Proc.
Nat.
Acad.
Sci.
USA
Vol.
72,
No.
7,
pp.
2545-2549,
July
1975
Biochemistry
Coupled
in
vitro
transcription
and
translation
of
vesicular
stomatitis
virus
messenger
RNA
(vesicular
stomatitis
virus
ribonucleoprotein
cores/virion-associated
RNA
polymerase/cell-free
protein
synthesis/slab
gel
electrophoresis)
MICHAEL
BREINDL
AND
JOHN
J.
HOLLAND
Department
of
Biology,
University
of
California,
San
Diego,
La
Jolla,
Calif.
92037
Communicated
by
E.
Peter
Geiduschek,
April
15,
1975
ABSTRACT
The
virion
transcriptase
(nucleosidetriphos-
phate:RNA
nucleotidyltransferase,
EC
2.7.7.6)
of
vesicular
stomatitis
virus
was
fully
active
when
ribonucleoprotein
cores
from
purified
virions
were
added
to
cell-free
protein
synthesizing
systems
of
eukaryotic
origin.
Synthesis
of
mRNA
was
linear
for
at
least
3
hr
and
the
newly
synthesized
viral
mRNA
was
efficiently
utilized
for
the
synthesis
of
viral
proteins
N
(nucleoprotein),
NS,
and
M
(matrix);
small
amounts
of
a
putative
G
(glycoprotein)
protein
precursor
and
several
unidentified
polypeptides
were
regularly
synthesized.
The
ratio
of
the
various
newly
synthesized
viral
proteins
was
identical
after
different
periods
of
coupled
mRNA
and
pro-
tein
synthesis.
Identical
proteins
were
obtained
when
the
cell-free
protein
synthesizing
systems
were
programmed
with
purified
VSV
mRNA
synthesized
in
vitro.
No
detectable
L
protein
was
synthesized,
even
though
transcripts
complemen-
tary
to
the
complete
viral
genome
were
detectable
in
the
mRNA
preparation
by
hybridization.
Vesicular
stomatitis
virus
(VSV)
is
a
rhabdovirus
containing
a
single-stranded
RNA
genome
with
a
molecular
weight
of
3.6
to
4
X
106
(1,
2).
The
five
viral
structural
proteins
(3-5)
are
the
glycoprotein
(G),
the
matrix
protein
(M),
the
nucleo-
protein
(N)
and
two
minor
proteins
(L
and
NS).
VSV
also
has
a
virion-associated
RNA
polymerase
(nucleosidetriphos-
phate:RNA
nucleotidyltransferase,
EC
2.7.7.6)
which
can
synthesize
RNA
complementary
to
the
viral
genome
in
vitro
(6,
7).
The
in
vitro
RNA
products
have
molecular
weights
ranging
from
0.2
to
1
X
106
(8)
and
are
polyadenylylated
(9,
10).
Polysomes
from
VSV-infected
cells
contain
two
size
classes
of
virus-specific
mRNA
(11,
12):
12-18S
RNAs,
which
are
similar
in
size
to
the
in
vitro
transcriptase
prod-
uct,
and
28S
RNA.
The
latter
class
is
not
found
in
the
in
vitro
product
RNA
(8,
13).
Morrison
et
al.
(14)
have
been
able
to
translate
polysomal
mRNA
isolated
from
VSV-infect-
ed
cells
into
virus-like
proteins
in
cell-free
extracts
of
eukar-
yotic
origin.
The
12-18S
RNA
directed
the
synthesis
of
pro-
teins
co-migrating
on
polyacrylamide
gels
with
the
viral
N,
NS,
and
M
proteins,
and
the
28S
RNA
coded
for
the
L
pro-
tein.
No
unequivocal
evidence
for
the
synthesis
of
the
viral
G
protein
was
presented
by
these
authors.
While
this
work
was
in
progress
it
was
shown
by
Both
et
al.
(15)
that
the
12-18S
RNA
synthesized
in
vitro
can
func-
tion
as
mRNA.
In
cell-free
extracts
of
wheat
embryos,
this
RNA
directed
the
synthesis
of
proteins
similar
to
the
viral
N,
NS,
and
M
proteins
and
possibly
a
nonglycosylated
G
pro-
tein
precursor.
