JOURNAL
OF
VIROLOGY,
Aug.
1969,
p.
154-161
Vol.
4,
No.
2
Copyright
@
1969
American
Society
for
Microbiology
Printed
in
U.S.A.
Ribonucleic
Acid
Synthesis
of
Vesicular
Stomatitis
Virus
I.
Species
of
Ribonucleic
Acid
Found
in
Chinese
Hamster
Ovary
Cells
Infected
with
Plaque-forming
and
Defective
Particles
MARTHA
STAMPFER,
DAVID
BALTIMORE,
AND
ALICE
S.
HUANG
Department
of
Biology,
Massachusetts
Institute
of
Technology,
Cambridge,
Massachusetts
02139
Received
for
publication
9
June
1969
Plaque-forming
B
particles
of
vesicular
stomatitis
virus
(VSV)
induce
the
synthesis
of
virus-specific
ribonucleic
acid
(RNA)
in
Chinese
hamster
ovary
cells,
whereas
defective
T
particles
do
not.
Infection
with
low
input
multiplicities
of
B
results
in
the
formation
of
four
species
of
RNA.
During
infection
with
high
multiplicities,
RNA
synthesis
begins
with
mainly
these
four
species
of
RNA
but
gradually
shifts
to
a
new
pattern
of
RNA
synthesis
involving
five
other
species
of
RNA.
The
change
can
also
be
induced
by
superinfection
with
T
at
2.5
hr
after
infection
with
a
low
multiplicity
of
B.
T
added
at
the
same
time
as
B
prevents
virtually
all
RNA
synthesis.
Synthesis
of
the
first
group
of
RNA
species
correlates
with
the
formation
of
B
par-
ticles,
whereas
synthesis
of
the
second
group
correlates
with
the
formation
of
T
particles.
The
various
species
of
RNA
formed
after
infection
with
VSV
particles
include
single-stranded
RNA,
a
completely
double-stranded
RNA,
and
RNA
with
partially
double-stranded
regions.
These
observations
begin
to
establish
a
molecular
basis
for
understanding
the
ability
of
T
particles
to
interfere
with
the
growth
of
B
particles.
Biochemical
studies
on
the
replication
of
vesicular
stomatitis
virus
(VSV)
are
complicated
by
the
fact
that
crude
virus
preparations
contain
not
only
bullet-shaped,
infectious
B
particles
but
also
defective
T
particles
which
interfere
specifi-
cally
with
the
growth
of
B
(5,
8).
Separation
of
the
two
particles
by
rate
zonal
centrifugation
has
shown
that
B
and
T
are
antigenically
identical
(9)
and
have
the
same
polypeptides
(11,
17);
however,
T
contains
only
one-third
the
length
of
ribonucleic
acid
(RNA)
found
in
B
(4,
7).
Previously
it
was
shown
that
T
particles
do
not
replicate
in
the
absence
of
B
but
do
arise
from
plaque-purified
clones
of
VSV
(18).
Depending
on
the
relative
input
ratio
of
B
to
T
added
to
cell
cultures,
T
can
either
partially
inhibit
the
produc-
tion
of
B
while
causing
a
large
yield
of
T,
or
T
can
completely
prevent
the
synthesis
of
both
B
and
T
particles.
Because
interference
by
T
is
an
intracellular
event
requiring
functional
T
RNA
(8),
we
examined
virus-specific
RNA
synthesis
to
understand
better
the
interference
phenomenon
on
a
molecular
level.
In
a
previous
report,
Schaffer
et
al.
(16)
showed
that
VSV-specific
RNA
syn-
thesis
can
be
detected
in
infected
cells
and
they
partially
characterized
the
RNA
species
which
are
formed.
MATERIALS
AND
METHODS
Cells
and
media.
Chinese
hamster
ovary
(CHO)
cells
(15),
obtained
from
T.
T.
Puck,
were
used
for
all
of
the
experiments.
In
this
laboratory
CHO
cells
were
grown
in
continuous
suspension
culture
maintained
at
105
to
4
X
105
cells/ml
in
Eagle
medium
modified
for
Spinner
culture
with
added
nonessential
amino
acids
and
7%
fetal
calf
serum.
Monolayer
cultures
of
CHO
cells
were
made
by
seeding
-7.0
X
105
cells
per
60-
mm
plastic
Falcon
petri
plate
and
incubating
them
at
37
C
in
a
humidified
atmosphere
of
5%
CO2
for
24
hr
prior
to
use.
The
modified
Eagle
medium
with
added
CaCI2
at
1.8
mm
was
used
for
monolayer
cultures
and
for
all
infections.
Viruses.
The
strain
of
VSV
used
in
all
these
experi-
ments
was
the
large
plaque
variant
of
the
Indiana
serotype
isolated
and
described
by
Wagner
et
al.
(18).
Plaque
assay
of
VSV
was
done
by
established
methods
(18)
with
the
appropriate
medium
and
serum
on
CHO
monolayers.
To
prepare
virus
stocks,
CHO
cells
were
concen-
trated
to
4
X
106
to
40
X
106
cells/ml
and
infected
with
-0.01
plaque-forming
unit
(PFU)/cell
for
diluted
passage
or
-20
PFU/cell
for
undiluted
passage.
After
a
30-min
attachment
period,
the
cells
were
diluted
to
1.2
X
106
cells/ml
and
incubated
at
37
C.
VSV
was
harvested
from
the
medium
at
10
to
12
hr
after
infection
for
a
diluted
passage
stock
and
at
16
to
20
hr
after
infection
for
an
undiluted
passage
stock.
Diluted
passage
stocks
contained
0.5
X
109
to
2
X
154