JOURNAL OF VIROLOGY, May 1977, p. 340-345
Copyright©1977 AmericanSocietyforMicrobiology Printed inU.S.A.
Mitochondrial DNA Synthesis
in Adenovirus Type 2-Infected
HeLa
Cells
PAUL B. FISHER' AND MARSHALL S. HORWITZ*
Departments ofMicrobiology-Immunology,*Cell Biology, and Pediatrics, Albert Einstein College of
Medicine,
Bronx,
NewYork 10461Receivedforpublication17November1976
Mitochondrial DNA synthesis inadenovirus type 2-infected HeLa cellswas
measuredat various timesfrom0 to 24 hpostinfection. Althoughviral infection
effectively turned off host chromosomal DNA synthesis, mitochondrial DNA
synthesiswasnotinhibited. Thesefindingsindicateadissociation between the
regulation of host andmitochondrial DNA synthesis after infection with
adeno-virustype2.
Anasymmetrical model of bidirectional
rep-lication has beenproposed for adenovirus DNA
(5, 11, 12, 29, 31). This model of displacement
replication is similartomodels proposed for the
replication ofmitochondrial DNA (1, 17, 27).
Adenovirusandmitochondrial DNAsyntheses
have also been found to be similar in their
resistance to the effect of protein inhibitors (13,
30). Individual molecules ofadenovirus DNA
arebothinitiated and completely replicatedin
the presence of concentrations of cyclohexi-mide, which inhibit up to 97% of HeLa cell
proteinsynthesis (13).Similarly,mitochondrial
DNA synthesis in HeLa cells is within 75%
of normal for45minand approximately50%of
theratesofcontrols foranadditional3hafter
addition of cycloheximide (30). In contrast,
HeLa cell chromosomal DNA synthesis is
re-duced more than 90% within 10 min of the
addition of cycloheximide (13). This
dissocia-tionbetween protein synthesis and DNA
syn-thesisdemonstrated by both mitochondria and
adenovirus is notfound invarious other DNA
viruses infecting eucaryotic cells. Inhibition of
proteinsynthesisincells infected with polyoma
virus (3), simian virus 40 (SV40) (19), rabbit
poxvirus(18), orpseudorabiesvirus (16) results
inarapid cessation of viral DNA synthesis.
The effects of three DNA viruses, SV40 (21), polyoma (33), and herpes simplex type 1 (25,
26), onmitochondrial DNA synthesis have been
studied. SV40 infection of confluent African
green monkey kidney or mouse embryo 3T3
cultures results in a stimulation of both
nu-clearand mitochondrialDNAsynthesis; in
con-trast, SV40 infection of BSC-1 cells does not
stimulate nuclear or mitochondrial DNA
syn-1Presentaddress: Institute of Cancer Research, Colum-biaUniversity College of Physicians and Surgeons, New York,NY10032.
thesis (21). A stimulatory effect on
mitochon-drial DNA synthesis also results when
con-fluent 3T3 cultures are infectedwiththe DNA
tumor virus, polyoma (33). In type 1 herpes
simplex-infected HeLa cells, chromosomal
DNA synthesis is inhibited during 1 to 5 h
postinfection (p.i.), whereas mitochondrial
DNAsynthesis wasstimulated (26). Similarly,
aherpes-inducedenhancement of DNA
synthe-sis in isolated HeLa mitochondria has been
demonstrated (25).
Because of the similar mechanism of
dis-placement synthesis and resistance to protein
inhibitorsduring the replication of both
adeno-virusandmitochondrialDNA,wehave
investi-gatedthe effect of adenovirus type 2 (Ad2)
in-fection on mitochondrial DNA synthesis in
HeLacells.
MATERIALS AND METHODS
Cells and virus. Thesourcesof HeLa S3 cellsand
Ad2 have beenpreviously described(22). Cellswere grown in suspension culture with Eagle medium
supplementedwith 5%fetalcalfserum. Cellswere infected withpurifiedvirionat aninputmultiplicity
of4,000 particles/cell as previously described (10). Mock-infectedcells weresimilarly washedand con-centrated, butno virus wasadded.
