ArticlePDF Available

Inhibition of SV40 T antigen formation by interferon

Authors:

Figures

Content may be subject to copyright.
INHIBITION
OF
SV40
T
ANTIGEN
FORMATION
BY
INTERFERON*
BY
MICHAEL
N.
OXMAN
AND
PAUL
H.
BLACK
LABORATORY
OF
INFECTIOUS
DISEASES,
NATIONAL
INSTITUTES
OF
HEALTH,
BETHESDA,
MARYLAND
Communicated
by
Robert
J.
Huebner,
March
29,
1966
The
mechanism
of
action
of
interferon
has
not
yet
been
determined.
However,
a
number
of
studies'
have
made
it
clear
that
its
inhibitory
effect
on
virus
replication
is
exerted
at
a
stage
following
the
uncoating
of
the
viral
nucleic
acid
but
prior
to
the
replication
of
the
viral
genome.
Thus
it
was
of
interest
to
determine
the
effect
of
interferon
on
the
early
prereplicative
function
of
the
input
viral
genome.
The
oncogenic
DNA
virus
SV40,
which
is
capable
of
initiating
both
lytic
and
nonlytic-
infections
in
mammalian
cells,
offers
a
unique
opportunity
to
study
this
question
and
also
to
explore
the
early
events
in
viral
oncogenesis.
Cells
infected
with
the
SV40
virus
or
purified
SV40
DNA2
develop
a
specific
antigen
(T
antigen),
detectable
by
complement
fixation
and
fluorescent
antibody
(FA),
which
is
distinct
from
the
SV40
virion
antigen;
it
is
present
in
SV40
tumors,
transformed
cells,
and
cells
acutely
infected
with
SV40
virus.36
In
cytolytic
infections,
the
SV40
T
antigen
is
produced
early
in
the
infectious
cycle
prior
to
the
appearance
of
infectious
virus
or
SV40
virion
antigen,6
and
inhibitor studies
show
that
T
antigen
is
produced
in
undiminished
amounts
in
the
presence
of
inhibitors
of
viral
and
cellular
DNA
synthesis.7
Thus
the
SV40
T
antigen
appears
to
be
coded
for
by
a
portion
of
the
input
viral
DNA
and
to
be
formed
prior
to,
and
independent
of,
its
replication.
Therefore,
studies
were
carried
out
to
determine
the
effect
of
interferon
on
the
forma-
tion
of
SV40
T
antigen.
In
the
present
studies
the
3T3
mouse
cell
line
was
employed.8'
9
With
high
mul-
tiplicities
of
virus,
nearly
100
per
cent
of
these
cells
are
infected
and
form
T
antigen,
and
transformation
rates
as
high
as
50
per
cent
occur
without
the
synthesis
of
any
infectious
virus
or
virion
antigen.'0
The
absence
of
viral
replication
in
these
cells
eliminates
the
complications
attending
secondarily
infected
cells."'
12
Inhibition
of
SV40
transformation
of
this
cell
line
by
interferon
has
already
been
demonstrated
by
Todaro
and
Baron."
In
addition,
an
interesting
ancillary
finding
is
presented,
namely,
that
many
of
the
SV40-infected
cells
which
initially
form
T
antigen
lose
their
ability
to
produce
T
antigen
with
subsequent
cell
divisions,
while
nondividing
cells
retain
the
T
antigen
for
many
days.
Only
transformed
cells
appear
to
retain
their
T
antigen-forming
capacity
through
many
generations.
Materials
and
Methods.-Tissue
culture:
The
origin
and
properties
of
the
continuous
line
of
mouse
fibroblasts
(3T3)
have
been
described.8'
9
The
line
was
obtained
from
Dr.
G.
Todaro
(New
York
University)
and
was
used
after
11-19
passages
in
our
laboratory.
Except
where
otherwise
noted,
the
cells
were
grown
and
maintained
in
Eagle's
minimal
essential
medium's
in
Earle's
balanced
salt
solution,
containing
four
times
the
usual
concentrations
of
vitamins
and
nonessential
amino
acids,
2
mM
glutamine,
and
penicillin
and
streptomycin
at
concentrations
of
100
units
and
100
gug
per
ml,
respectively
(3T3M),
and
10%
unheated
fetal
calf
serum.
In
all
experiments
the
cells
were
incubated
at
370C
in
a
humidified
atmosphere
with
5%
C02.
Primary
cultures
of
African
green
monkey
kidney
(AGMK)
and
the
continuous
line
of
Cerco-
pithecus
kidney
cells
(BSC-1)
were
obtained
from
Microbiological
Associates,
Inc.,
and
Flow
Laboratories,
Inc.,
respectively.
