptthat at the times
indicated in Fig. 2 two cultures were shifted from
incubators ofonetemperatureto theother. At 96 hr
postinfection (PI),all cultureswerefrozen and thawed
oncepriortoharvestingcellsandsupematantfluid.
Complement fixation(CF) test.Preparation ofcell extractsanddemonstration ofSV40Tantigen bythe
CFtesthave been described (13, 17).
IF test. The presence ofSV40 T antigen in cells
transformed by SV40 was demonstrated by indirect
IF (12) by using sera from hamsters bearing SV40 transplant (virus-free) tumors andfluorescein-labeled
I09F
SV40Cl307L(38.7C)
1081
/08+ >< SV40Cl 307L (33.5C)
1071 P SV40tsTNG-I(335C)
IO6~~~~~~~~~~~~~~~~~~~~~~~~~~~
LO
U106~~~~~~~~~~~~~~~~~'
I0
CL SV40tsTNG-1(38.7C)
104
103-1021
O 20 40 60 80 00 20 140
HR AFTERSV40INFECTION
FIG. 1. Growth ofparental SV40 clone 307L antd
mutantSV40tsTNG-1inCV-J cellsat33.5and 38.7 C.
At the times indicated, two infectedculturesfor each
pointwerefrozen.Bothviruseswereassayedat33.5C.
goat antihamster globulin. V antigen was
demon-stratedbydirect IF byusingfluorescein-labeled
SV-40-hyperimmune monkeysera(20).
Preparation and assay of infectious SV40 DNA.
SV40 DNA was extracted from infected CV-1 cells
andseparated from cellular DNAby the method of
Hirt (8). Extracts were deproteinized with an equal
volume of phenol and then dialyzed against 0.15 M
sodium chloride, 0.015 M sodium citrate, pH 7.0
(1 X SSC), until thedialysatewas free from phenol,
as determined by absorption measurements at 260 nm. Unfractionated DNA samples and samples
fractionated by equilibriumorvelocitycentrifugation wereassayed for infectivityby the diethylaminoethyl-dextranmethodonCV-1 monolayers (15).
FractionationofDNApreparations. Closed-circular double-stranded SV40 3H-DNA (form I) was
sepa-rated from nicked-circular (form II) or from linear
double-stranded DNA by nitrocellulose chromatog-raphy (17),orbyequilibriumcentrifugation in cesiuni chloride (CsCl) solutions (density of 1.57 g/cm3) containing 300 pig ofethidium bromide (EtBr) per
ml (9). Samples studied by the dye-buoyant density methodwerecentrifugedat20C for44 hrat 120,000
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[image:2.494.278.424.62.381.2]288
KIT ET AL.100
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HR AFTER SV40 INFECTION
FIG. 2. Temperature shift-up experiment. CV-1 cultures were infected with SV40tsTNG-1 and incu-bated at 33.5 C. For curve X, two infected cultures
were shlifted to 38.7 C at the timnes indicated and
in-cuibated until 96 hr PI. Forcurve 0, ilfectedcultures
werefrozenat thetimes indicated. The virus yieldsare
presentedasthepercentyield of conttrol SV40tsTNG-J-infected cultures incubated 96 hrat33.5 C.
X gby usingrotor5OTiandaSpinco L2centrifuge. EtBr was removed from DNA solutions by
extract-ing two times with an equal volume of isoamyl
al-chohol, followed by ether extraction. The extraction procedure with isoamyl alcohol and ether was
re-peated,andthe DNAsolutionsweredialyzed against
1 X SSC containing 0.001 M
ethylenediaminetetra-aceticacid (EDTA),and thenagainst 1 X SSC
with-out EDTA. Control experiments demonstrated that the EtBr concentration wasreduced belowthe level detectableby spectrophotometricmeasurementat476
or284nmand that theinfectivityofSV40DNAafter
removalofEtBrwasthesame asthatofDNAwhich
hadnotbeenexposedtoEtBr.
Heavy and light DNA fractions obtained by the
J. VIROL.
dye-buoyant density procedure and freed ofEtBr as described above were in some instances further analyzed by centrifugation through 5 to 30%70 (w/v) sucrose gradients. Sucrose solutions were dissolved in 0.1 M sodium chloride, 0.001 M EDTA, 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochlo-ride buffer, pH 7.2. DNA samples (0.1 ml) were layered on top of thegradient and centrifuged at 5 C for210 minat 100,000 X gby usingan SW39or an SW50 rotor and a Spinco L2 centrifuge.