Isolation of mitochondria. Mitochondria were isolated fromcells that were washed twiceinEagle
medium without serum and suspended in hypotonic bufferconsisting of 50mMNaCl,1.5mMMgCl2,and 10 mMTris-hydrochloride,pH8.0(9). Thecells (5x 106/ml) were ruptured by homogenization (16
strokes) with a Dounce homogenizer. Approxi-mately95% of the cells were disrupted, as monitored by phase-contrast microscopy. The cytoplasmic su-pernate was freed of nuclei and debris by three
centrifugationsat2,000 rpm for5mininthe inter-national PRJ centrifuge. Mitochondria were sedi-mented from the supernate by centrifugation at 10,000 rpm for30 min in the angle30 rotorof the 340
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tracting the aqueous phase twice with chloroform-isoamyl alcohol, as described by Schildkraut and Maio (28). The RNase step included in their proce-dure (28) was omitted because Hela cell mitochon-drial DNA containsa small number of ribonucleo-tides and is, therefore, nicked by RNase A andT, (34).
Characterization of mitochondrial DNA. Mito-chondrial DNApurifiedfromAd2-infected cultures ormock-infected HeLa cell controls wasanalyzedby sedimentation in neutral sucrose (12) and cesium
chloride-ethidium bromide (CsCl-EtBr) (24)
gra-dients. Foranalysis of DNA in neutral sucrose, a 0.5-mlsample ofpurified mitochondrial DNA was
layered on a 16.5-mlgradientcontaining 5 to 20% neutral sucrose in 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA with a 0.5-ml cushion of 60%sucrose. Sampleswere centrifugedfor 18hat 24,000 rpm inthe SW27.3 rotor of the L350Beckman
ultracentrifuge. Gradients were fractionated by
pumping equal portions (approximately 0.6 ml)
throughaprobeplaced1.5 cmabove the bottom of the tube. Determination ofacid-precipitable radio-activity was performed by methods previously
re-ported(10). ForCsCl-EtBranalysisofpurified mito-chondrialDNA,a0.75-mlsamplewasaddedto3.2 g ofCsCl,0.2mlof EtBr solution (10mg/ml),41Lgof calf thymusDNA, and2.25 ml of0.Olx SSC. The refractive index was adjusted to 1.3888 (density,
1.580 g/cm8) with 0.01x SSC. Samples were then
centrifugedinaSpincoangle40rotor at36,000rpm for 40to48h,and0.1-mlfractionswerecollectedby
puncturing the bottom of the tube. Refractive in-dexes of every fifth samplewere determined, and
acid-precipitablematerialwasquantitated.
Band sedimentation of DNA on alkalinesucrose gradients. Whole cells wereplaced onalkaline su-crosegradientsandsedimentedat24,000rpmfor14 h withanSW27.3 rotor,aspreviously described (10).
Gradientswere fractionated, and the incorporated radioactivity in the fractions and the sonically
treatedpellet (2ml)wasquantitated. Under these
conditions,cell DNAwaspelletedintothecushion,
and viral DNAappearedinthegradient.
Radioactivelabelingof DNA.Marker HeLa
mito-chondrialDNAwaslabeled with['4C]thymidine by
growingcells for3daysinmediacontaining3,uCiof
[I4C]thymidine (54 mCi/mmol) per 100 ml.
Mito-chondrial DNA from mock- andAd2-infected cells wasisolated from1 x 108to1.5x 108cells grown for 4 h with 1.5 mCi of [3Hlthymidine (15 Ci/mmol)
addedattime0, 4, 8,12, 16,or20hp.i.Ad2-infected cells (1.25x 106),used forquantitationof viral DNA at various times, were labeled with 25 ,ACi of
[3H]thymidine (16Ci/mmol)for1hfollowedbya 30-minchase withunlabeledthymidine(10-sM).
and relaxed-circular (form II) mitochondrial
DNA, whichsedimentat37Sand 26S,
respec-tively (14), were separated from mock- and
Ad2-infected HeLacells by centrifugation ina
5 to 20%neutral sucrose gradient (Fig. 1). By
using 14C-labeled uninfected cells mixed with
3H-labeled mock- or Ad2-infected cells, it was
possibletoquantitatemitochondrial DNA
syn-thesis until 12h p.i. Total mitochondrial DNA
synthesis, monitoredfor 4-hperiods,wasfound
tobe similar in4-, 8-, and 12-h Ad2- or
mock-infected cultures (Fig. 1 and Table 1). Theuse
ofneutral sucrose gradients to monitor
mito-chondrialDNA synthesisbeyond 12 hp.i. was
not possible, because large amounts of Ad2
DNA(31S)contaminated the mitochondrial
re-gion of gradients. We and others have found
that most of theencapsidatedadenoviralDNA
leaksfrom the intact nucleiprepared by 0.5%
Nonidet P-40lysis of cells and isfound inthe
cytoplasm (M. S. Horwitz, personal
observa-tion; reference 4). Unencapsidated viral DNA
remainsinthenucleus. Since thereis
approxi-mately a 6-h lag between the onset of viral
DNA synthesis and its encapsidation into
virion, we were ableto use the sucrose
gradi-entmethod until12hp.i. The DNAinvirions,
whichcontaminated the cytoplasm, sedimented
with themitochondriaat10,000 xgfor30min.