Both
et
al.
(15)
also
separated
the
12-18S
RNA
on
sucrose
gradients
into
fractions
with
different
cod-
ing
capacity,
suggesting
that
the
in
vitro
RNA
consists
of
several
monocistronic
mRNAs
each
of
which
can
code
for
an
individual
protein.
In
this
publication
we
report
the
translation
of
VSV
in
vitro
mRNA
in
cell-free
extracts
from
wheat
embryos
(con-
firming
the
results
of
Both
et
al.,
ref.
15),
Krebs
II
ascites
cells,
and
rabbit
reticulocytes.
We
also
show
that
the
tran-
scription
and
translation
processes
can
be
coupled
in
one
in
vitro
system
by
adding
VSV
ribonucleoprotein
cores
directly
to
cell-free
protein
synthesizing
systems.
The
proteins
syn-
thesized
under
direction
of
purified
VSV
in
vitro
mRNA
as
well
as
in
the
coupled
transcription-translation
system
are
mainly
the
viral
N,
NS,
and
M
proteins,
and
several
imiden-
tified
bands,
including
small
amounts
of
a
possible
precursor
of
the
viral
G
protein.
These
proteins
represent
only
about
50%
of
the
genetic
information
of
the
virus.
Although
tran-
scripts
of
the
residual
50%
can
be
detected
by
hybridization
in
the
in
vitro
synthesized
mRNA,
no
detectable
amounts
of
the
viral
L
protein
are
synthesized
in
the
different
cell-free
systems.
MATERIALS
AND
METHODS
Cells
and
Virus.
The
Indiana
serotype
of
VSV
was
grown
in
BHK
21
(baby
hamster
kidney)
cells
and
purified
by
su-
crose
gradient
centrifugation
and
subsequent
potassium
tar-
trate
gradient
centrifugation,
as
described
elsewhere
(16).
The
purified
virus
was
stored
at
00
in
10
mM
Tris-HCl
buff-
er,
pH
7.6.
Preparation
of
Ribonucleoprotein
Cores.
Purified
virus
at
a
concentration
of
about
1
mg/ml
was
treated
with
0.5%
Nonidet
P
40
in
50
mM
Tris-HCl,
pH
7.6,
5
mM
Mg-acetate,
0.1%
2-mercaptoethanol,
and
250
mM
KCI
at
00,
layered
immediately
on
a
discontinuous
glycerol
gradient
[lower
layer:
1.5
ml
of
75%
(v/v)
glycerol,
upper
layer:
1.5
ml
of
25%
(v/v)
glycerol,
containing
10
mM
Tris1HCl,
pH
7.6],
and
centrifuged
at
20
in
the
Spinco
Ti
50
rotor
at
40,000
rpm
for
60
min.
The
ribonucleoprotein
cores
in
the
pellet
were
free
of
the
virion
G
and
M
proteins,
and
recovery
of
transcriptase
activity
was
uosually
80-100%
(not
shown).
The
cores
were
resuspended
in
a
minimal
volume
of
10
mM
Tris-
HCl,
pH
7.6,
and
used
immediately
for
in
vitro
synthesis
of
VSV
mRNA
or
for
coupled
in
vitro
transcription
and
trans-
lation.
In
Vitro
Synthesis
and
Purification
of
VSV
mRNA.
Ri-
bonucleoprotein
cores
were
incubated
in
100
mM
KC1,
50
mM
Tris-HCl,
pH
7.6,
5
mM
Mg-acetate,
0.1%
2-mercap-
Abbreviations:
VSV,
vesicular
stomatitis
virus;
viral
structural
pro-
teins:
G,
glycoprotein;
M,
matrix
protein;
N,
nucleoprotein;
L
and
NS,
minor
proteins.
2545
2546
Biochemistry:
Breindl
and
Holland
toethanol,
and
1
mM
each
of
the
four
ribonucleoside
triphos-
phates
at
280
for
3
hr.
The
reaction
was
terminated
by
the
addition
of
EDTA
(15
mM
final
concentration)
and
nu-
clease-free
CsC1
to
a
density
of
1.36,
and
the
mRNA
was
iso-
lated
by
pelleting
it
at
40,000
rpm
and
20
for
16-22
hr
in
the
Spinco
SW
50.1
rotor
(J.