Ten-dropfractionswerecollected from the gradients on Whatman no. 4 filter paper. The papers were washed with cold 5%' trichloroacetic acid and cold ethyl alcohol and assayed for radioactivity by liquid scintillation counting (17).
Enzyme assay. Thymidine kinase activity of un-infected and SV40-un-infected CV-1 cells was assayed with 3H-deoxyuridineasnucleoside substrate (13).
Materials. Optical grade CsCl was obtainedfrom the Harshaw Chemical Co., Cleveland, Ohio, and thymidine-methyl-3H (14 to 18 Ci/mM) from New England Nuclear Corp., Boston, Mass. Nitrosoguani-dine(N-methyl-N'-nitro-N-nitrosoguanidine) was pur-chased from the Aldrich Chemical Co., Inc., Mil-waukee,Wis.
RESULTS
Treatment of SV40-infected cell cultures with
nitrosoguanidine. Nitrosoguanidine treatment of
SV40-infected CV-1 cellswasusedtoinduce SV40
mutants. Selection of this mutagen was based
upon the observations of Cerda-Olmedo and
Hanawalt (1) that nitrosoguanidine inflicts very high levelsofmutagenesis withlittle lethalityand preferentially mutagenizes the region of replica-tionofthebacterialchromosome.Also,Kao and Puck (10, 11) had found thatnitrosoguanidineis
an efficient mutagen for mammalian cells. Cell
cultures infected with parental SV40 clone 307L
at an input multiplicity of five PFU/cell were
treatedat2hr after infectionwith 15 ,ig of
1-3-D-arabinofuranosylcytosine (ara-C) per mlto
syn-chronize thereplication ofSV40DNA.At26hr,
the medium was removed, the cultures were
washed, and fresh mediumcontaining
deoxycyti-dine (45 ,g/ml) was added to reverse the ara-C
block of DNA replication. Nitrosoguanidine (100 ,ug/ml) was added to replicate cultures for
30 minat0, 30, 60, 90, and 120 min,respectively,
afterthe addition ofdeoxycytidine. After the
30-min nitrosoguanidine pulse, the cultures were
again washed, fed with fresh growth medium
containing deoxycytidine, and incubated at 37 C
until 56hrPI. The SV40 yields (assayedat 37C)
of nitrosoguanidine-treated cultures were 8 to
16% of the yields obtained from untreated
cul-tures. The SV40 mutant described in this report
was derived from cultures pulsed with
nitroso-guanidine at120 to 150 minafter the reversal of
the ara-C block. 0.1L
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[image:3.494.80.224.66.412.2]SV40 TEMPERATURE-SENSITIVE MUTANT
Isolation ofvirus mutants. To select virus
mu-tants, harvests from cultures mutagenized with
nitrosoguanidine weretreated by sonicoscillation
for 1 minat10kcat4 C and wereplatedonCV-1
monolayers. They were overlaid with
agar-me-dium and incubated at 33.5 C until smallplaques
(1 mm indiameter) developed. Theperiphery of
eachplaquewasmarked, and then theplateswere
shifted to 38.7C for 4 days. The majority of
plaques enlarged at the high temperature. Those
plaques whichfailed to enlarge (12of1,137) were
picked with a capillary pipette and resuspended
in 2 ml of medium. After dispersion by
freeze-thaw and sonic oscillation for 1 min, 0.1-ml
samplesofundiluted fluidwereplatedoneach of
four CV-1 monolayers. Two of these were
incu-batedat33.5 C andtwoat38.7 C.Sampleswhich
produced confluent destructionat33.5 C, andless
than 100 plaques at 38.7 C, were titrated at the
two temperatures. Fortheinitialsample ofvirus
mutant SV40tsTNG-1, the ratio of plaques
formed at 33.5 to 38.7 C was 120. The mutant
virus was plaque-purified a second time, and
virus poolswereprepared. Afterfour passages in
CV-1 cells, the titer ofSV40tsTNG-1 was 1.7 X
108PFU/ml at33.5 C, and less than 103PFU/ml
at 38.7 C. Parental SV40 clone 307L gives the
sametiterwhenassayedateithertemperature.