Maintenance
medium
for
AGMK
cultures
consisted
of
Eagle's
basal
medium
plus
2%
heated
(560C
for
30
min)
agamma
calf
serum
(Hyland
Laboratories,
1133
1134
MICROBIOLOGY:
OXMAN
AND
BLACK
PROC.
N.
A.
S.
Calif.)
with
antibiotics.
BSC-1
cultures
were
maintained
with
3T3M
plus
10%
unheated
fetal
calf
serum.
Virus:
SV40
strain
77714
was
grown
in
BSC-1
cells.
At
the
time
of
maximum
cytopathogenic
effect
(CPE),
the
cultures
were
frozen
and
thawed
three
times;
the
crude
lysate
was
clarified
by
centrifugation
at
1500
g
for
15
min
and
stored
in
1-dram
vials
at
-60'C.
The
pool
used
in
these experiments
titered
2
X
101
tissue
culture
infectious
doses5o
(TCID50)
per
ml
in
AGMK
roller
tube
cultures.
Vesicular
stomatitis
virus
(VSV)
Indiana
strain
was
grown
in
primary
mouse
embryo
tissue
culture.
The
pool
used
titered
106.2
TCID5o
per
ml
and
was
stored
at
-60oC.
Interferon:
Mouse
serum
interferon,
produced
in
vivo
by
the
intravenous
inoculation
of
New-
castle
disease
virus
(NDV)
and
held
at
pH
2
for
7
days,15
was
kindly
supplied
by
Dr.
Samuel
Baron
(National
Institutes
of
Health).
Three
batches
were
used
in
these
experiments;
they
titered
between
1000
and
3000
units
per
ml
by
a
VSV
plaque
reduction
assay
previously
described."5
They
were
retitrated
in
each
of
the
present
experiments
by
pretreating
tube
cultures
of
3T3
cells
with
serial
half-log
dilutions of
interferon
for
18
hr
and
then
challenging
with
103
TCID5o
of
VSV
per
tube.
The
tubes
were
observed
daily
for
7
days.
One
unit
of
interferon
is
here
defined
as
the
amount
that
protects
50
%
of
3T3
tube
cultures
infected
with
103
TCID50
of
VSV.
Titers
of
each
batch
of
interferon
by
this
method
agreed
with
the
titers
obtained
by
the
plaque
reduction
assay
performed
in
Dr.
Baron's
laboratory
within
the
limits
of
reliability
(threefold
over-all)
reported
for
the
latter.16
Control
preparations
consisted
of
uninfected
mouse
serum
treated
at
pH
2
in
parallel
with
interferon
preparations.
Detection
of
SV4O
T
antigen
in
3T3
cells:
Confluent
monolayer
cultures
of
3T3
cells
in
50-mm
plastic
Petri
dishes
(Falcon
Plastics,
Los
Angeles)
containing
11
X
22-mm
glass
coverslips
were
washed
with
3T3M
and
infected
with
a
multiplicity
of
103
TCID50
of
SV40
virus
per
cell.
After
2
hr
adsorption
at
370C,
4.5
ml
of
3T3M
with
5%
heated
agamma
calf
serum
were
added
and
the
cultures
returned
to
370C.
Control
cultures
received
medium
alone.
At
appropriate
times
after
infection,
coverslips
were
rinsed
twice
and
fixed
in
cold
acetone
for
10
min.
In
studies
to
determine
the
effect
of
interferon
on
T
antigen
formation,
confluent
monolayer
cultures
were
drained
and
washed.
They
were
then
incubated
with
appropriate
dilutions
of
in-
terferon
or
control
preparations
for
18
hr,
washed
3
times
with
3T3M,
and
infected
as
above.
In
addition,
tube
cultures
of
3T3
cells
were
incubated
for
18
hr
with
1.5
ml
of
the
same
dilutions
of
interferon
and
control
solutions
and
challenged
with
103
TCID5o
of
VSV
to
provide
a
parallel
titration
of
the
interferon
preparations.
In
one
experiment,
interferon
was
added
for
various
time
intervals
before
or
after
infection
with
SV40.
Some
cultures
were
pretreated
for
18
hr
as
above,
while
other
cultures
received
interferon
after
the
2-hr
period
of
virus
adsorption
or
at
later
time
intervals.
In
the
latter
instances,
the
virus
inoculum
was
removed
before
the
addition
of
the
interferon
solution
and
the
interferon
was
left
on
for
the
remainder
of
the
experiment.