Perrault,
unpublished).
VSV
mRNA
purified
by
this
simple
one-step
procedure
was
free
of
detectable
amounts
of
template
RNA
(J.
Perrault,
unpub-
lished)
and
contained
only
minimal
amounts
of
free
ribonu-
cleoside
triphosphates.
The
RNA
was
resuspended
in
H20
and
used
either
directly
or
after
reprecipitation
with
ethanol
for
cell-free
protein
synthesis.
Globin
mRNA.
Globin
mRNA
was
extracted
from
rabbit
reticulocyte
polyribosomes
with
sodium
dodecyl
sulfate-
deoxycholate
as
described
by
Stewart
et
al.
(17)
and
purified
by
sucrose
gradient
centrifugation
(18).
Cell-Free
Protein
Synthesis.
The
protein
synthesizing
systems
used
in
these
experiments
were
prepared
from
Krebs
II
ascites
cells
and
rabbit
reticulocytes
as
described
be-
fore
(18),
and
from
wheat
embryos
by
modifications
of
pro-
cedures
described
by
others
(19-21).
The
Krebs
II
ascites
system
was
supplemented
with
initiation
factors
prepared
from
rabbit
reticulocytes
or
rat
liver
by
a
modification
of
previously
described
methods
(18,
22).
Standard
reaction
mixtures
of
100
ul
contained
10
Ml
of
S-30
extract,
50
,gg
of
initiation
factors
(ascites
system
only),
mRNA
or
ribonucleoprotein
cores
as
indicated,
1
mM
each
of
the
four
ribonucleoside
triphosphates,
15
mM
creatine
phosphate,
10
Mg
of
creatine
kinase,
80
mM
KC1
(ascites
and
reticulocyte
systems)
or
55
mM
K-acetate
(wheat
embryo
system),
5.5
mM
Mg-acetate,
40
mM
Tris-HCl,
pH
7.6,
and
0.1%
2-mercaptoethanol.
To
follow
protein
synthesis,
we
added
either
5
MCi
of
[35S]methionine
(Amersham/Searle
Corp.,
specific
activity
150-300
Ci/mmol)
or
5
ACi
of
[3H]valine
(Schwarz/Mann,
specific
activity
16
Ci/mmol);
the
concentrations
of
the
appropriate
unlabeled
amino
acids
were
30
uM.
Incubation
was
at
280.
At
the
indicated
times
aliquots
were
precipitated
on
Whatman
3MM
filter
paper
strips
in
10%
trichloroacetic
acid,
washed,
and
analyzed
in
a
liquid
scintillation
counter
as
described
(18).
When
RNA
synthesis
was
followed
the
radioactive
amino
acids
were
re-
placed
by
the
appropriate
unlabeled
amino
acids,
the
UTP
concentration
was
0.1
mM,
and
0.5
uCi/100
Mil
of
3H-labeled
UTP
(Schwarz/Mann)
was
added.
Aliquots
were
spotted
on
Whatman
DE
81
paper
discs,
washed,
and
analyzed
for
ra-
dioactivity
according
to
the
method
of
Blatti
et
al.
(23).
Polyacrylamide
Gel
Electrophoresis
and
Autoradiogra-
phy.
Sodium
dodecyl
sulfate
(final
concentration
1%)
and
2-mercaptoethanol
(1%)
were
added
to
10
,l
aliquots
of
the
protein
synthesis
assays.
The
samples
were
then
heated
for
2
min
at
1000,
and
subjected
to
electrophoresis
on
analytical
10%
polyacrylamide-dodecyl
sulfate
slab
gels
following
the
procedure
of
Laemmli
(24).
Electrophoresis
was
at
10
mA
for
4
hr,
and
the
gels
were
stained,
destained,
and
dried
in
vacuo.
Autoradiograms
were
obtained
by
exposure
to
Kodak
RP
Royal
X-Omat
medical
x-ray
film
for
the
times
indicated
in
the
figure
legends.
RNA-RNA
Hybridization.
