JOURNAL OF VIROLOGY, Jan. 1975, p. 27-3f5
Copyright01975 AmericanSocietyforMicrobiology
Vol.15,No. 1 Printed inU.S.A.
Transcription of
the
Genome
of
Adenovirus Type 12
I.
Viral mRNA in Abortively Infected and Transformed Cells
JUAN ORTIN ANDWALTER DOERFLER*
InstituteofGenetics, University of Cologne, Cologne, Germany Received forpublication22July 1974
In
baby hamster kidney
(BHK-21)
cells
abortively infected
with adenovirus
type
12,
polysome-associated,
virus-specific
RNA could
be detected starting
5 to
7
h
after infection.
The
amount
of
this
RNA
reached
a
maximum between
10
to
12
h after infection
and
continued to
be synthesized
at a
reduced level
until late
in
infection
(48
to
50
h).
In BHK-21
cells transformed
by adenovirus
type
12(HB
cells),
0.26% of
the
polysome-associated mRNA
was
virus
specific.
The
size of
the
virus-specific
mRNA isolated
from
polysomes
of BHK-21
cells
abortively
infected
with,
or
transformed
by, adenovirus
type 12 was
determined
by
electrophoresis
in
polyacrylamide gels
in
98%
formamide, i.e., under
conditions
which eliminated
secondary
structure or
aggregation of
RNA.
In
abortively
infected
hamster cells viral mRNA size classes of
molecular
weights 0.9 x
10'
and
0.65
x
10.
to 0.67
x
106 were
predominant.
A minor fraction of 1.5 x 10
daltons
was
consistently
found and
increased
with time after infection. Late after
infection (24 to 26 h), viral mRNA of 1.9 x
106
daltons was
also
observed.
The
size
distribution
of
adenovirus
type
12-specific
mRNA from transformed hamster
cells
(HB line)
wasvery
similar to that in
abortively
infected
cells,
except that
the relative amount of the viral mRNA fraction of
1.5x 10'
daltons
wasmuch
higher.
It is
uncertain whether the viral mRNA of
high-molecular-weight
represents
mixed transcripts derived from
integrated
viral
genomes and
adjacent
host genes.
Baby
hamster kidney
(BHK-21) cells
infected
with
adenovirus
type 12
(Adl2)
represent
amodel
system
of abortive virus infection which
leads
to
malignant transformation in a small
proportion of
the infected cells (27).
In
nonpro-ductive
infection BHK-21
cells
with Adl2
(4,
24,
25), the
block
in
virus
production
is
located
in
anearly
function, since viral DNA
replication
cannot
be detected
(4, 5, 6, 21), and
synthesis
of
viral
capsid
proteins is not apparent
(20).
Start-ing
atapproximately 8 h after
infection,
the
virus-specific
T-antigen is
synthesized. The
time of onset
of
T-antigen synthesis
depends
onthe actual
multiplicity
of infection. At
24h after
infection with
high
multiplicities, 100% of the
cells contain
T-antigen (24).
The
block for
Adl2
replication does not seem to be due to a
diffusi-ble
repressor-like function, since upon
infectionof
heterokaryons
of BHK-21cells and
permis-siveHEp2
cellswith
Adl2,
viral
antigens and
viral DNAcan be
detected in both types
ofnuclei
(31).
In
cells arrested
inGl phase by serum
deple-tion, cellular
DNAsynthesis
iselicited
starting
at 12h after infection withAdl2,
and reaches
amaximum
at 21 to 24h after infection
(22). In
growing
BHK-21 cells, cellular DNA
synthesis
continues after
infection with Adl2
at anin-creased
rate(4).
The
cellular
DNA
synthesized
starting
24h
after
infection is
cleaved
to,
orsynthesized in, fragments of 5 x 10.
to10
x 106
daltons (4).
Beginning
10to
12h after
infection,
fragments of the
parental viral DNA
areob-served which sediment at a rate of
18S (2).
The DNA of Adl2
becomes
integrated by
covalent
linkage,
probably in the form of
frag-ments, into the genome
ofBHK-21 cells
(3, 5).
This integration is
first detectable at 6 h after
infection and is not
dependent on DNA
replica-tion,
i.e., the viral genome integrates
evenif
DNA
replication is inhibited
>96%
by
cytosine
arabinoside
(5). Inhibition of
protein synthesis
does
notaffect
integration to a great
extent(5).
This
finding
suggests that the function(s)
re-sponsible
forintegration preexists
in the cell or inthe
virion.
Transcription of the DNA of
Adl2
in BHK-21
cells
wasinvestigated by Raska et al. (22) and
by Raska and Strohl (21). Virus-specific RNA
wasfound by
these authors between 10 and 18
27
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h postinfection.