Virion could notbe separated from
mitochon-dria by sedimentation through a 15 to30%
su-crosegradientat24,000 rpm for30min(Fisher,
personal observations). The inability to
subse-quently separate mitochondrial from
adeno-virus DNA on sucrose gradients necessitated
the useofCsCl-EtBrequilibrium gradientsto
separate mitochondrial DNA from adenovirus
DNA when the cells were infected for more
than 12h.
CsCI-EtBr gradient analysis of
mitochon-drial DNA synthesis. Form I mitochondrial
DNAwas, therefore,quantitatedincells either
mock or Ad2 infected from 4 to 24 h earlier
(Fig. 2and Table 1). Between0and16h,
mito-chondrial DNA synthesis in both mock- and
Ad2-infectedcultures wassimilar, whereas
be-tween 16 to 20 and 20 to 24 h, mitochondrial
DNA synthesis in mock-infected cells was
ap-proximately twice that found in Ad2-infected
cultures. This apparent decrease in
mitochon-4
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0 1
3-
2-0 10 20 30 0 10 20 30
FRACTION NUMBER
FIG. 1. Neutral sucrosegradientanalysis ofmitochondrial DNA synthesis in mock-infectedand Ad2-infectedHeLa cells. (A) Mitochondrial DNA wasisolatedfromamixtureof108mock-infectedHeLa cells labeled with[3H]thymidinebetween 0 and4hand 5 x 107[14C]thymidine-labeledHeLa cells(seetext).A 0.5-ml sampleofpurifiedDNAwas run on a5to20%neutralsucrosegradientfor18hat24,000rpm.Gradients werefractionated,andacid-precipitable radioactivity fromeachfractionwasdetermined. (B)Mitochondrial DNA wassimilarly isolatedfromamixtureof108Ad2-infectedcells labeled with[3HWthymidine between 0 and4 hand 5 x 107[14C]thymidine-labeled HeLa cells. Thepurified DNA was run on neutralsucrose
gradientsas describedabove.(Thepeak of formImitochondrial DNA is infraction 8,andformII isin
fraction13.Thepeak oftheAd2 DNAmarker(31S)sedimented infraction10.Sedimentationisfrom
right
to left in allgradients.)80 A B 9
0101.61021
60 -30 -.9
jl.57
40-20
.-a550
10-~~~~
xT
5-4 4
3-3I
2 -2
0 10 20 30 40 0 10 20 30 40 FRACTION NUMBER
FIG. 2. CsCl-EtBrgradient analysis ofmitochondrial DNA synthesis in mock- andAd2-infectedHeLa cells.(A) Mitochondrial DNAwasisolatedfrom108[3Hthymidine-labeledmock-infectedHeLacells labeled for4-hperiods between 0 and24 hp.i.The cellsweremixed with5 x 107[14C]thymidine-labeledHeLacells
beforepurificationofthe mitochondria and the DNA. Thesampleswere runon4-mlCsCl-EtBrgradients for
48hat36,000 rpm.Onehundred-microliterfractionswerecollectedbypuncturing the bottomofthetube,and
acid-precipitable material was determined. Refractive indexes of everyfifth sample were measured. The
samplelabeledfrom0 to 4hp.i. is shown.(Thepeak of formImitochondrial DNA isinfraction10;other linear DNAs bandinthe upperfractions.) (B) Mitochondrial DNAwasisolatedfromAd2-infectedcells and processedidenticallyto(A). Thesample labeled from 0to 4hp.i.isshown,andthe resultsfromother time pointsaresummarizedinTable1.