Annealing
reactions
were
car-
ried
out
in
sealed
glass
capillaries
at
70°
for
20
hr
as
de-
scribed
elsewhere
(16)
except
that
the
phosphate
buffer
con-
centration
was
0.48
M.
After
annealing,
10
Mug/ml
of
pancre-
atic
ribonuclease
and
5
units/ml
of
T1
ribonuclease
were
added
for
30
min
at
37°.
Aliquots
were
then
spotted
on
Whatman
DE
81
paper
discs,
washed,
and
analyzed
accord-
1
2
3
4
5
6
7
8
L_
NS-
ANE
N
S-
1
_
_
M-
W
FIG.
1.
Analysis
of
proteins
synthesized
in
response
to
VSV
in
vitro
mRNA
in
different
protein
synthesizing
systems.
VSV
mRNA
was
transcribed
in
vitro,
purified,
and
translated
in
cell-
free
protein
synthesizing
systems
from
Krebs
II
ascites
cells,
rabbit
reticulocytes,
and
wheat
embryos,
as
described
in
Materials
and
Methods.
Aliquots
were
analyzed
by
sodium
dodecyl
sulfate-poly-
acrylamide
slab
gel
electrophoresis
(24)
and
autoradiography.
The
results
of
different
experiments
are
summarized
in
this
figure.
The
slots
contain:
[14C]valine-labeled
VSV
proteins
extracted
from
in-
fected
cells
(1),
proteins
synthesized
in
the
Krebs
II
ascites
extract
without
(2)
and
with
(3)
addition
of
VSV
in
vitro
mRNA,
proteins
synthesized
in
the
rabbit
reticulocyte
extract
without
(4)
and
with
(5)
VSV
in
vitro
mRNA,
and
proteins
synthesized
in
the
wheat
embryo
extract
without
(6)
and
with
(7)
VSV
in
vitro
mRNA.
The
autoradiograms
shown
in
slots
1-7
were
exposed
for
2
days.
Slot
8
shows
the
same
in
vitro
product
as
slot
7,
but
exposed
for
10
days.
The
position
of
the
putative
G
protein
precursor
is
indicated
by
the
dotted
arrows.
This
band
was
not
seen
in
the
control
(without
mRNA)
exposed
for
10
days.
Globin,
the
main
endogenous
product
of
the
reticulocyte
system,
runs
off
the
gels
under
these
conditions
of
electrophoresis.
RESULTS
Translation
of
purified
VSV
in
vitro
mRNA
in
cell-free
extracts
of
different
eukaryotic
cells
VSV
mRNA
was
synthesized
in
vitro
by
virus
ribonucleo-
protein
cores
and
purified
as
described
in
Materials
and
Methods.
Cores
prepared
from
5
mg
of
purified
virus
con-
tained
about
50
Mg
of
template
RNA
and
yielded
approxi-
mately
150
,g
of
mRNA
in
3
hr.
This
mRNA
was
translated
in
cell-free
extracts
prepared
from
Krebs
II
ascites
cells,
wheat
embryos,
and
rabbit
reticulocytes,
and
the
resulting
proteins
were
analyzed
by
dodecyl
sulfate-polyacrylamide
slab
gel
electrophoresis.
Fig.
1
shows
that
VSV
mRNA
syn-
thesized
in
vitro
principally
directed,
in
all
three
cell-free
systems,
the
synthesis
of
three
of
the
five
viral
proteins
(the
NS,
N,
and
M
proteins),
and
a
reproducible
pattern
of
pro-
teins
not
corresponding
in
size
to
any
of
the
major
viral
pro-
teins.
The
relative
proportions
of
the
different
proteins
syn-
thesized
in
vitro
were
similar
in
the
three
cell-free
extracts,
except
that
the
NS
protein
was
synthesized
less
efficiently
in
the
wheat
embryo
extract
(Fig.
1,
compare
slots
3,
5,
and
7).
The
NS
protein
did
not
co-migrate
exactly
with
the
authen-
tic
viral
protein,
but
it
has
been
further
identified
as
NS
by
using
polyacrylamide
gels
containing
sodium
phosphate
buffer
instead
of
Tris-glycine.