Coverslips
were
bisected
and
stained
for
the
SV40
T
antigen
with
a
serum
pool
from
hamsters
bearing
SV40
tumors.
An
indirect
fluorescent
antibody
(FA)
procedure,
the
specificity
of
which
has
been
well
established,
was
used
as
previously
described.4
Half
coverslips
from
confluent
dishes
were
found
to
contain
approximately
50,000
cells
and
were
read
as
negative
only
after
all
cells
were
visualized.
Positive
coverslips
were
scanned
under
low
power
(10OX)
to
verify
the
uniform
distribution
of
positive
nuclei:
100
high-power
fields
(each
containing
approximately
50
cells)
evenly
distributed
over
the
coverslip
were
then
examined.
Positive
cells
were
counted
in
each
field
and
total
cells
counted
in
every
fifth
field
to
give
an
estimate
of
the
total
number
of
cells
counted.
These
data,
representing
high-power
visualization
of
approximately
5000
cells
(a
ran-
domly
distributed
10%
of
the
total
cell
population),
were
used
to
compute
the
per
cent
cells
with
T
antigen.
Transformation
assay:
The
colonial
morphology
transformation
assay
of
SV40-infected
3T3
cells
has
been
described
in
detail.9'
10
In
experiments
to
determine
the
effect
of
interferon
on
viral
transformation,
confluent
cultures
of
3T3
cells
were
pretreated
with
various
dilutions
of inter-
feron
for
18
hr
at
370C,
then
washed
three
times
and
infected
with
103
TCID50
per
cell
of
SV40
virus.
After
a
2-hr
period
of
adsorption,
4.5
ml
of
3T3M
with
5%
heated
agamma
calf
serum
was
added,
and
the
cultures
were
incubated
for
20
additional
hr
at
370C.
The
cells
in
each
dish
VOL.
55,
1966
MICROBIOLOGY:
OXMAN
AND
BLACK
1135
were
then
dispersed
with
trypsin,
counted,
and
1000
cells
in
5
ml
3T3M
+
10%
fetal
calf
serum
inoculated
into
each
of
10
Falcon
Plastic
dishes,
while
10
dishes
were
seeded
with
200
cells
each.
These
were
incubated
at
370C,
and
fixed
and
stained
with
1%
Mayer's
hematoxylin
at
17
days.
In
the
dishes
seeded
with
1000
cells,
transformed
foci
could
easily
be
identified
against
the
back-
ground
of
confluent
normal
cells
as
dense,
darkly
stained
colonies
of
piled-up
epithelioid
cells.
Normal
3T3
cells
were
triangular
to
fibroblastic
and
stopped
dividing
when
confluence
was
achieved.
Total
colonies
as
well
as
transformed
colonies
were
counted
in
the
dishes
seeded
with
200
cells
to
determine
plating
efficiency.
Transformation
rates
are
expressed
as
the
ratio
of
trans-
formed
colonies
to
both
the
total
number
of
colonies
that
appeared
(t-col)
and
the
number
of
cells
that
were
plated
(t-cell).
Since
transformation
rates
were
approximately
the
same
in
both
groups,
the
transformation
rates
in
200
and
1000
cell
dishes
were
pooled
for
presentation.
Un-
infected
cultures
were
trypsinized
and
similarly
plated
for
determination
of
plating
efficiency.
No
transformed
colonies
were
seen
in
uninfected
cultures.
Results.-Effect
of
interferon
on
the
time
course
of
SV40
T
antigen
formation:
In
Figure
1,
the
time
course
of
SV40
T
antigen
formation
in
control
and
interferon
pretreated
3T3
cells
is
shown.
Cells
containing
T
antigen
were
first
observed
at
6
hr
in
control
cultures.
The
staining
was
characteristic
of
the
SV40
T
antigen
in
that
it
was
granular,
confined
to
the
nucleus,
and
spared
the
nucleoli.4
5
The
per
cent
positive
cells
was
at
or
near
maximum
by
48
hr
and,
in
these
confluent
non-
dividing
cultures,
remained
maximum
without
visible
loss
of
intensity
for
at
least
7
days.
In
contrast,
cells
pretreated
with
100
units
per
ml
of
mouse
serum
inter-
feron
had
no
detectable
T
antigen
at
12
hr
after
infection.
As
judged
by
the
dis-
placement
of
the
curve
of
T
antigen
formation,
there
was
a
delay
of
10-12
hr
in
T
antigen
production
in
the
interferon-pretreated
cultures.
Also,
there
was
approxi-
mately
an
84
per
cent
reduction
in
the per
cent
of
T-antigen-positive
cells
at
48
hr
and
even
7
days
later
there
was
a
significant
reduction
in
the
number
of
cells
con-
taining
T
antigen
in
the
interferon-treated
cultures.