Growth ofSV40clone 307L andSV40tsTNG-1
at two temperatures. Parental SV40 clone 307L
grows equally well at 33.5 and 38.7 C, reaching
titers greaterthan 108PFU/106cells (Fig. 1).At
33.5C,theeclipseissomewhatlonger, and
maxi-mumyieldswereobtained about 10to20hr later
thanat38.7 C. Growth ofmutantSV40tsTNG-1
resembled that of SV40 clone 307L at the low
temperature (33.5 C), but very little increase
in SV40tsTNG-1 titer was observed at 38.7 C
(Fig.
1).
Temperature-shift experiments. To determine
the time at which replication of mutant
SV40tsTNG-1 was blocked at thehigh
tempera-ture (38.7C), cultures infected with the mutant
virusat amultiplicityof 5 PFU percellwere
incu-batedat33.5 C. At various times afterinfection,
two cultures were shifted to 38.7 C. All of the cultureswereharvestedat96hrPI.The results of this experiment are shown in Fig. 2, along with thegrowth curveofSV40tsTNG-1 at33.5 C.No
virusgrowthoccurredwhen infected cultureswere
shifted from 33.5 to 38.7 C before 40 hr PI. A
comparison of thetwo curvesindicates that when
cultures were shifted between 40 and 96 hr, no
newvirusmaturationoccurred,for the virusyields
of the shifted cultureswerealmost identicalwith
those of cultures frozen at the indicated times.
These results suggest (i) that virus replication is
blocked in a late function, and
(ii)
that theproduct of thelate function does not accumulate,
but rather, the product is used as rapidly as it is
synthesized.
Shift-down experiments were also performed.
Cultures infected with SV40tsTNG-1 were
incu-bated at 38.7 C. Atvarious times after infection,
two cultures were shifted to the lower
tempera-ture (33.5 C). When cultures were shifted to
33.5 C at any timebetween 2 and 66 hr PI,
maxi-mum virusyields wereobtained by 96 hr PI.
How-ever, whencultures were shifted from the high to
the low temperature at 76 hr PI, about 15 ofthe
maximum virusyield wasobtained. These results
also indicate that a late function is blocked at the high temperature.
Induction of T antigen and thymidine kinase activity. Two earlySV40 functions are induction of Tantigenandthymidinekinase activity (4,13,
17). The capacity of SV40tsTNG-1 to induce
these functions wasstudied by infecting confluent
monolayer cultures of CV-1 cells at 33.5 and
38.7 C. Enzyme and T antigen assays were then
performed atvarious timesfrom 16 to 66 hrPI.
Both T antigen and thymidine kinase activity
were induced by the mutant at either 33.5 or
38.7C.
SynthesisofSV40 DNAat33.5and 38.7C. To
determine the totalamount of SV40 DNA made
atthehigh and low temperatures, CV-1cellswere
infected with parental SV40 clone 307L or SV40tsTNG-1 ateither33.5or38.7 C.Atvarious
times after infection, theDNA was labeled with
3H-thymidine for 4 hr, and then theSV40DNA
wasseparated fromthe bulkofthe cellularDNA
by theselective precipitation methodofHirt (8). Supernatant fractions from the Hirtextractwere assayed for totalinfectious DNAat 33.5C (Fig. 3),andat38.7C(Fig. 4).Inaddition,thesamples
were studied by nitrocellulose column
chroma-tography (17) to demonstratethatheat-resistant, double-stranded 3H-DNA, similar to form I
SV40 DNA,had beensynthesized.
The 33.5-C assays (Fig. 3) show that parental
SV40 clone 307L and mutant SV40tsTNG-1
DNA species were synthesized somewhat earlier
when cells were infected at 38.7 than at 33.5 C.
Also,
the total infectious DNA reached amaxi-mumby about50hrafterinfectionat38.7 C and
then declined somewhat. However, with
infec-tions at 33.5 C, the total infectious SV40 DNA
continuedto increase from 50to 72 hr after
in-fection.
Figure 4 shows that parentalSV40 clone 307L
DNA synthesized at either 38.7 or33.5 C could
be assayed at 38.7 C. In contrast, the infectious
DNAsynthesizedby SV40tsTNG-1 ateither
tem-perature formed few plaques on CV-1
mono-layers whenassayedat38.7 C.
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KIT ET AL.
INFECTIOUS SV40 DNA ASSAYED AT 33.5C SV40 tsTNG-I(33 50)
.