The size ofthis RNA
wasestimated
tobe
approximately 18S (22). From
the results of experiments using the
technique
of competition
hybridization,
it wasconcluded
that the exclusively early RNA is transcribed
inthis
system(21). About 60% of the RNA
se-quencestranscribed
early
in thelytic
system(21)
arepresentin
Adl2-infected BHK-21 cells.
These
sequencescorrespond
tothose which
aretranscribed exclusively early but
notlate in the
productive infection (11, 21). In cells
produc-tively infected with adenovirus
type2,the
early
virus-specific
RNA represents 8 to 45% of theviral
genome(11,
30), corresponding
toRNA ofmolecular
weight
of 0.9 x10'
to 5.0 x10'.
A
review of the
eventsoccurring
inAdl2-infected
hamster cells
waspublished recently
by Strohl (26).
In
the
presentreport,the
kinetics of
appear-ance
of
thepolysome-associated,
Adl2-specific
mRNA
in BHK-21 cells
isdescribed. The size
ofthe
Adl2 mRNAwasdetermined
both inabor-tively infected BHK-21 cells and in the
Adl2-transformed BHK-21 cell line, the HB cells.
The
mainsize
class of viral mRNA both inabortively infected
BHK-21 cellsand
in HBcells
had a molecularweight
of9.0 x105,
butsizeclasses of1.5 x 106,6.5 x 105,and 3.0x 105 to 4.0 x 105 were
also
apparent.The
relative
amount
of
Adl2-specific mRNA
inthe
size-class
of
1.5 x 106daltons increased
with time afterinfection, and reached
amaximum
inthe
trans-formed
HB
cells.
MATERIALS AND METHODS
Cells. KB (CCL17) and BHK-21 (CCL10) cells
were obtained from the American Type Culture
Collection. BHK-21 cellswerealsoobtainedasagift
from P.Faulkner. Human embryonic kidney (HEK) cells used for plaque assays were purchased from
Flow Laboratories. HB cells, a line of BHK-21 cells
transformed by Adl2, were isolated by W. Strohl et al. of Rutgers University and were obtained from
P.Choppin of RockefellerUniversity, New York. Virus. Seed preparations of human Adl2 and adenovirus type 2 were a giftofW.Rowe, National Institutes ofHealth,Bethesda, Md.
Media and sera. The media used in our
experi-mentswere Eagle medium (10), the modification of
Eagle medium forsuspension cultures, and the Dul-becco modification ofEagle medium (1). Calfserum
and fetal calf serum were purchased from Flow
Laboratories, andtryptosephosphate brothwasfrom
Difco Laboratories.
Solutions.PBS, phosphate-bufferedsaline(9),and PBS-d, the same solution without Ca2+ and Mg2+, were used. TE was 0.01 M Tris-hydrochloride (pH 7.5) and0.001 MEDTA. Isotonic bufferfor cell frac-tionation contained 0.15 M NaCl, 0.0015 M MgCl2, and0.01 MTris-hydrochloride (pH 8.5).Thesucrose
solutions for polysome isolation were prepared as follows. To destroy ribonuclease contaminations,
10and 50% solutionsofsucroseindistilled water were
incubated overnight with 0.1% diethylpyrocarbonate
at 37C and subsequently for 30 min at 100 C to eliminate the residualdiethylpyrocarbonate.
The sucrose solutions were adjusted to 7 and 47%
by adding sterile solutions of NaCl, MgCl2, and
Tris-hydrochloride tothe final concentrations of the isotonic buffer. Polyuridylic acid sepharose buffer I
(PUSbufferI)was25%formamide, 0.7 M NaCl, 0.01 M EDTA, and 0.05 M Tris-hydrochloride (pH 7.5).
PUS buffer II was 90% formamide, 0.01 M EDTA, 0.2% Sarkosyl, and 0.01 M Tris-hydrochloride (pH 7.5). SSC was 0.15 MNaCl, 0.015 M sodium citrate.
Sarkosyl buffer consisted of 0.1 M NaCl, 0.01 M Tris-hydrochloride(pH7.5), 0.001 MEDTA, and 1%
Sarkosyl.
Chemicals. Sepharose 4B and Ficoll wereobtained from Pharmacia Uppsala, Sweden.