Thus
interferon
appears
to
reduce
the
number
of
cells
that
produce
T
antigen
after
high-multiplicity
infection
with
SV40
virus
and
delay
T
antigen
production
in
those
cells
which
ultimately
do
form
T
antigen.
Sensitivity
of
SV4O
T
antigen
production
to
interferon:
Table
1
presents
the
results
of
three
groups
of
experiments
in
which
3T3
cultures
were
pretreated
with
varying
doses
of
interferon
and
control
preparations,
and
subsequently
infected
with
103
TCID50
SV40
virus
per
cell.
It
is
clear
that
in
all
experiments,
and
at
all
time
in-
tervals
examined,
pretreatment
with
interferon
markedly
inhibited
T
antigen
pro-
duction.
The
data
obtained
48
hr
after
infection
in
experiment
2
are
plotted
as
per
cent
inhibition
versus
interferon
dose
(Fig.
2,
curve
A).
It
is
noteworthy
that
50
per
cent
inhibition
of
T
antigen
formation
occurred
at
interferon
concentrations
of
approximately
1
unit
per
ml,
indicating
that
this
expression
of
the
SV40
genome
is
approximately
as
interferon-sensitive
as
is
VSV
lytic
infection.
I,,
-ir-
Conro/
OFIG.
1.-Effect
of
interferon
on
the
time
IC!
/
K
course
of
T
antigen
formation
in
3T3
cells
in-
Z/
fected
with
SV40
virus.
Monolayer
cultures
f
I
iv
<_~~1,1&,eon
Peleoted
of
3T3
cells
were
pretreated
for
18
hr
at
370C
z
with
100
units
of
mouse
serum
interferon
per
ml
or
control
preparations.
The
cultures
were
infected
at
time
0
with
a
multiplicity
of
103
L
/
,
TCID6o
of
SV40
virus
per
cell.
Coverslips
were
0OT-
acetone-fixed
at
the
indicated
times
and
stained
for
the
SV40
T
antigen
by
indirect
FA.
They
NEGATivE
L
t6
2
were
read
as
negative
when
no
T-antigen-
TIME
(hows)
days
positive
cells
were
seen
in
105
cells
(<0.001%).
1136
MICROBIOLOGY:
OXMAN
AND
BLACK
PROC.
N.
A.
S.
TABLE
1
INHIBITION
OF
SV40*
T
ANTIGEN
PRODUCTION
IN
3T3
CELLS
BY
INTERFERON
Interferon
Expt.
Interferon
doset
Per
Cent
Cells
with
T
Antigen$
-
Per
Cent
of
Control---
no.
preparation
(units/nil)
24
hr
48
hr
88
hr
7
days
24
hr
48
hr
7
days
1
A
100
1
10
1
10
30
5 6
10
7
8
3
12
14
Control§
84
97
95
100
100
2
B
100
4
8
7
15
30
8
13
10
12
20
3
13
21
1
23
39
Control§
61
56
100
100
3
C
100
1
10
25
3
16
36
Control§
37
64
70
100
100
100
*
Infected
at
time
0
with
103
TCID5o
of
SV40
virus
per
cell.
t
3T3
cultures
pretreated
with
3
ml
interferon
solutions
for
18
hr
at
370C.
I
Uninfected
controls
were
always
negative
when
stained
for
T
antigen.
§
pH
2
treated
and
untreated
mouse
serum
controls
produced
no
inhibition
of
T
antigen.
Inhibition
of
SV4O
T
antigen
by
interferon
applied
for
various
time
intervals
before
and
after
infection:
The
results
of
experiments
involving
the
addition
of
100
units
per
ml
interferon
at
different
times
before
and
after
SV40
infection
of
confluent
non-
dividing
3T3
cultures
are
presented
in
Table
2.
Interferon
produced
significant
inhibition
of
T
antigen
formation
when
added
prior
to,
or
up
to
2
hr
after,
SV40
infection.
However,
there
was
no
detectable
inhibition
when
interferon
was
added
4
hr
after
infection
or
at
any
time
thereafter.
The
time
required
for
exogenous
interferon
to
establish
the
antiviral
state
within
the
cells
is
not
known
for
this
sys-
tem.
However,
the
fact
that
100
units
per
ml
of
interferon
added
2
hr
after
infec-
tion
produced
a
degree
of
T
antigen
inhibition
(39%)
less
than
that
seen
when
cul-
tures
were
pretreated
for
18
hr
with
only
1
unit
per
ml
(Fig.
