of 3
JOURNAL
OF
VIROLOGY,
Mar.
1971,
p.
409-411
Copyright
©
1971
American
Society
for
Microbiology
Vol.
7,
No.
3
Printted
in
U.S.A.
Absence
of
Interference
During
High-Multiplicity
Infection
by
Clonally
Purified
Vesicular
Stomatitis
Virus
MARTHA
STAMPFER,
DAVID
BALTIMORE,
AND
ALICE
S.
HUANG'
Department
of
Biology,
Massachusetts
Institute
of
Techniology,
Cambridge,
Massachusetts
02139
Received
for
publication
3
December
1970
Stocks
of
vesicular
stomatitis
virus
free
of
defective
interfering
particles
were
pro-
duced
by
serial
clonal
isolation.
High-multiplicity
infections
with
these
stocks
led
to
no
interference
or
formation
of
defective
interfering
particles.
Defective
interfering
particles
were
generated
by
three
successive
passages
at
high
multiplicity.
Replication
of
the
Indiana
serotype
of
vesicular
stomatitis
virus
(VSV)
produces
standard
plaque-
forming
B
particles
and
defective
interfering
(DI)
particles
(5,
8,
11).
The
major
DI
particle
found
in
our
stocks
is
one-third
the
length
of
standard
B
particles
and
is
referred
to
below
as
DI-T
rather
than
"T
particles"
as
has
been
the
previous
convention
(8).
Previous
work
has
shown
that
the
sucrose
gradient
pattern
of
ribonucleic
acid
(RNA)
species
synthesized
by
cells
infected
with
VSV
varies,
depending
upon
the
amount
of
DI-T
present
(12).
In
the
absence
of
interfering
particles,
the
predominant
effects
are
synthesis
of
B
particles
and
of
group
I
RNA
species
(mainly
28S
and
13S
RNA).
When
cells
are
co-infected
with
DI-T
and
B,
synthesis
of
DI-T
and
group
II
RNA
(mainly
19S
and
6S)
predominates.
High-multiplicity
infections
with
B
particles
purified
by
rate
zonal
centrifugation
will
also
yield
group
II
RNA
and
DI-T.
Production
of
DI-T
and
group
II
RNA
species
at
high
multiplicities
could
be
either
an
intrinsic
property
of
cells
multiply
infected
by
B
particles
or
the
result
of
contamination
by
undetectable
amounts
of
DI-T
in
B
preparations
leading
to
co-
infection
of
cells
by
DI-T
and
its
helper,
standard
virus.
To
distinguish
between
these
possibilities,
several
clonal
plaque
isolations
of
VSV
have
been
made.
Because
initial
passages
appeared
to
be
free
of
DI
particles,
successive
high-multiplicity
passages
were
analyzed
for
the
production
of
DI
particles.
To
detect
DI
yields
in
this
study,
we
used
radioactive
labeling
and
separation
on
sucrose
gradients,
as
well
as
analysis
of
the
species
of
RNA
produced
in
infected
cells.
A
typical
clonal
isolation
of
B
particles
and
the
1
Present
address:
Channing
Laboratory,
Boston
City
Hospital,
Boston,
Mass.
02118.
initial
detection
of
DI
particles
are
presented
here.
Repetition
of
this
procedure
has
led
to
similar
results.
To
isolate
cloned
preparations
of
VSV,
a
monolayer
of
Chinese
hamster
ovary
(CHO)
cells
infected
with
6
plaque-forming
units
(PFU)
of
VSV
was
overlaid
with
agar
as
previously
described
(12).
A
well
isolated
plaque
of
ap-
proximately
1.5
mm
in
diameter
was
picked
by
inserting
a
capillary
tube
into
the
agar
directly
above
the
plaque,
withdrawing
the
capillary
tube,
and
blowing
the
occluded
agar
into
1
ml
of
medium.
After
disrupting
the
agar
by
using
a
vortex
supermixer,
the
solution
was
diluted
and
assayed
for
plaques
on
fresh,
CHO
cell
mono-
layers.