Polyvinylpyrroli-done and bovine serum albumin, fraction V, were purchased from Sigma Chemical Co., St.Louis, Mo., and Nonidet P-40 (NP-40) was a gift from Shell Chemical Co. Acrylamide and N,N'-methylenebisac-rylamide, obtained from Serva, Heidelberg,
Ger-many, were recrystallized as described previously (15). Polyuridylic acid [poly(U)
I
was obtained fromCalBiochem, Los Angeles, Calif., and nitrocellulose
filters, type BA 85, werebought fromSchleicherand
Schiill, Dassel, Germany. Sucrose, RNase free, was purchased from Schwarz-Mann, Orangeburg, N.Y.; diethylpyrocarbonate, Genetron 113
(1,1,3-tri-chlioro-trifluoro-ethane)
and sodium dodecylsarcosin-ate (Sarkosyl) were bought from Serva, Heidelberg. Sodium dodecyl sulfate (Merck, Darmstadt, Ger-many) was recrystallized from 95% ethanol.Form-amide, analytical grade (Merck) was purified as de-scribedpreviously (29).Thescintillators
2,5-diphenyl-oxazole (PPO), and 1,4-bis-(5-phenyloxazolyl)
benzene(POPOP) were purchased from Merck, and toluene and methanol were from Baker, Doventer, Holland. All other chemicalsandreagents were
ana-lytical grade.
Radioisotopes. [5-8Hluridine (specific activity 24 to 28 Ci/mmol) and
["4CIsodium
formate (50 to 60mCi/mmol) were obtained fromThe Radiochemical Center,Amersham, England.
Propagation of cells. Cell stocks were stored frozen inEaglemedium with 10%calf serum and 10% glycerol in liquid N2. KB cells were grown inEagle
medium supplemented with 10% calf serum, and BHK-21 and HB cellsweregrown inDulbecco medium
supplemented with 10% tryptose phosphate broth and 10%calfserum.Cellsweresubculturedroutinely
twice a week withsplitratiosof 1 to10.
Propagation of Adl2. The methods for cultivation ofAdl2 in suspension cultures have been described elsewhere (7). Suspension cultures of KB cells were infected with Adl2 at a multiplicity of infectioi. (MOI) of 20 PFU per cell and harvested after60hat 37C. The infected cells wereresuspendedin 0.02M
Tris-hydrochloride (pH 8.0),and crude extracts were prepared by ultrasonictreatment.Thecellular debris was pelleted by centrifugation at 4C for 15 min at
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Adl2-SPECIFIC RNA IN BHK-21 CELLS
5,000 x g. The extracts were further purified by
extraction with Genetron 113, and the virus was
purified by threecycles of equilibrium centrifugation in cesium chloride density gradients. The virus was
stored frozenas crude extractand purified immedi-ately priortouse.
AssayforAdl2.Plaqueassayswerecarriedouton
HEK cells in secondary passage as described
previ-ously (18). Adl2 (p = 1.328g/cm') wasidentified by
its characteristic buoyant density by equilibrium sedimentation in CsCl density gradients (4) in a
model E analytical ultracentrifuge (Beckman) orin an SW56 rotor of the Spinco L2-65B preparative ultracentrifuge. Ad2(p = 1.334g/cm8) wasusedasa
density marker.
Assayformycoplasma.At leastonceweekly, cell
cultures were tested for mycoplasma contamination
as described by Hayflick (13) with minor
modifica-tions. Furthermore, the cellswereroutinely screened
formycoplasma by electron microscopy. The cultures used forthe experimentstobedescribedwerefound
free ofmycoplasma by these methods.
Preparation of labeled rRNAas marker.
Expo-nentially growing BHK-21 cells were labeled for 2 days with
[14C]sodium
formate (10ACiIml)
in Dul-becco mediumsupplemented with 10%tryptose phos-phate broth and 10% fetal calfserum(DTFC). Radio-activemediumwasremoved, and the monolayerwaswashed and incubated with fresh DTFC foran
addi-tional 6-h period. Subsequently, the cellswere
har-vested and fractionated intonuclei and cytoplasm as
described below. The cytoplasmic fraction was
ex-tracted withphenol-chloroform-isoamylalcoholas
de-scribed by Weinberg et al. (32), and the RNA was
precipitated with ethanol overnight at-20C. Infection of BHK-21 cells with Adl2. BHK-21 cellsgrowninDTFCwereinoculated with
CsCl-puri-fied Adl2atanMOI of 100to200PFUpercell. The
medium was removed and the monolayers were
washed with PBS. The cells were inoculated with
Adl2 diluted in PBS or mock-infected with PBS.
Aftera2-h adsorption periodat37C, theinoculum
was removed, the cells werewashed withPBS, and
freshDTFCwasadded.
Labeling of infectedcells.Atdifferent timesafter infection, as shown in each experiment, infected or
mock-infected cellswerelabeledfor2hat37C with [5-'H]uridineat400gCi/mlinDTFC.