2),
and
the
failure
of
interferon
added
at
4
hr
to
produce
any
detectable
inhibition,
suggests
that
the
interferon-sensitive
biochemical
reaction
in
the
synthesis
of
T
antigen
is
completed
sometime
between
2
and
4
hr
after
infection.
,00
A
FIG.
2.-Effect
of
interferon
on
T
antigen
formation
and
Intferwon-r
transformation
in
3T3
cells
infected
with
SV40
virus.
All
O
8a
\
cultures
were
pretreated
with
mouse
serum
interferon
or
5
80
C\
/nterferon-
~
t
control
preparations
for
18
hr
at
37°C
and
then
infected
with
a
multiplicity
of
103
TCID50
of
SV40
virus
per
cell.
Curve
A
60
\x
use
(-
),
per
cent
inhibition
of
T
antigen
formation
by
mouse
serum
interferon;
curve
B
(
U),
per
cent
in-
°0
60
-
\
\
hibition
of
transformation
by
mouse
serum
interferon
(t-
cell);
curve
C
(0
0);
per
cent
inhibition
of
trans-
Z
E4.^.
\
>
\
formation
by
mouse
serum
interferon
(t-col);
curve
D
\
(O---
0),
per
cent
inhibition
of
T
antigen
formation
by
in-
40-
56e
terferon
heated
56°
1
hr;
curve
E
(A..
.
A),
per
cent
in-
hibition
of
T
antigen
formation
by
interferon
heated
600
1
hr;
60
'
\
curve
F
(V-.-.-V),
per
cent
inhibition
of
T
antigen
formation
60'T
by
mouse
serum
interferon
in
heterologous
cells
(BSC-1).
20
ntereeron-T
\
\
One
unit
of
interferon
is
defined
as
that
amount
producing
a.
\
X
50%
inhibition
of
VSV
lytic
infection
in
the
same
experi-
ment.
Titer
of
interferon
preparation
used
was
1000
units/ml.
IntefeOnr'
Titer
after
560
heating
was
100
units/ml
and
after
600
heat-
Fw
{SSC~1
3
'-
ing
was
10
units/ml.
0
NtEo
30
10
3
C
0
INTERFERON
CONCENTRATION
(unitsftl)
VOL.
55,
1966
MICROBIOLOGY:
OXMAN
AND
BLACK
1137
TABLE
2
INHIBITION
OF
SV40
T
ANTIGEN
IN
3T3
CELLS
BY
INTERFERON
APPLIED
FOR
VARIOUS
INTERVALS
OF
TIME
BEFORE
AND
AFTER
VIRUS
INOCULATION
Interval
of
Contact
with
100
Units
per
ml
Interferon*
Time
interferon
added
Time
interferon
removed
Per
cent
T
antigen
(hr)
(hr)
at
48
hr
Per
cent
inhibition
-18t
48
9
87
-18
0 10
85
-1
0
37
44
+2
48
40
39
+4
48
70
0
+6
48
67
0
+8
48
70
0
+10
48
71
0
+16
48
67
0
Control
66
Uninfected
control
-
0
*
Interferon
(100
units
per
ml)
added
in
3
ml
of
3T3M
+
5%
heated
agamma
calf
serum.
3T3
cultures
infected
at
time 0
with
103
TCIDso
of
SV40
virus
per
cell.
Experiment
terminated
with
fixation
of
cover-
slips
at
+48
hr.
t
Interferon
removed
from
these
cultures
at
time
of
infection
(time
0)
and
re-added
at
+2
hr.
Comparison
of
the
effect
of
interferon
on
T
antigen
production
and
transformation
in
3TS
cells
infected
with
SV40:
The
results
of
transformation
assays
done
in
parallel
with
T
antigen
determinations
(Table
1,
expt.
2)
are
presented
in
Table
3.
Preincu-
bation
for
18
hr
with
100
units
of
interferon
per
ml
produced
79
per
cent
inhibition
of
transformation
in
cultures
infected
with
103
TCID50
SV40
virus
per
cell.
It
is
again
evident
that
1
unit
per
ml
of
interferon
produced
approximately
50
per
cent
inhibition.
The
data
for
T
antigen
inhibition
and
inhibition
of
transformation
from
the
same
experiment
are
plotted
in
Figure
2
(curves
A,
B,
and
C)
as
per
cent
inhibi-
tion
versus
interferon
dose.
It
is
noteworthy
that
the
sensitivity
to
interferon
and
slope
of
the
interferon
dose
response
curves
of
transformation
and
T
antigen
produc-
tion
are
quite
similar,
suggesting
that
interferon
inhibits
a
single
function
necessary
for
both
these
expressions
of
the
SV40
viral
genome.