This
plaque
isolation
was
repeated
four
successive
times.
To
build
up
an
initial
stock
of
this
plaque-
purified
VSV
with
minimal
cross-infection
of
the
cells,
four
CHO
cell
monolayer
cultures
on
60-
mm
plastic
petri
dishes
were
each
infected
with
200
PFU
of
virus
from
the
fifth
successive
iso-
lation.
After
1
day,
when
most
of
the
plaques
were
not
overlapping,
virus
was
harvested
by
collecting
the
agar
overlay
from
each
of
the
four
plates.
Each
plate
was
then
rinsed
with
1
ml
of
medium.
This
medium
and
5
ml
of
fresh
medium
were
combined
with
the
agar,
and
the
suspension
was
centrifuged
at
27,000
X
g
at
4
C
for
10
min
in
the
Sorvall
RC2-B
centrifuge.
This
procedure
concentrated
the
agar,
and
a
total
of
15
ml
of
supernatant
was
collected
which
contained
108
PFU/ml.
From
this
initial
stock,
another
passage
of
VSV
was
made
by
infecting
suspended
CHO
cells
at
a
multiplicity
of
0.4
PFU
per
cell
and
harvesting
the
virus
at 7.5
hr
after
infection.
This
diluted
passage
was
then
used
for
the
fol-
lowing
infections
at
high
multiplicities.
Details
409
J.
VIROL.
of
VSV
infections
and
growth
of
CHO
cells
have
been
described
(12).
To
determine
the
species
of
viral
RNA
and
types
of
VSV
particles
synthesized
at
high
mul-
tiplicities,
suspended
CHO
cells
were
infected
for
three
successive
passages
at
multiplicities
of
100,
50,
and
50,
respectively.
After
each
infec-
tion,
labeled
viral
particles
from
the
medium
and
viral
RNA
from
the
cytoplasm
were
ana-
lyzed
by
sucrose
gradient
centrifugation
as
previ-
ously
described
(12).
Under
these
conditions
of
infection,
incorporation
of
radioactive
uridine
into
virus-specific
RNA
was
complete
by
5
hr
after
infection,
and
production
of
viral
particles
ceased
by
6
to
7
hr
after
infection.
Unlabeled
infected
cells,
harvested
in
parallel
with
the
labeled
cells,
provided
virus
for
the
subsequent
passage.
The
first
high-multiplicity
passage
yielded
1.5
X
109
PFU/ml,
the
second
7.3
x
108
PFU/ml,
and
the
third
7.8
X
107
PFU/ml.
The
decrease
in
VSV
titer
after
the
third
suc-
cessive
high-multiplicity
passage
shows
the
in-
terference
phenomenon
which
was
first
described
by
Bellett
and
Cooper
in
1959
(2).
The
VSV
particles
produced
during
the
three
high
multiplicity
passages
and
the
species
of
viral
RNA
which
were
synthesized
are
shown
in
Fig.
1.
In
the
first
passage
at
a
multiplicity
of
100,
only
the
group
I
cytoplasmic
RNA
species
were
detected
(Fig.
la),
and
only
labeled
stan-
dard
B
virions
were
made
(Fig.
ld).
This
shows
that
high-multiplicity
infections
can
be
carried
out
with
purified
B
preparations
in
the
absence
of
detectable
DI
production.
In
the
second
passage,
at
a
multiplicity
of
50,
again
only
group
I
RNA
species
were
present
in
the
cytoplasm
(Fig.
lb),
and
labeled
B
particles
were
found
in
the
supernatant
(Fig.
le)
along
with
some
B
aggregates
sedimenting
ahead
of
the
main
peak.
During
the
third
successive
passage,
at
a
mul-
tiplicity
of
50,
extensive
production
of
group
II
RNA
species,
as
well
as
some
group
I
species,
was
observed
(Fig.
ic).
Also,
RNA
between
19S
and
28S
was
present
which
had
not
been
seen
previously
(12).