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SV40tsTNG-I (38.7C)
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SW1OC1307L(335C)
1,
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0 20 40 60 80 0 20 40 60 80
HRAFTERSV40 INFECTION OFCV-1AT33.5COR 38.7C
FIG. 3. Synthesis of infectious DNA in CV-1 cells (8.2 X 106 cellsper culture) infected with parental SV40 clone 307LorSV40tsTNG-1 (input multiplicity
of20PFU/cell) at33.5or38.7 C.DNAextractswere
preparedfrom infected cellsatthe indicatedtimesand assayedat33.5 ConCV-Imonolayer cultures.
Radioactivity measurements provided further
evidence that SV40 DNA was synthesized by
SV40tsTNG-1 atthenonpermissivetemperature.
The Hirtsupernatantfractionsobtainedfrom the
cultures that hadbeenlabeledwith 3H-thymidine
for 4hrpriortoharvest wereboiled for 10min,
cooled, and applied to nitrocellulose columns
(17). The heat-resistant, superhelical SV40 DNA
which is double-strandedwas notretainedby the
nitrocellulose. Itwasshownthatduring infection
at 38.7 C with either parental SV40 or mutant
SV40tsTNG-1, the rate of synthesis of
heat-re-sistant DNA increased to a maximum at about
44to52 hr afterinfection and thendeclined. After
infectionsat33.5C,radioactivityinheat-resistant
DNAincreased from 24 to 52hr, but therateof
synthesisremainedhigh through72 hrafter
infec-tion. Very little radioactivity was found in the Hirt supernatant fractions from uninfectedcells.
Properties of the labeled DNA in the Hirt
ex-tracts. We wondered whether SV40 DNA ab-normalinsizeorformmight be generatedduring
infection ofcells bymutantSV40tsTNG-1 under
nonpermissiveconditions. SinceinfectiousDNA,
butnotinfectiousvirus,wasmade, it seemed
pos-sible that the processing of the DNA might be
abnormal.Therefore,theproperties ofthelabeled
DNAinthe Hirtextractswereexamined further. Chosen forstudywas aperiodin infection when
therate ofSV40 DNA synthesis was rapidly
in-creasing and when maturation ofinfectious
par-ticles would normally be occurring. Replicate
cultures of CV-1 cells were infected for26 hr at
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INFECTIOUSSV40 DNA ASSAYEDAT38.7C 107t
SV40Cl307L
(33.5 C )/ "
SV40 Cl307L
®/y1 (38.7C)
/f
104
V40tsTNG-II) A SV40tsTNG-I
(387C)-II
\f (33.5C)103
II II
0 20 40 60 80
HR AFTERSV40INFECTION OF
CV-1 AT335COR387C
FIG 4. Synithesisof infectious DNA in CV-1 cells infected with parental SV40 clonze 307L or
SV40ts-TNG-1 at33.5or38.7C.DNAextractswereprepared
from infectedcells at the indicated times andassayed
at38.7ConCV-1mon0olayercultures.
33.5 or38.7 C. Thecultureswerethenlabeledfor
4 hr with3H-thymidine. The mediumwaschanged
andcycloheximide (25 ug/ml) wasaddedtohalf
of the culturestoinhibitproteinsynthesis. Itwas
anticipatedthatcycloheximidewould stopcapsid
proteinformationin treatedcultures, atthesame
timereducing further SV40 DNA synthesis (16).
If abnormalSV40 DNAweresynthesizedat
non-permissive temperatures in mutant-infected
cul-tures,the alteredproperties mightbeaccentuated
whencapsid protein synthesiswas shutoff.
Incu-bationwas continuedfor a 5-hrchase period in
the absence of3H-thymidine. The
low-molecular-weightDNAwasthenextracted from the cellsby
the Hirtprocedure (8). About1.3 to 1.8 times as
muchDNAwas labeledby the26-to30-hrpulse
and 5-hr chaseat38.7C,as at33.5C. The
cyclo-290
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[image:5.494.52.243.72.245.2]SV40 TEMPERATURE-SENSITIVE MUTANT
heximide treatment, however, did not signifi-cantly change the total amount of radioactivity in
theHirt supernatant fractions.