Characterization
of
the
inhibitor
of
SV4Ot
T
antigen
as
interferon:
Since
all
inter-
feron
preparations
were
treated
at
pH
2
for
7
days,
the
inhibitor
of
T
antigen
forma-
tion
studied
here
was
pH
2
stable.
In
the
experiments
described,
cultures
were
preincubated
with
interferon
and
then
thoroughly
washed
prior
to
SV40
infection.
This
precluded
inhibition
by
an
inhibitor
that,
like
antibody,
interacts
directly
with
extracellular
virus.
In
addition,
data
presented
in
Table
2
show
that
the
interferon
preparations
were
able
to
inhibit
T
antigen
formation
when
added
2
hr
after
infec-
tion.
TABLE
3
INHIBITION
OF
SV40*
TRANSFORMATION
OF
3T3
CELLS
BY
INTERFERON
Interferon
doset
Plating
efficiency
Per
Cent
Control
(units/ml)
t-Cellt
t-Col
§
(%)
Based
on
t-Cell
Based
on
t-Col
100
0.8
6
15
16
21
10
1.8
10
18
34
37
1
2.6
13 21
51
49
0
5.1
26
21
100
100
Uninfected
control
0
0
26
-
*
3T3
cultures
infected
with
103
TCIDso
of
SV40
virus
per
cell.
t
3T3
cultures
pretreated
with
3
ml
interferon
solution
for
18
hr
at
371C.
$
t-Cell
represents
the
per
cent
of
cells
plated
which
yielded
transformed
colonies.
Each
point
represents
data
from
12
X
10'
plated
cells.
§
t-Col
represents
the
per
cent
of
total
colonies
formed
that
were
transformed.
1138
MICROBIOLOGY:
OXMAN
AND
BLACK
PROC.
N.
A.
S.
Samples
of
interferon
preparation
B
heated
at
560C
or
600C
for
1
hr,
preincubated
with
antibody
to
NDV,
or
centrifuged
at
105,000
g
for
3
hr
were
assayed
in
parallel
for
their
ability
to
inhibit
T
antigen
formation
and
to
protect
against
VSV
lytic
infection.
The
preparations
treated
with
antibody
to
NDV
and
centrifuged
for
3
hr
at
105,000
g
showed
no
loss
of
inhibitory
activity,
either
against
SV40
T
antigen
or
VSV.
However,
the
ability
of
the
interferon
preparation
to
inhibit
both
T
an-
tigen
formation
and
VSV
lytic
infection
was
partially
destroyed
by
heating
for
1
hr
at
560C
(Fig.
2,
curve
D)
and
more
extensively
inactivated
by
heating
at
600C
for
1
hr
(curve
E).
It
was
also
found
that
mouse
serum
interferon
failed
to
inhibit
T
antigen
formation
by
SV40
virus
in
heterologous
cells
(i.e.,
BSC-1
line
of
Cerco-
pithecus
kidney
cells)
(Fig.
2,
curve
F).
Several
pH
2
treated
and
untreated
normal
mouse
sera
were
used
as
controls.
None
of
these
produced
any
reduction
in
T
anti-
gen
formation.
These
results
demonstrate
that
the
factor
in
the
mouse
serum
in-
terferon
preparations
that
inhibits
SV40
T
antigen
formation
apears
to
have
the
properties
of
interferon.
Persistence
of
T
antigen
in
dividing
and
nondividing
cells:
In
conjunction
with
some
of
the
above
experiments,
cells
were
trypsinized
20
hr
after
SV40
infection,
seeded
on
coverslips
at
concentrations
of
103
and
106
cells
per
dish,
and
FA
stained
at
various
times
for
T
antigen.
At
time
of
trypsinization,
a
high
but
not
maximal
per cent
of
cells
were
positive
for
T
antigen
(compare
Table
1
and
Fig.
1)
and
24
hr
later,
both
sets
of
tryp~inized
cells
showed
the
same
per
cent
T
antigen
staining
(approximately
85%)
as
untrypsinized
infected
controls.
However,
7
and
11
days
after
trypsinization
the
heavily
and
lightly
seeded
cultures
were
markedly
different.
Those
seeded
with
106
cells,
in
which
there
was
little
or
no
cell
division,
had
un-
diminished
per
cent
and
intensity
of
T
antigen
staining.
In
contrast,
the
lightly
seeded
cultures,
which
had
undergone
approximately
10
generations,
showed
no
detectable
T
antigen
except
in
occasional
focal
areas
which
appeared
to
represent
transformed
colonies.