Labeled
particles
produced
during
the
third
passage
gave
a
heterogeneous
distribution
with
peaks
in
the
regions
expected
for
standard
B,
as
well
as
what
appears
to
be
shorter
defective
particles
(Fig.
lf).
If,
during
the
third
successive
passage,
co-infection
of
cells
was
reduced
by
using
a
multiplicity
of
one
or
less,
the
results
resembled
those
seen
during
the
first
high-multiplicity
passage.
Therefore,
it
is
most
likely
that
DI
particles
are
detectable
by
the
third
high-multiplicity
passage
because
of
coinfection
of
cells
with
standard
virus
and
DI
particles
which
were
presumably
generated
in
undetectable
amounts
during
the
first
and
second
high-multiplicity
passages.
However,
we
cannot
rule
out
the
possibility
that
the
third
high-mul-
tiplicity
passage
differs
in
some
indeterminate
way
from
the
first
and
second
by
its
ability
to
generate
large
amounts
of
DI.
The
heterogeneity
of
the
viral
particles
pro-
duced
after
the
third
high-multiplicity
passage
and
the
markedly
reduced
yield
in
virus
titer
indicated
that
DI
particles
were
generated.
Although
these
results
and
similar
results
ob-
tained
by
Crick
et
al.
(4)
do
not
yield
an
ab-
solute
number
for
the
frequency
of
appearance
of
DI
particles
among
a
population
of
VSV,
the
rate
of
generation
of
DI
particles
is
similar
to
reovirus
where
DI
particles
are
detected
upon
8
6
4
2
8
"I
6
2
2
28
19
13
RELATIVE
S-VALUE
10
20
FRACTION
NUMBER
0.2
0.1
FIG.
1.
Sucrose
gradient
patterns
of
viral
RNA
species
and
types
of
VSV
particles
synthesized
in
three
successive
high-multiplicity
passages
with
clonally
purified
VSV.
Suspensions
of
CHO
cells
at
4
X
106/ml
were
infected
with
purified
B,
and
10
,ug/ml
actinomycin
D
was
added.
After
a
30-min
attachment
period,
cells
were
diluted
twofold
and
ex-
posed
to
0.3
,uCi
of
14C-uridine/ml
(New
England
Nuclear
Corp.,
Boston,
Mass.,
55.6
mCi/mM).
At
7.5
hr
postinfection
the
cells
were
harvested
to
obtain
the
cytoplasmic
extract
and
the
released
viral
par-
ticles.
For
panels
(e)
and
(f),
viral
particles
were
obtained
by
centrifugation
of
the
medium
as
previously
described
(10).
For
panel
(d),
the
cell-free
medium,
was
layered
directly
on
the
sucrose
gradient.
The
data
in
each
panel
represent
acid-precipitable
radio-
activity
from
107
cells.
(a)
and
(d)
First
passage
at
a
multiplicity
of
100
PFU/cell;
(b)
and
(e)
second
passage
at
a
multiplicity
of
50
PFU/cell;
(c)
and
(f)
third
passage
at
a
multiplicity
of
50
PFU/cell.
CYTOPLASMIC
VIRAL
RNA
EXTRACELLUAR
VIRAL
PARTICLES
*/
\
.,.+.11w,,..
(ab)
(d
e
,,.i
;.,-
/
*_
37
28
0
20
(b)
Ce)
38
28
14
10
20
.c:
I
T.4I
X
410
NOTES
3
2
T
2
x
VOL.
7,
1971
the
third
or
fourth
successive
high-multiplicity
passages
after
clonal
isolations
(10).
In
contrast,
poliovirus
requires
18
successive
high-multiplicity
passages
to
generate
detectable
levels
of
DI
particles
(C.
N.
Cole,
D.
Smoler,
E.
Wimmer,
and
D.
Baltimore,
J.
Virol.,
in
press).