The radioactive DNA extracts were fraction-ated by equilibrium centrifugation in cesium chloride density gradients containing ethidium bromide.Figure 5 shows the properties of extracts from cells infected with mutant SV40tsTNG-1. Similar results were obtainedwith extracts from cells infected with parental SV40 clone 307L. "Heavy" and "light" fractions, respectively,
represent superhelical DNA and nicked-circular
plus linear DNA. Of the total 3H-DNA, 77 to
89% was found in the heavy density peak
(super-helicalDNA). This distribution ofradioactivity
inDNA was foundinculturesincubated at33.5
and 38.7 C, with or withoutcycloheximide
addi-tionduringthe 5-hrchase period.
The sizesof the DNA species in theheavy and
25 (a)
387CINFECTION t
20-15
25-(b)
387C INFECTION (PLUSCYCLOHEXIMIDE)
20-15
-light peaks were next analyzed by velocity sedi-mentation in neutral5to 30% sucrosegradients.
This was done to determine whether oligomeric
circular formsof SV40 DNA weresynthesizedby mutant SV40tsTNG-1. Analyses of the heavy
fractions(fractions14to19 of Fig. 5)obtainedby
dye-buoyant density centrifugation showed that
this 3H-DNA sedimented with the samevelocity
as anSV40 form I marker DNA (21S). Thus,the
form I DNA synthesized at 38.7 or 33.5 C by SV40tsTNG-1 consisted almost entirely of monomeric circles. The same result was obtained withparentalSV40 DNA.
[image:6.494.53.241.273.517.2]When the DNA fromthe lightpeaks shownin
Fig. 5 was centrifuged in neutral sucrose
gradi-ents,two radioactivitypeaks wereobserved (Fig.
6). Theslowpeaksedimentedatabout 16S. The faster peak was heterogeneous and had an
average sedimentation coefficient of about 36S.
The faster (36S) peak, butnotthe 16Speak,was also obtained when DNA from Hirt extracts of uninfected CV-1 cellswas centrifuged in sucrose gradients (Fig. 6d). Both peaks were found in DNAsamples obtainedfrom cells infectedat38.7 or 33.5C by either SV40tsTNG-1 or parental SV40 clone 307L. Theradioactivity ofthe faster
10 10
5-
5-5 10 15 20 25 30 5 10 15 20 25 30
159c) 15 (d)
335C INFECTION 335CINFECTION (PLUSCYCLOHEXIMrE)
10-
lo-5L
5-5 10 15 20 25 30 5 10 15 20 25 30 FRACTION NUMBER
FIG 5. Radioactivity distributionafterdye-buoyant
density gradient centrifugation ofsodium dodecyl
sul-fate-extracted DNA. Replicate cultures ofCV-J cells
wereinfected withmutantSV40tsTNG-1ateither38.7
C(Fig. Sa,Sb) or33.5 C(Fig. 5c,Sd)for26hr, then
incubatedwith 'H-thlymidine(5 &Ciand0.5 iAg/ml) for
4 hr. The media were removed andfresh media were
added. Cycloheximide (25 ,ug/ml) wasaddedtohalf of
thecultures(Fig. Sb, Sd),andincubationwascontinued for 5 hrat38.7or33.5C.Low-molecular-weightDNA wasextractedbythemethod ofHirt(8). Sampleswere
centrifuged at 20 C in CsCI-EtBr. Twenty-drop
frac-tionswerecollected and 20-jiliter portionswereassayed forradioactivity.
Sv4O n TNG -' DNA tFor0A)
8 _ (INFECTiON AT 38 7C,CycLOHEAMIDIE)
7-
5-a._
5 0 a1 20 25 30
4- 10C0OEIC3215
2-0il
9SV40C1307LDNA lTom2)g
8_(INfECONAT8 N
7
5
4
3
2 iW
,,
5 10 15 20 25 30
6- (d) 900w01CVOMIE)CIM 5
3 UO
5 10 15 20 25 30
FIG. 6. Sedimentation velocityanalysisin S to30%
(w/v) neutralsucrose gradientsofnickedcircular and linear DNAfrom uninfectedandSV40-infected CV-1 cells. Fractions 20 to 26ofthe"light" DNAfraction
purified by CsCI-EtBr equilibrium centrifugation (see
Fig. 5) were centrifuged. The distributions of
radio-activityoffractions from CV-1 cellsinfectedat38.7 C with mutant SV40tsTNG-1 are shown in Fig. 6a and 6c; radioactivity distribution of cells infected with parental SV40 clone307L isshowninFig. 6band6d. The radioactivity distribution of low-molecular-weight
DNAfrom uninfected CV-1 cellsisalso shown inFig. 6d. Thearrowmarks the positionofmarkerSV40form
IDNA(21S).