Thus,
SV40
T
antigen
appears
to
be
lost
from
dividing
cells
unless
some
particular
event,
perhaps
the
integration
of
the
SV40
genome,
occurs
to
perpetuate
the
T
antigen
producing
potential
in
the
cell.
Discussion.-A
number
of
observations
indicate
that
T
antigen
formation
reflects
the
presence
and
function
of
SV40
genetic
material,
rather
than
derepression
of
the
host
cell
genome,2-6
17-20
and
that
T
antigen
formation
is
an
early
function
of
the
input
SV40
genome
independent
of
its
replication.2
7
Thus
the
finding
that
inter-
feron
inhibits
SV40
T
antigen
production
demonstrates
that
the
action
of
interferon
involves
inhibition
of
the
early
function
of
the
input
SV40
DNA.
Recent
work
with
RNA
viruses2'
has
shown
that
interferon
(or
more
correctly
the
interferon-induced
intracellular
protein
mediating
its
action)
22
inhibits
the
associ-
ation
of
input
viral
RNA
with
host
cell
ribosomes,
preventing
the
formation
of
the
viral
polysomes
presumably
required
for
synthesis
of
early
viral
proteins.
Thus,
for
RNA
viruses,
there
is
also
evidence
of
inhibition
by
interferon
of
the
early
func-
tion
of
the
input
viral
genome.
The
fact
that
both
SV40
T
antigen
production
and
SV40
transformation
in
3T3
cells
are
sensitive
to
interferon
with
very
similar
dose-response
curves
suggests
the
inhibition
by
interferon
of
a
single
event
necessary
for
both
these
expressions
of
the
viral
genome.
It
should
also
be
noted
that
both
these
expressions
of
the
SV40
viral
genome
are
approximately
as
sensitive
to
interferon
as
is
VSV
lytic
infection.
VOL.
55,
1966
MICROBIOLOGY:
OXMAN
AND
BLACK
1139
Although
this
similarity
may
be
coincidental,
it
suggests
that
the
mechanism
of
action
of
interferon
is
the
same
in
these
widely
different
cell-virus
interactions.
It
seems
probable
from
the
data
presented
above
that
interferon
produces
its
antiviral
action
either
by
inhibiting
the
synthesis
of
virus-specific
messenger
RNA
(mRNA)
on
the
input
viral
template
or
by
blocking
the
function
of
the
virus-specific
mRNA.
The
similar
interferon
sensitivity
of
an
RNA
virus
(VSV)
and
a
DNA
virus
(SV40)
observed
in
these
experiments
favors
the
latter
mode
of
action.
Although
the
interferon
sensitivity
of
T
antigen
formation
and
transformation
are
similar in
confluent
cultures
of
3T3
cells,
the
effect
of
cell
division
on
these
two
expressions
of
the
SV40
viral
genome
and
on
their
sensitivity
to
interferon
is,
at
least
superficially,
quite
different.
It
has
been
demonstrated
by
Todaro
and
Green
that
cell
growth
is
necessary
for
the
establishment
of
transformation
in
SV40-in-
fected
3T3
cells
and
that
the
interferon
sensitivity
of
transformation
is
lost
within
18
hr
in
dividing
cultures
but
persists
for
more
than
4
days
in
confluent
nondividing
cultures.23
In
contrast,
even
in
confluent
nondividing
cultures,
T
antigen
forma-
tion
becomes
completely
insensitive
to
interferon
4
hr
after
SV40
infection;
also,
SV40
transformed
cells
that
are
T-antigen-positive
remain
so
throughout
hundreds
of
generations
in
tissue
culture,
even
in
the
continuous
presence
of
interferon.24
Finally,
SV40
T
antigen
persists
for
more
than
7
days
in
confluent
nondividing
cells
but
is
entirely
lost
from
dividing
cells
(except
for
occasional
colonies
of
probably
transformed
cells)
within
several
divisions.
These
data
indicate
that the
SV40
genome
has
both
an
integrated
and
a
non-
integrated
relationship
with
3T3
cells.
T
antigen
is
produced
when
the
viral
genome
is
in
either
the
integrated
or
the
nonintegrated
state.
In
contrast,
transformation
appears
to
occur
only
when
the
SV40
genome
is
integrated.
In
the
majority
of
acutely
infected
(T-antigen-positive)
cells,
the
viral
genome
is
not
integrated
and
is
lost
with
cell
division,
the
progeny
being
T-antigen-negative
and
not
transformed.
In
a
proportion
of
infected
cells,
however,
the
viral
genome
becomes
integrated,
presumably
at
the
time
of
DNA
replication
or
cell
division.23
25
With
integration,
T
antigen
formation
becomes
stable
and
heritable,
and
the
cells
are
transformed.