342
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[image:3.504.111.403.56.267.2] [image:3.504.119.409.373.580.2]8 to 12 1.06 0.98
12 to 16 - 0.97
16 to 20 - 0.46c (0.93)
20 to 24 _ 0.49" (0.96)
aAtotal of 108to1.5 x 108mock-orAd2-infected
HeLa cells were labeled with 1.5 mCi of
[3H]thymidine(15Ci/mmol)for4-h periods.
bMitochondrial DNA synthesis was expressed as the ratio of [3H]thymidine-labeled mitochondrial DNA from adenovirus-infected cells divided by
[3H]thymidine incorporated into HeLa mitochon-drial DNAinuninfected cells. Both numbers were
corrected for losses during processingby addinga constant amount of ['4C]thymidine-labeled HeLa cells to each sample. The "4C-labeled mitochondrial
DNA served as a reference point for the recovery of
purified DNA. Only form I DNA (37S in neutral sucrose or1.595g/cm3inCsCl-EtBrgradients) was used for the calculations. Numbers reflected the average ratios ofAd2-infected to mock-infected cells from two experiments.
cThese numbers reflect the valuesobtainedwhen
the "control" wasmock-infected cells. However, by
16 hafter mock infection, the cell number had dou-bled inthe uninfected cell culture but was
essen-tiallyunchangedinAd2-infected cells. If the values wereeithercorrected for cell numberorcompared
withcells mockinfectedforlessthan 16 h, the val-uesshown inparentheseswereobtained.
drial DNA synthesis in the infected culture
appears toresult fromanincreasedcell number
inthe uninfected controls that had dividedby
16 to 20 h after mock infection. Due to the
feedingschedule ofourHeLacells,theytendto
becomepartially synchronized and divideover
a4-to5-hperiod. This results inanabrupt rise
incell number rather thanagradual doubling
over 18 to 24h (Table 1). Thus, the amountof
[3H]thymidine-labeled mitochondrialDNA
dur-inga4-hpulse increasesat 16hinuninfected
HeLa cells, but remains constant in
adenovi-rus-infectedcells.
Figure 3 compares the species of DNA
syn-thesized and associatedwiththemitochondrial
pelletinadenovirus-infectedandmock-infected
HeLa cells from 16 to 20h p.i. The
mitochon-drial DNAs from both cellsaresuperimposable
(fraction 12), but thereis ashift ofdensity in
thenonmitochondrial DNA atthetopsofthese
gradients. The densityof the DNAfrom
adeno-virus-infected cells (fraction 31) is 6 mg/cm3
greaterthan that ofuninfected cells.Thus,the
C.,
0
Ix
rI
100-
10-
5-I
1 10 20 30 40
FRACTION NUMBER
FIG. 3. CsCl-EtBrgradient analysis of mitochon-drial DNAsynthesisinHeLacellsinfected for16to 20h.Mitochondrial DNAwas isolatedfroma
mix-ture of 108 Ad2-infected HeLa cells labeled with
[3H]thymidine between 16 and 20 h p.i. andmixed
with 5 x 107[14C]thymidine-labeleduninfectedHeLa cells.Samples wereassayedonCsCl-EtBrgradients
asdescribedinFig.2and thetext.
greater density of native adenovirus DNA in
comparisontoHeLaDNAismaintainedinthe
presenceofethidium bromide.
Rates of viral, chromosomal, and
mito-chondrial DNA synthesis. Ad2 DNA can be
separatedfrom intact HeLa cell DNAas a
con-sequence of differences in the sizes of these
molecules (8). With alkaline sucrosegradients
(10), Ad2 DNA isfound inthegradient (34S),
and HeLa cell DNA is largely found in the
pellet (_77to 80S). Theeffect of Ad2 infection
on host chromosomal and mitochondrial DNA
is shown in Fig. 4. Host DNA synthesis was
markedly reduced by10hp.i. andcontinuedto
decline during the course of Ad2 infection,
whereas Ad2 DNAwasdetectedat6hp.i. and
was at a maximum rate at 10 to 14 h p.i. In contrast to thesechanges, mitochondrial DNA
synthesis remainedat aconstantrate
through-outthecourse(0to24h) of Ad2infection (Fig.4
and Table1).