Cell fractionation and isolation of polysomes. After the labeling period, the monolayers were
cooled to 0 C and washed three times with PBS-d. Thecells were scraped off the plate and lysed with
isotonic buffer containing 0.5% NP-40 for 10 min at
DC.Suspensionsofnuclearwerevortexedfivetimes
for10s,and nucleiwerepelleted by centrifugationfor 3min at 2,000 x g. Thenuclei pellets were washed oncebythe same procedure, and both supernatants werepooledtoyield thecytoplasmicfraction.To iso-late the polysomes, the cytoplasmic fraction was
layered ontopofa7to47%sucrosedensity gradient
inisotonic buffer andcentrifugedat4C for120to150 min at 35,000rpm in an SW41 rotor. In Fig. 1 the resultofatypical polysome isolation experiment is
presented. Routinely, fractions corresponding to S
6'-,4'
--CM
.
0.10 (D
C\j
10 20
FRACTION NUMBER
FiG. 1. Isolation of polysomes from BHK-21 cells infected with Adl2. BHK-21 cells grown on 60-mm
plastic dishes were labeled with [5-'H]uridine (400
uCi/ml) between 10 and 12 h postinfection. Subse-quently, the cells were fractionated into nuclei and
cytoplasm as described in Materials and Methods.
The cytoplasmic fraction was centrifugedat 1 C for 120minat35,000rpmon a7to47%sucrosegradient
inisotonic buffer.Inportions ofeachfractionthe ab-sorbancy at 260 nm (0) and trichloroacetic
acid-insolubleradioactivity (0)weredetermined.
valueshigherthan 100werepooled, and the RNAwas
extracted and subjected to affinity chromatography
onpoly(U) sepharose.
Poly U sepharose chromatography. Poly(U) sepharosewasprepared by the method described by
Lindberg and Persson (14) with minormodifications. Dissociation of polysomes and chromatography on
poly(U) sepharose was performed as reported by
Lindbergetal. (15).RNAmolecules (atleast in part mRNA) containing sequences of polyadenylic [poly
(A)]
acid and RNA molecules lackingsuchsequencescould be separated by affinity chromatography on
poly(U) sepharose columns. The data presented in Fig. 2 show the elution profile of the (3H]uridine-labeled RNA extracted from polysomes from an
experiment similar to the one shown in Fig. 1.
Subsequently, both the poly(A)-containing RNA (PUS I fractions) and the RNA without poly(A) sequences (PUS I fractions) were precipitated with ethanol before they were analyzed by velocity
sedi-mentation on sucrose density gradients or by
poly-acrylamide gelelectrophoresis.
Analysis of RNA on sucrosedensity gradients. Theethanolprecipitatesof the PUSIIfraction were
resuspendedin 0.1 x SSC andcentrifugedon5to20%
sucrosedensity gradients in Sarkosyl bufferat4C for 6h at35,000rpminan3W41rotor.Portions ofeach fractionwerecounteddirectly,and theRNA from the remaining volumewashybridizedtoAdl2 DNAfixed
onnitrocellulose filtersasdescribedbelow.
Polyacrylamide gel electrophoresis. Polyacryl-amide gel electrophoresis in the presence of 98% formamidewascarriedoutessentiallyasdescribedby
Duesberg and Vogt (8). Upper and lower bufferswere
0.02 M sodium phosphate (pH 6.8) in water and formamide, respectively. RNA samplesin0.2 ml ofa
mixturecontaining1volume of bufferedformamide,3
VOL. 15, 197fi
29
0
0
*~~*" *
a~~~
I.a....~~~~~~~~~~~~~~~~I/~~~~~~~~~~~~~~~~~~~
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[image:3.504.253.446.60.220.2]PUS
n
buffer
10-w~ v
E
a
u
2-I
1 2 3 4 5 6 7 8 9 10
[image:4.504.62.254.60.217.2]Elution steps
FIG. 2. Poly(U)sepharosechromatography
of
poly-somal RNA. Polysomes were isolated as described inFig.
1, dissociated by the addition of Sarkosyl andEDTA to final concentrations of 1% and 0.03
M,
respectively, and subsequently diluted five times withPUSIbuffer. This RNAsolution was passed over a
poly(U) sepharose column of 1-ml bed volume at a
flowrateof 0.2 to 0.4ml/minat 4 C. Thecolumn was washed three times with 2 ml of PUS Ibuffer, and the
boundRNA was eluted six times with 1 ml of PUS II buffer. The first bar in the graph represents the 3H radioactivity in the percolate of the column. volumes ofglycerol, and 6volumes of formamide were
appliedto 8-cmgels consisting of 3.5% polyacrylam-ide in bufferedformamide. The samples were overlaid with buffered formamide and subjected to electropho-resis for 16 h at 100 V at room temperature. After electrophoresis, the gels were cut into 2-mm slices, andthe RNA from eachslicewas eluted for 24 h at 37Cin1ml ofhybridization buffer,andhybridizedto Adl2 DNAonnitrocellulosefiltersasdescribed below. DNA-RNAhybridization. Adl2DNAwasbound tonitrocellulose filtersbyfilteringthe DNAsolution in4 x SSCat arateof <5ml/min. The filters were kept overnightunder vacuum and heated for 2 h at 80C beforeuse.