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[image:6.494.257.449.344.516.2]peakrelative to the slower peak was reduced when infected cultures were treated with cycloheximide
at 30 to 35 hr after infection. Possibly, the
addi-tion of cycloheximide for 5 hr caused some of the 36S DNA to be degraded to smaller pieces.
Thelight DNA fractions (Fig. 5) that had been
fractionated by sucrose gradient centrifugation
werealso assayedonCV-1 monolayersat33.5 C.
The peaks of infectivity, representing 73.2%o of
the total infectivity, were found in fractions
cor-responding to the 16S peaks (Fig. 6). Another
20.5%o
ofthe infectivity was found in theinter-mediatefractions (about 21S) and may have been
part of the leading front of the 16S peak or
replicative intermediates. Few plaques were
found in thefractions from the fast peak. These
results suggest that the DNA in the fast (36S)
peak from the light density DNA was mostly
cellular and that the DNA from the slow (16S)
peak contained minimally nicked forms of SV40
DNA.
Formation of V antigen. The formation of V
antigen by SV40tsTNG-1-infected cells at the
permissive and nonpermissive temperatures was
demonstrated by direct immunofluorescence by
using SV40-immune green monkey serum
con-jugated with fluorescein isothiocyanate. Under
the conditions used, the antiviralmonkey serum
failedtostain SV40-transformedmousecells
con-tainingSV40 Tantigen.CoverslipsofCV-1 cells
infected with mutant SV40tsTNG-1 were
incu-batedateither 33.5 or38.7 Cfor 2to72hr. Ata
multiplicity of 50 PFU/cell, essentially all of the
cells were positive for both T and V antigen at
72 hr PIat 33.5C, andat48hrPIat 38.7 C. At
multiplicities of 5.6, 1.3, and 0.3 PFU/cell,onlya
fraction of the cellswas positiveforTantigenat
either33.5 or38.7C,butapproximatelythesame
proportionofcellsateither temperaturewas
posi-tive for V antigen. The staining for V antigen
could not be diminished or abolished by
pre-treating the SV40tsTNG-1-infected cells for 1 hr withunconjugatedSV40 hamstertumor serum.
Ara-Chas been shownto inhibitthesynthesis
of SV40 DNA (13) and SV40 coatprotein
anti-gen,butnotthesynthesis of SV40Tantigen(20).
To substantiate the fact that SV40 V antigen is
synthesized in SV40tsTNG-1-infected cells atthe
nonpermissive temperature, the effect of ara-Con
the induction of SV40 T and V antigen was
studied at 33.5 and 38.7 C. CV-1 cells on cover
slipswereinfected withSV40tsTNG-1 at a multi-plicity of about one PFU/cell. After 1 hr of
ad-sorptionat36.5C, medium either withorwithout
15 ,ug of ara-C per mlwas added. Then the
cul-tures were incubated at either 33.5 or 38.7 C. At
thetimes indicatedinTable 1, cellson coverslips
werefixedin acetone and usedforIF.Theresults
TABLE 1. Inzductioni ofTanid Vanitigent synzthesisin CV-1 cellsintfected with mutantSV40tsTNG-1
(It33.5 anid38.7 Ca
Times
post-infection
hr
2 20 28 48 72
2 20 28 48 72
Incubation temp
C
33.5
38.7
Percentofcellsb positivefor
Tantigen Vantigen
Noara-C
0 0.4 4.2 12.6 15.6
0 8.5 19.9 24.7 18.7
With
ara-C
0.9 7.0 16.7 11.4
10.9 17.4 13.8 11.4
No ara-C ~,ara-CWith
0 0 0 5.6 13.3
0 1.6 3.4 15.9 24.8
0 0 0.1c 0.05c
0.08c 0.05c 0.1c 0.09e
a Petri dishes of CV-1-containing cover slips
wereinfected at a multiplicity of about 1 PFU/ cell with SV40tsTNG-1. Virus was allowed to adsorb for 1 hr at 36.5C. Medium (5 ml) either with orwithout ara-C (15
Ag/ml)
was added, and dishes were incubated for indicated times at 33.5 or38.7 C.IAt least 500 cells were scored for each
deter-mination.
cVery faint staining.