DISCUSSION
Mitochondrial DNA replicationwas
investi-gated in adenovirus-infected cells because of
several unusual similaritiesin the
replication
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[image:4.504.252.447.69.319.2] [image:4.504.46.240.74.338.2]344
IN
LU
50
I-I "
\NVIRUS
,
HOST-2 6 10 14 18 22 24
TIME (hours)
FIG. 4. Mitochondrial, Ad2, and host chromo-somal DNA synthesis in Ad2-infected HeLa cells. Mitochondrial DNA synthesis was determined by
both neutralsucroseand CsCl-EtBrgradient
analy-sis between0 and 24 hp.i.asdescribed in thetext
and Table 1.Ad2 andtotalHeLa cell DNAsynthesis after infection withAd2wasdeterminedby alkaline sucrose gradient analysis. 1.25 X 106Ad2-infected
cellsatdifferent times p.i.werelabeled with 25 uCi [3H]thymidine for1 h andchased with cold thymi-dine (10-5 M) foranadditional 30min. Cells were
pelleted, suspended, and centrifugedasdescribedin thetext.One hundredpercentsynthesis for mitochon-drial DNA refers to the radioactivity of mitochon-drial DNA made in uninfected HeLa cells. The
amount of radioactivity in HeLa cell chromosomal DNA between4and 8 hp.i.wasthe 100%reference
point for both chromosomal and Ad2 DNA synthe-sized in theinfected cells.
of these DNAs. Recently it has been demon-strated thatsynthesis of the linear adenovirus DNA begins at or near the left end for one
strand andtheright end for thecomplementary strand. Replication proceeds in the 5' to 3' di-rectiononeach ofthese strands (12). This mode
ofreplication generates large regions of single-stranded DNA inadisplacement reactionvery
similar to the D-loop displacement shown for mitochondrial DNA (27). In addition, both of thesemammalian DNAsarepeculiarly
resist-ant to the effect of protein inhibitors, which rapidly shut down the synthesis ofmostother eucaryoticaswellasprocaryoticDNAs (13, 30).
The inhibition ofchromosomal DNA
synthe-sis inadenovirus-infected cells isnot well
un-derstood (23); however,it maybesecondaryto
theprofound shutoff of host protein synthesis.
Similar controls do not affect mitochondrial
DNAsynthesisinadenovirus-infected cells,
al-though mitochondrial DNA synthesis is
pre-sumablydependentonenzymessynthesizedon
cytoplasmic polyribosomes. Perhaps the
mito-chondrial DNA is resistant to shutoff because
each round ofreplication does not require the
synthesis of proteins de novo, or the effect of
adenovirus infection on chromosomal DNA
mayinvolvethe inhibition ofselectedproteins
andleaves theproduction of factors needed for
mitochondrial DNA synthesis intact. Although
adenovirus DNA issynthesized in the nucleus
and mitochondrial DNAinthecytoplasm, itis
possible that these two processes share some
replicativeenzymes. There is noevidence that
adenovirus induces the synthesis ofa newDNA
polymerase (2, 15). However, the virion does
code for its own DNA-binding protein (32).
Since adenovirus infection inhibitsnotonly the
synthesis ofchromosomal DNA but also that of
SV40 (13) or vaccinia DNA (6) in cells coin-fected with either of these viruses, the mainte-nance ofmitochondrial DNAsynthesisat
nor-mal levels in adenovirus-infected cells is also
unique inthisregard.
Inall of these experiments, the incorporation
of[3H]thymidineinto an appropriately
charac-terized chromosomal, mitochondrial, or viral
DNA isassumed to correlate with therate of
synthesis of that macromolecule. Since adeno-virus genes do not code for a viral thymidine
kinase (20) and thechange in levels of
thymi-dine kinase in exponentially growing
unin-fectedcells varies less than30%afterinfection
(7), there is probably a good correlation
be-tween [3H]thymidine incorporation and DNA
synthesis. Since chromosomal,
mitochondrial,
andviral DNAweredeterminedunder identical
conditions inthecell, anomalies oflabelingof
mitochondrial DNA could only arise ifit
ob-tained [3H]TTP fromapooluniquetothe
syn-thesis of that macromolecule. Aseparate
thy-midine kinase has beenreported for
mitochon-drial DNA, but its enzymatic activitydid not
change after adenovirus infection (20). Since
adenovirus-infected cells are notorious for the
artifactual translocation of macromolecules
uponcell fractionation (e.g., virion is isolated
inthe cytoplasm asmentioned above), we did
not attempt to measure intramitochondrial
pools of TTP after infection. It is furthermore
unlikely thatcompensatingartifacts oflabeling
inourexperimentswouldcombineto yield con-stantratesof[3H]thymidineincorporation into
mitochondrial formIDNA fora 24-hperiod.
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[image:5.504.68.251.55.321.2]LITERATURE CITED
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