The DNA-RNA hybridization reactions were car-riedoutwithAdl2DNAboundtonitrocellulose filters (13or25 mmindiameter) in 0.3to1.6ml of the RNA solution in 50% formamide, 5 x SSC, and 0.1% sodium dodecyl sulfate, for 3 days at 37C. After
hybridization,the filterswerewashed with2 xSSC, treated with RNase A(heated for10minat90C),20 Mg/mlin2 x SSC, for 1hat roomtemperature, and washed again with2 x SSC.
For DNAsaturationexperiments, aseries of filters containing 0.5, 1, 2, and4
Ag
ofAdl2DNAwasused, and thehybridizationdatawereplottedasdescribed by Lucas andGinsberg (17). The data presented inFig.3show theextentofhybridizationofthe
poly(A)-containing RNA isolatedfromAdl2-infected BHK-21 cells at various times after infection andfrom HB
cells, an Adl2-transformed BHK-21 cell line, to increasingamounts ofAdl2DNA. Fromthe plot, the saturation hybridization value and the amount of DNArequired for saturation were calculated. In all experiments describedinthis report, DNA-RNA
hy-bridization wascarriedoutunder conditions of DNA excess, using5 ugofAdl2DNAper filter.
Determinationofradioactivity.For direct count-ing, sampleswerediluted inascintillatorcontaining
5.0 g ofPOP and 0.3 gofPOPOP per liter ofa 1:1
toluene-methanolmixture. Insome
experiments,
por-tions of each sample werespotted
ontoglass
fiberfilter disks,
dried,
and counted in a scintillator containingonly
toluene and POP and POPOP asdescribed.
To determine the trichloroacetic acid-insoluble
radioactivity, samples were
precipitated
in 10%tri-chloroaceticacidfor 10 minat0Cin the presence of 50
Ag
ofbovineserumalbuminpermlascarrier.The sampleswerepassed
throughglass
fiberfilters and thefilterswerewashedwithcold5%trichloroacetic acid and ethanol.
Subsequently,
thefiltersweredriedandcounted inthetoluene-based scintillator.
Physical determinations. Radioactive
samples
werecounted in a 3385Tri-Carb
liquid
scintillationspectrometer. The optical density of
samples
wasmeasured in a Zeiss
PMQ
II spectrophotometer. AnalyticalcentrifugationswereperformedinaSpinco model E analytical ultracentrifuge (Beckman, Inc.,Palo Alto, Calif.)
equipped
with UV optics and a monochromator.RESULTS
Kinetics
of
transcription
of
Adl2 DNA in
BHK-21 cells.
In earlier work(21,
22),
itwasdemonstrated
thattranscription
ofAdl2 DNAin
BHK-21 cells
startsapproximately
10 h
C-0
20
'0
z
0
2
DNA (zig/fiIter)
FIG. 3. Saturation hybridization of viral mRNA withAdl2DNA. The dataarepresentedasreciprocal
plots and are derivedfrom saturation hybridization experiments usingpoly(A)-containingRNAlabeledin
Adl2-infected
BHK-21 cellsat 7 to 9hpostinfection(PI)(circles), 10 to 12hpostinfection(triangles),and 24 to 26 hpostinfection(squares). The RNAfromHB cells, an Adl2-transformed BHK-21 cell line (crosses), was also analyzed. The RNA inputs per filter wereasfollows: 7to9hPi, 134,300
counts/min;
10 to 12 h PI, 129,400counts/min;
24 to 26 hPI,
115,600counts/min, and HBcells, 60,350
counts/min.
The values on the ordinateare expressed as micro-grams ofDNA per counts per minute at saturation (reciprocalplot).
30
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[image:4.504.296.427.377.540.2]Adl2-SPECIFIC RNA IN BHK-21 CELLS
postinfection,
and that only the exclusively early
genes
of
Adl2
are
transcribed.
In
this
report,
the
transcription of
the
Adl2
genome in
abortively
infected cells was
further investigated, and the
time
course of appearance of
viral mRNA on the
polysomes was
determined.
Virus-specific
RNA containing poly(A)
se-quences was first
detected in association with
polysomes between 5 and 7 h after infection.
Viral RNA was
clearly demonstrable between 7
and 9 h after
infection, and the amount of viral
RNA reached a maximum
between
10
and 12 h
(Fig. 4). At later times after infection, there was
a
steady decline in the amount of viral RNA
synthesized;
however,
small
quantities
of
virus-specific
RNA
continued
tobe
produced late,
i.e., 48 to 50 h after infection. As pointed out
above,
all experiments were
performed under
saturation conditions for DNA-RNA
hybridiza-tion.