(Table 1)indicate that(i)Vantigen issynthesized
ateither 33.5 or 38.7 C in the absence of ara-C,
but is notsynthesizedateither temperature in the
presenceof ara-C; (ii) T antigen is synthesized at
both temperatures whether ara-C is present or
not; (iii) the per cent of T antigen-positive cells
increases between 20 and 72 hr PI at33.5 C and
between 20 and 48 hr PIat38.7 C; (iv)T
antigen-positive cellsare moreprevalent thanV
antigen-positivecellsat20to28hr PI; and(v) the per cent
ofV antigen-positive cells at72hrPI equals the percentof Tantigen-positivecellsat48hr PI.
Induction of cellular DNA synthesis. In primary
mouse kidney cultures infected with parental
SV40, little or no viral DNA replication takes
place. Nevertheless, cellular DNA synthesis is
enhanced (17). To determine whether mutant
SV40tsTNG-1 also induced cellular DNA
syn-thesis, 5-day-old cultures of primary mouse kidney cells (3.3 X 106 cells per culture) were
infected at input multiplicities of 150 PFU/cell
withparentalandmutantSV40strains and incu-bated at 33.5 and 38.7 C. The cultures were pulse
labeled with 3H-thymidine from 40 to 44 hr and
from64 to68hrPI; thetotalDNAwasextracted
and hydrolyzed, and the incorporation of
on November 11, 2019 by guest
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[image:7.494.256.448.72.303.2]SV40 TEMPERATURE-SENSITIVE MUTANT
thymidine into DNA was determined. After infec-tion by either parental or mutant SV40, the
for-mation of3H-DNAwas stimulated 2.5- to4.3-fold
at 33.5 and at 38.7 C. These resultsindicate that
mutant SV40tsTNG-1 can induce cellular DNA synthesis at the nonpermissive and permissive temperatures.
Transformation of mouse kidney cells by SV40tsTNG-1. Parental SV40 clone 307L is
capable of transforming mouse kidney cells in
culture (4). To demonstrate that mutant
SV40-tsTNG-1 alsohad this ability, primaryculturesof mouse kidney cells were infected with virus, incu-batedat 36.5 C for 18 hr, and subcultured. The cultures were fed twice weekly for 2 to 3 weeks until transformed colonies developed. Thereafter, transformed cells, mKS(tsTNG-1), were sub-cultured at 36.5 C at 4 to 7 day intervals as de-scribed previously (3, 4). Nine clonal lines of mKS(tsTNG-1) cells were isolated. The clonal
lines allcontained theSV40 Tantigen, but
infec-tiousvirus was notdetected in cell-freeextracts.
To determine whether SV40 could be rescued
from the clonal mKS(tsTNG-1) cells, theywere fused with CV-1 cells in the presence of ultra-violet-irradiated Sendai virus. Cellmixtures were then plated with freshly trypsinized CV-1 cells, overlaid withagar, andfurtherincubated at either
33.5 or 38.7 C
[frequency
ofinduction test(3)].
When rescue experiments were performed with
mKS-U13, a mouse kidney line transformed by
SV40 clone 307L,thesamenumber ofinfectious
centers were produced at the two temperatures.
However, SV40 infectious centers were not
de-tected when the rescue experiments with
mKS-(tsTNG-1)
cloneswereperformedatthe nonper-missive temperature of 38.7 C. Nevertheless, SV40 was rescued atthepermissive
temperature (33.5C) from seven of ninemKS(tsTNG-1)
clones. Thefrequencyof inductionvaried from 2
to 60infectious centersper105 transformed cells
inthe fusion mixture.Infectiouscenters
appearing
at 33.5 C in rescue
experiments
of each of theseven
mKS(tsTNG-1)
clones werepicked
andassayedforSV40onCV-1
monolayers
atthetwotemperatures. The rescued virus formed
plaques
at 33.5 C but failed to form
plaques
at 38.7 C.These results demonstrate that the
mKS-(tsTNG-1)
cells were indeed transformedby
themutantSV40tsTNG-1 and not by a revertant or
contaminating
wild-type
SV40. Moreover,they
provideanother
example
thatSV40 rescued from a transformed cell resembles the virus used toinitiate thetransformation.