In
such
cells,
T
antigen
formation
cannot
be
blocked
by
interferon.
It
is
striking
that
a
prereplicative
viral
synthetic
process
(T
antigen
production)
whose
initiation
is
interferon-sensitive,
becomes
resistant
to
interferon
when
the
viral
genome
is
in
the
integrated
state.
However,
it
is
not
yet
known
whether,
once
initiated,
T
antigen
formation
by
3T3
cells
with
virus
in
the
nonintegrated
state
can
be
blocked
by
interferon.
If
preformed
T
antigen
is
relatively
stable
in
non-
dividing
cells,
subsequent
inhibition
of
its
synthesis
by
interferon
may
be
obscured.
The
question
of
the
stability
of
preformed
T
antigen
is
also
of
central
importance
to
an
understanding
of
the
apparent
disparity
between
the
findings
that
in
nondividing
3T3
cells
the
interferon
sensitivity
of
T
antigen
formation
is
lost
by
4
hr,
while
SV40
transformation
remains
sensitive
to
interferon
for
at
least
4
days.
It
is
clear
that
interferon
does
block
integration
of
the
SV40
genome.
Whether
it
does
this
without
also
blocking
the
T
antigen
synthetic
activity
of
the
nonintegrated
SV40
genome
is
presently
under
study.
Summary.-Pretreatment
of
mouse
fibroblast
(3T3)
cells
with
interferon
reduced
the
number
of
cells
that
produced
SV40
T
antigen
after
infection
with
SV40
virus
and
resulted
in
a
delay
of
T
antigen
formation
in
ultimately
positive
cells.
This
1140
MICROBIOLOGY:
OXMAN
AND
BLACK
PROC.
N.
A.
S.
sensitivity
to
interferon
was
lost
by
4
hr
after
viral
infection.
Transformation
of
3T3
cells
by
SV40
was
as
sensitive
to
the
inhibitory
action
of
interferon
as
T
antigen
formation,
suggesting
that
interferon
inhibits
a
single
function
necessary
for
both
of
these
expressions
of
the
SV40
viral
genome.
The
results
indicate
that
interferon
acts
by
inhibiting
an
early
function
of
the
input
viral
DNA.
The
SV40
T
antigen
was
found
to
be
stable
in
nondividing
and
tranformed
cells,
but
was
lost
with
division
in
nontransformed
cells.
The
hypothesis
was
advanced
that
two
alternative
virus-cell
associations
may
exist
in
SV40-infected
3T3
cells,
with
the
SV40
genome
coding
for
T
antigen
in
both.
In
one
case
the
viral
genome
is
integrated
and
interferon-resistant,
resulting
in
transformation
and
perpetuation
of
T
antigen
formation
through
succeeding
generations.
In
the
other,
the
SV40
genome
is
not
integrated
and
remains
interferon-sensitive;
transformation
does
not
occur
and
T
antigen
formation
is
lost
with
cell
division.
The
authors
are
greatly
indebted
to
Dr.
Wallace
P.
Rowe
for
his
many
helpful
dis-
cussions
throughout
the
course
of
these
studies
and
for
his
critical
review
of
the
manuscript.
They
also
wish
to
thank
Dr.
Samuel
Baron
for
his
advice
and
criticism,
and
for
his
generous
gifts
of
interferon.
*
This
work
was
partially
supported
by
the
National
Cancer
Institute
Field
Studies.
Wagner,
R.
R.,
Virology,
13,
323
(1961);
Grossberg,
S.
E.,
and
J.
J.
Holland,
Federation
Proc.,
20,
442
(1961);
Ho,
M.,
Proc.
Soc.
Exptl.
Biol.
Med.,
107,
639
(1961);
De
Somer,
P.,
A.
Prinzie,
P.
Denys,
and
E.
Schonne,
Virology,
16,
63
(1962);
Isaacs,
A.,
Advan.
Virus
Res.,
10,
1
(1963).
2
Black,
P.
H.,
and
W.
P.
Rowe,
Virology,
27,
436
(1965).
3
Black,
P.
H.,
W.
P.
Rowe,
H.
C.
Turner,
and
R.
J.
Huebner,
these
PROCEEDINGS,
50,
1148
(1963).
4Pope,
J.
H.,
and
W.
P.
Rowe,
J.
Exptl.
Med.,
120,
121
(1964).
6Rapp,
F.,
J.
S.
Butel,
and
J.
L.
Melnick,
Proc.
Soc.
Exptl.
Biol.