Characterization
of
virus-specific
mRNA
from Adl2-infected
BHK-21
cells: analysis by
velocity sedimentation on sucrose density
gradients. The size
distribution
of
Adl2-specific mRNA
synthesized in BHK-21 cells
was
determined
by velocity sedimentation on
sucrose density
gradients. At various times after
z
0
N
20 40
TIME AFTER INFECTION (HOURS)
FIG. 4. Time course of synthesis ofAdl2-specific
RNA in BHK-21 cells. BHK-21 cells inoculated with
Adl2 at a multiplicity of infection of about 200 PFU/cell or mock-infected with PBS were pulse-labeled with
[5-'H]uridine
at different times afterinfection as indicated. The cells were fractionated
into nuclei andcytoplasm, the RNA wasextracted, and mRNA molecules were selectedby PUS chroma-tographyasdescribedbefore. mRNAwasanalyzed by saturation hybridization with increasingamounts of
Adl2
DNA on nitrocellulose filters. The 3H-labeled RNA bound at saturation levels was calculated as described in Materials and Methods and is presented hereaspercentageof the inputradioactivity applied to the filters. Background values of hybridization (usually <0.01%) derived from experiments with mock-infected cells were subtractedfrom all experi-mental values.infection,
the RNA
waslabeled with [3H
uri-dine,
and the
polysome-associated
mRNA
wasisolated and sedimented
on sucrosedensity
gradients. The
virus-specific RNA was
identi-fied
by DNA-RNA
hybridization.
At
the three time
points
investigated,
7 to9,
10 to 12,
and
24 to26h after infection
(Fig.
5),
the
major
species
of
Adl2-specific
RNA
sedi-mented
at
17 to18S. This result confirmed the
-n o 01
C
-I
C
F-I
[image:5.504.259.447.180.419.2]Fraction Numbpr
FIG. 5. Analysis ofAdl2-specific RNA by velocity sedimentation on sucrosedensity gradients. BHK-21 cells grownin 100-mmplasticdisheswereinoculated with Adl2at a multiplicity of infection ofabout200
PFUIcell
or mock-infectedwith PBS. Subsequently,the cells were pulse-labeled with [5-3H]uridine at time points as indicated. The poly(A)-containing
RNA waspurified from thepolysomes as described andanalyzed by sedimentation on 5 to20osucrose
density gradients in Sarkosyl buffer. The samples
weresedimentedfor6hat35,000rpm and4C inan
SW41rotor.Asmarker, ['4C]rRNAwasaddedtoeach
gradient;thepositions ofthe18Sand28SrRNA in the
gradientsareindicatedbyarrows.Totalradioactivity
wasdetermined in
20-,gliter
portions ofeachfractionand theremainingRNAwashybridizedtoAdl2 DNA under conditionsofDNA excess (5
Mtg
perfilter) on nitrocellulose filters, as described in Materials and Methods. The values of percent countsper minute hybridized, expressed on the ordinate, were calcu-latedaccordingtotheformulaasfollows: (counts per minute hybridized in each fraction x 100)/(totalcountsperminuterecoveredfromgradient).(a) RNA from uninfected control cells. (b) RNA from
Adl2-infectedcells labeled7 to 9hpostinfection. (c) RNA fromAdl2-infectedcellslabeled10to 12h postinfec-tion. (d) RNAfromAd12-infectedcellslabeled24 to 26hpostinfection.
31
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[image:5.504.52.245.367.511.2]finding of
Ragka
etal.(22).
Early
andlate
afterinfection,
minorfractions
ofmRNA
werefound
sedimenting heterogeneously between
25Sand
35S. Since the RNA used
inthis
study had been
denatured
by
90%formamide
during
theelution
frompoly(U) sepharose, it
isunlikely
that the
fast-sedimenting
RNA could be
accounted for
by aggregation of
17S RNA molecules.
How-ever, torule
outthis
possibility
and
toobtain
amore
precise estimate of the molecular
weight
of
these RNA
species,
the viral RNA isolated
fromabortively infected cells
wasalso
analyzed
by
polyacrylamide
gel
electrophoresis
in 98%formamide.
Analysis
by
polyacrylamide gel
electropho-resis in 98%
formamide. The
polysome-associated viral mRNA from
Adl2-infected
BHK-21cells
wasalso
analyzed
under
condi-MOCK INFECTED
10
2 10 12 HP
10
tions
that
completely denatured RNA. The
major size classes
ofviral
mRNA were deter-minedby electrophoresis in polyacrylamide gels
in98%
formamide(Fig.
6). The majorcompo-nents had molecular
weights of 0.9 x 106and
0.67x
106 to0.65
x 105 at all time
periodsafter
infection that
wereinvestigated.