How-
ever,
it
should
be
noted
that
in
certain
cells
successive
high-multiplicity
passages
do
not
lead
to
progressive
interference
for
influenza
(3),
VSV
(6),
or
Sendai
virus
(9).
Data
from
the
first
passage
show
that
a
high-
multiplicity
passage
per
se
with
VSV
does
not
result
in
the
production
of
large
amounts
of
DI
particles
and
that
the
stock
used
to
initiate
the
high-multiplicity
passages
was
free
of
de-
tectable
DI
particles.
Thus,
biochemical
experi-
ments
(7)
can
be
performed
with
VSV
without
any
interference
at
high
multiplicities
if
the
first
few
passages
from
clonally
purified
virus
are
used.
Furthermore,
very
high
titer
VSV
stocks
(1)
can
be
grown
using
virus
from
the
early
passages.
This
work
was
supported
by
an
American
Cancer
Society
research
grant
(E-512).
M.
S.
was
supported
by
a
National
Science
Foundation
predoctoral
fellowship
and
D.
B.
by
an
American
Cancer
Society
Faculty
Research
Award.
LITERATURE
CITED
1.
Batimore,
D.,
A.
S.
Huang,
and
M.
Stampfer.
Ribonucleic
acid
synthesis
of
vesicular
stomatitis
virus.
II.
An
RNA
polymerase
in
the
virion.
Proc.
Nat.
Acad.
Sci.
U.S.A.
66:572-576.
2.
Bellett,
A.
J.
D.,
and
P.
D.
Cooper.
1959.
Some
properties
of
the
transmissable
interfering
component
of
VSV
prepara-
tions.
J.
Gen.
Microbiol.
21:498-509.
3.
Choppin,
P.
W.
1969.
Replication
of
influenza
virus
in
a
con-
tinuous
cell
line:
high
yield
of
infective
virus
from
cells
inoculated
at
high
multiplicity.
Virology
39:130-134.
4.
Crick,
J.,
B.
Cartwright,
and
F.
Brown.
1969.
A
study
of
the
interference
phenomenon
in
vesicular
stomatitis
virus
replication.
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27:221-235.
5.
Hackett,
A.
J.,
F.
L.
Schaffer,
and
S.
H.
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1967.
The
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infectious
and
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in
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virus
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Virology
31:114-
119.
6.
Huang,
A.
S.,
and
D.
Baltimore.
1970.
Defective
viral
particles
and
viral
disease
processes.
Nature
(London)
226:325-327.
7.
Huang,
A.
S.,
D.
Baltimore,
and
M.
Stampfer.
1970.
Ribo-
nucleic
acid
synthesis
of
vesicular
stomatitis
virus.
I11.
Multiple
complementary
messenger
RNA
molecules.
Virology
42:946-957.
8.
Huang,
A.
S.,
J.
W.
Greenawalt,
and
R.
R.
Wagner.
1966.
Defective
T
particles
of
vesicular
stomatitis
virus.
I.
Prepa-
ration,
morphology,
and
some
biologic
properties.
Virology
30:161-172.
9.
Kingsbury,
D.
W.,
and
A.
Portner.
1970.
On
the
genesis
of
incomplete
Sendai
virions.
Virology
42:872-879.
10.
Nonoyama,
M.,
Y.
Watanabe,
and
A.
F.
Graham.
1970.
Defective
virions
of
reovirus.
J.
Virol.
6:226-236.
11.
Petric,
M.,
and
L.
Prevec.
1970.
Vesicular
stomatitis
virus-
a
new
interfering
particle,
intracellular
structures,
and
virus-specific
RNA.
Virology
41:615-630.
12.
Stampfer,
M.,
D.
Baltimore,
and
A.
S.
Huang.
1969.
Ribo-
nucleic
acid
synthesis
of
vesicular
stomatitis
virus.
1.
Species
of
ribonucleic
acid
found
in
Chinese
hamster
ovary
cells
infected
with
plaque-forming
and
defective
particles.
J.
Virol.
4:154-161.
411
NOTES