DISCUSSION
Atemperature-sensitivemutant,SV40tsTNG-1,
has beenisolated after
nitrosoguanidine
treatmentof SV40-infected CV-1 cultures. The mutant replicates in a normal manner at the permissive
temperature of 33.5 C but not at the
nonpermis-sive temperature of 38.7 C. For the induction of
the mutant, SV40 DNA synthesis was first
syn-chronized by ara-C treatment ofinfected cultures.
Deoxycytidine was then added to reverse the
ara-C blockand to initiate DNA replication. At
various times after SV40 DNA synthesis had started, theinfected cultures were treated for 30
min with nitrosoguanidine. This procedure was
based upon the finding that nitrosoguanidine preferentially mutagenizes the replication region
ofDNA (1). The procedure used here may be
useful in inducingsequential mutations at various
sites ontheSV40 DNA.
In the present study, nitrosoguanidine
muta-genesis was used to obtain a conditional lethal
mutant defective in a late function. The data that
have been presented showthat the mutant
SV40-tsTNG-1 was capable ofreplicating its DNA at
38.7 C andthat theearly functions of inducing T
antigen, thymidinekinase, andcellularDNA syn-thesis were expressed. The DNA synthesized at 38.7Cwas fullyinfectious at 33.5 C but did not plaquewellat 38.7 C. Theviral DNA synthesized in SV40tsTNG-1-infected cells was studied by
various biophysical methods. It was similar to
DNA from parental SV40 in its resistance to
denaturation at 100 C and in dye-buoyant density
centrifugation. The possibility was considered
that the processing of the SV40tsTNG-1 DNA
might be abnormal at the nonpermissive
tem-perature. However, sucrosesedimentationvelocity
studies demonstrated that
practically
all of theform I DNA consisted of monomeric circles
(21S), andthatalmostallof theinfectivity of the
nickedforms sedimentedatabout 16 to 21S.
The DNA studies and the temperature-shift
experiments suggestthat a latefunctionis
defec-tive when SV40tsTNG-1
replicates
at 38.7C. Itdoes not appear, however, that formation of
capsid proteins isblocked.The IFstudies ofviral
antigens indicate that antigen(s) (coat protein)
reacting with viral antisera issynthesizedin
cells
infected at 38.7 Cwith SV40tsTNG-1. This
sug-gests that this mutant produces noninfectious
particlesat thenonpermissive temperature, since
antisera made against purified virus generally
reactwellonlywithparticles.Consistent with this
interpretation, electron microscopy studies of
thin sections ofSV40tsTNG-1-infectedcells have
shown that structures which resemble virus
par-ticles aremade at both 33.5 and 38.7C
(S. Kit,
D.
Zarling,
and D. R. Dubbs, unpublisheddata).
Furtherexperiments are neededtodetermine the
amounts and properties of theindividual
virion-peptide chains synthesized under
nonpermissive
293
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conditions in SV40tsTNG-1-infected cells and to determine whether these proteins are assembled
properly at 38.7C.
Mutant SV40tsTNG-1 transforms mouse
kid-ney cultures at 36.5 C. Heterokaryons of
inKS-(tsTNG-1) and CV-1 cells incubated atthe
per-missive temperature (33.5 C) yielded infectious
virus, butheterokaryons incubatedatthe
nonper-missive temperature (38.7 C) did not. However,
merely incubating mKS(tsTNG-1) cells alone at
33.5C does not lead to virus production. The
mousecellstransformed by the
temperature-sensi-tive SV40 mutant have different propertiesfrom
mousecells transformedbytemperature-sensitive
polyomamutants.Cellstransformed
by polyoma
mutant Ts-a initiate infectious virus formation
whenshiftedfromanonpermissiveto apermissive
temperature (24),butcellstransformedby
SV40-tsTNG-1 do not. The difference arises from the
factthatmouse cells are permissive for
polyoma
but not forSV40 replication. Therefore, in
addi-tiontothetemperatureshift-down,the
susceptible
monkey cell protoplasm must be supplied for
SV40rescue to occur.
ACKNOWLEDGMENTS
This investigation was aided by grants from theNational Science Foundation (GB 8469), the American Cancer Society (E291F), the Robert A. Welch Foundation (Q-163), and by Public Health Service grants CA-06656-08, 1-K6-AI-2352, and 5-K3-CA25,797 from the National Cancer Institute and the NationalInstituteofAllergy and Infectious Diseases.
WethankCarolyn Smith, MarjorieJohnson,andJudith
Rot-bein forabletechnical assistance.
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