There was aminor
viralmRNA
component of 1.5 x106
daltons whose proportion
increased slightly withtime
after infection. At late times after infection (24 to 26 h), a new size class of 1.9x
106
daltons could be detected.
The small amountof
low-molecular-weight
RNA of heter-ogeneoussize
wasprobably
dueto breakdown of viralRNA. It
should
beemphasized
that, due tothe sensitivity
ofthis
method to detect breaks inthe RNA
molecules
and the high specificradio-activity
ofthe
RNA,
any delay to perform theC)
-8 4
-106
3:
1056
as
EP
0n
_ lo
20 40 20 40
Fraction Number
FIG. 6. AnalysisofAdl2-specific RNA from Adl2-infected and mock-infected BHK-21 cellsby electrophore-sis onpolyacrylamidegels in98%formamide. Thepolysome-associated, poly(A)-containing RNAwasisolated fromAdl2-infectedBHK-21 cellsasdescribedinthelegendtoFig.5.TheRNAwasanalyzedbyelectrophoresis onpolyacrylamide gelsin98%formamideasdescribed in MaterialsandMethods.Asmarker,
"4C-labeled
18S and28SrRNAwasused.Electrophoresis wasperformedatroomtemperaturefor16hat100V.Subsequently,thegelswerecutinto2-mmslicesandthe RNAfromeach slicewaseluted with
50%o
formamide,5 xSSC,and 0.1%sodium dodecyl sulfatefor24h at37C.The 3Hand "4C activitieswere counted inoneportion ofeach fraction; anotherportion was used inDNA-RNA hybridization experiments with Ad12DNA (5ug/filter).
According tothedatapresented inFig.3, thisamountofDNA is well inexcessover the
viral-specific
RNA present in the gel. The values ofpercent countsper minute hybridized, expressed on theordinate,
werecalculatedasdescribed in thelegendtoFig.5.
on November 10, 2019 by guest
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[image:6.504.145.385.277.548.2]Adl2-SPECIFIC RNAIN BHK-21 CELLS
analysis
resulted
in an increase in the
low-molecular-weight
RNA
and
aconcomitant loss
of
the
high-molecular-weight molecules.
Characterization
of
Adl2-specific
mRNA
from HB
cells.
Growing
HB cells
werepulse-labeled
with
[5-3Hjuridine
(400 MCi/ml) for 2 h,
and the polysome-associated mRNA was
iso-lated as
described in Materials and Methods. In
DNA-RNA
hybridization experiments, 0.26% of
the
labeled,
poly(A)-containing RNA associated
with
polysomes
wasfound
to
be Adl2
specific.
The data
presented
in
Fig.
3(cf. curve
desig-nated by
crosses) relate the
degree
of
hybridiza-tion
of
Adl2-specific mRNA
from HB
cells
tothe amount of Adl2 DNA on the
filter. All
experiments
described
in
the following were
performed under conditions of DNA excess (5
Ag
per
filter).
The
Adl2-specific mRNA
from
Adl2-trans-formed BHK-21 cells
(HB
cells)
wasalso
ana-lyzed
by polyacrylamide gel electrophoresis
in
98%
formamide
and
by
DNA-RNA
hybridiza-tion of each
gel
fraction
(Fig. 7).
The main size
classes of viral RNA
corresponded
tomolecules
of
1.5 x106
and 0.88
x106 daltons with minor
peak fractions
at0.6
x
106
and 0.47 x 106
daltons.
DISCUSSION
Adenovirus
type
12infects BHK-21 cells
abortively. At
20min after
inoculation, viral
particles
can
be
readily detected
inthe
cyto-plasm
of
the
infected
cells
by
electron
micros-copy
(D.
T. Brown
and W.
Doerfler,
unpub-lished
results).
At
2h
postinfection,
anAdl2-specific complex
of
DNA, RNA,
and
protein
can
be
isolated
(7).
The data
presented
in
this
communication
showed
that
virus-specific
RNA
associated with
polysomes
could first be
detected
between
5and
7h
postinfection.
The
amount
of
newly
synthesized
viral mRNA
wasmaximal at
10 to 12h
postinfection and then
de-creased,
but
wasstill
detectable
aslate
as 48 to50
h
postinfection. The reasons for this
de-creasein
the rate of viral mRNA
synthesis are
not
understood. One possible explanation is
that the fragmentation of cellular DNA which
took
place late after infection (4) altered
cellu-lar functions
which may
be responsible for viral
transcription.
Thesynthesis
ofT-antigen starts
asearly as 8 h postinfection (24). Therefore, if
the
T-antigen
isindeed
a
virus-coded
function,
itstime
of
synthesis correlates fairly well with
the
onsetof viral
mRNA
synthesis.
Viral mRNA
hasbeen
characterized
by
poly-acrylamide gel electrophoresis
inthe
presence
of98%
formamide. This
technique
represents
thex -.1~~~~~~~~
.N
7o106
u
o 0
Fraction Number
FIG. 7.
Analysis of
Ad12-specific
RNA isolated from HB cells by electrophoresis onpolyacrylamide gelsin 98%formamide.RNApulselabeledfor2h in HB cells wasisolatedandanalyzedasdescribed in thelegendtoFig.6. TheRNA elutedfrom eachgelslice washybridized toAdl2DNA onnitrocellulosefilters (5 pg
of
DNA perfilter). As shown in Fig. 3, this amount of DNA is in excess over the viral RNA present in thegel. Thelinerepresented by trianglesshows the result of unspecific hybridization to calf thymusDNA (5
jg
perfilter). The valuesofpercent countsper minutehybridized, expressed onthe ordi-nate, were calculated as described in the legend toFig.5.
most
reliable method for the determination of
molecular
weights of RNA molecules, since both
intermolecular
aggregation and intramolecular
interactions are negligible under these
condi-tions
(8).
Viral mRNA has been analyzed both
in
abortively
infected BHK-21
cells at different
times after
infection
and in BHK-21 cells
trans-formed
by
Adl2,
the HB line.
This line has been
isolated by W. A. Strohl et al. (28). HB cells
contain many
genome
equivalents of
Adl2
DNA
asshown by the technique of reassociation
kinetics (E. Fanning and W. Doerfler,
unpub-lished results).
The
results of
gel
electrophoresis experiments
in
the presence of 98% formamide demonstrate
that the viral mRNA
inBHK-21 cells abortively
infected with or transformed by
Adl2
is very
similar in size. The main size
classes of viral
mRNA had molecular
weights of 0.88 x
106
to
0.90x
106and
0.65x
106
to 0.67 x 106. A minor
fraction
of 1.5 x
106
daltons consistently
oc-curred
inabortively infected cells and increased
with time
after
infection. At 24 to 26 h
postin-fection, there
wasalso a minor
peak at 1.9 x 106
daltons.
InAdl2-transformed
cells, the RNA
fraction of 1.5 x
106
daltons was
very
pro-nounced.
933
VOL.15,1975
on November 10, 2019 by guest
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[image:7.504.263.442.63.250.2]It
has
not yetbeen shown that
thedifferent
sizeclasses
ofviral mRNA
areindeed distinct
specieswith
nonoverlapping sequences.
Ifthisassumption
werecorrect,
about
30% of thetranscriptional
capacity
of
the
Adl2
genomewould
be expressed
incells
abortively
infected
with,
ortransformed
by,
adenovirus
type 12.The
patternof
Adl2-specific
mRNA isolated
from
KB
cells
atearly times after productive
infection
with
Adl2 is almost identical
tothe
distribution of
Adl2-specific
mRNA isolated
from HB cells
(K. H.
Scheidtmann,
J.
Ortin,
and W.
Doerfler,
unpublished results).
At this time, it is uncertain whether the
Adl2-specific transcripts in
abortively
infected
and transformed hamster cells
arederived from
integrated viral
genomes(3,
5)
orfromfree viral
DNA.
To
distinguish
between these
twopossibili-ties,
astudy has been initiated
todetermine the
specific
regions
ofthe viral
genome fromwhich
these RNA molecules have been transcribed.
Mulder
etal. (19) have demonstrated that
Adl2
DNA
iscleaved
tosixunique fragments
by
the
restriction
endonuclease Eco R R. These
frag-ments can
be
separated
by
electrophoresis
onpolyacrylamide
agarosegels.
Preliminary results
(J. Ortin and W.
Doerfler,
unpublished
results)
indicate that the
polysome-associated
RNA
from
Adl2-infected BHK-21 cells anneals
pref-erentially
with
fragments
A(left
molecular
end)
and
C
(right molecular
end)
ofAdl2 DNA.
Inthis
context it isinteresting
to notethat
Sambrook
etal.
(23)
have
reported
that,
in
several lines
ofadenovirus type
2-and
adeno-virus
type5-transformed
ratcells,
fragments
of
viral
DNAcorresponding
tothe
right
and left
molecular ends
ofthe viral
genomepersist.
Thus,
it willbe
interesting
tocomparethe
pat-terns of
transcription
of the viral
genomein
abortively
infected and
intransformed cells.
ACKNOWLEDGMENTS
Thisresearchwassupported bythe Deutsche Forschungs-gemeinschaft (SFB74).J. 0. was afellow of theEuropean
MolecularBiologyOrganization.
We thankElisabeth Marczinski-Verheugt and Marianne
Stuppforexcellent technicalassistance.
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VOL. 15, 1975