Under
these
conditions
the
NS
protein
characteristically
changes
its
position
relative
to
the
other
viral
proteins
and
migrates
faster
than
the
N
pro-
tein
(25).
An
extended
exposure
time
of
the
autoradiograms
showed
that
the
Krebs
II
ascites
and
wheat
embryo
systems
synthesized
small
quantities
of
a
protein
also
present
in
in-
fected
cell
extracts
labeled
with
radioactive
amino
acids
(Fig.
1,
slots
1
and
8).
This
protein
has
tentatively
been
iden-
Proc.
Nat.
Acad.
Sci.
USA
72
(1975)
ing
to
Blatti
et
al.
(23).
Proc.
Nat.
Acad.
Sci.
USA
72
(1975)
2547
Table
1.
Hybridization
of
[3H]UTP-labeled
VSV
virion
RNA
and
VSV
mRNA
synthesized
in
vitro
RNA
hybridized
%
ribonuclease
cpm
resistance
No
treatment
2285
Ribonuclease
resistance*
148
6.5
Self-annealingt
202
8.8
4,ug
of
mRNA
2294
100.4
1.2,jg
of
mRNA
2368
103.6
0.4gg
of
mRNA
2332
102.1
0.12,ug
of
mRNA
1517
66.4
0.04
mg
of
mRNA
797
34.9
3H-labeled
virion'RNA
obtained
from
purified
VSV
labeled
during
replication
with
[3H]uridine
was
.provided
by
Dr.
J.
Perrault.
Un-
labeled
VSV
in
vitro
mRNA
was
added
in
the
amounts
indicated
and
annealed
and
digested
with
RNAse
as
described
in
Materials
and
Methods.
*
No
mRNA
added,
sample
was
kept
at
00
prior
to
ribonuclease
digestion.
t
No
mRNA
added.
tified
as
the
nonglycosylated
precursor
of
the
viral
G
protein
(15).
It
has
very
recently
been
found
by
Both
et
al.
(26)
that
a
protein
with
similar
electrophoretic
mobility
is
coded
for
by
mRNA
extracted
from
membrane-bound
polyribosomes
of
VSV-infected
cells.
This
protein
has
a
similar
tryptic
pep-
tide
pattern
as
the
viral
G
protein,
justifying
the
assumption
that
it
is
indeed
the
G
protein
precursor.
The
putative
G
protein
precursor
was
not
detected
in
the
reticulocyte
in
vitro
product
because
endogenous
proteins
migrate
at
the
same
position
in
the
polyacrylamide
gel
(Fig.
1,
slots
4
and
5).
These
results
generally
confirm
the
findings
of
Both
et
al.
(15).
The
viral
L
protein
was
not
synthesized
in
detectable
quantities,
although
hybridization
studies
(Table
1)
showed
that
the
transcription
of
the
viral
genome
was
complete
under
the
conditions
employed
here,
i.e.,
virtually
100%
of
the
template
RNA
was
protected
from
RNAse
digestion
by
product
RNA.
This
is
in
agreement
with
results
of
others
(refs.
7
and
16,
J.
Perrault,
unpublished).
Coupled
in
vitro
transcription-translation
system
VSV
ribonucleoprotein
cores
in
the
presence
of
cell-free
pro-
tein
synthesizing
extracts
of
Krebs
II
ascites
cells
or
wheat
embryos
were
active
in
RNA
synthesis
by
the
virion-associ-
ated
RNA
polymerase
for
at
least
3
hr
(Fig.
2a).
The
newly
synthesized
RNA
was
used
as
mRNA
by
these
extracts
and
translated
into
trichloroacetic-acid-precipitable
proteins
(Fig.
2b).
We
usually
observed
a
slight
stimulation
of
tran-
scriptase
activity
by
the
cell-free
extracts,
possibly
due
to
a
stabilization
of
the
ribonucleoprotein
cores.
Sucrose
gradient
analysis
of
the
product
RNA
synthesized
in
the
presence
of
the
wheat
embryo
extract
revealed
that
it
was
similar
in
size
(10-18
S)
to
that
synthesized
by
VSV
cores
alone
or
deter-
gent
activated
virus;
however,
in
the
presence
of
the
Krebs
II
ascites
extract
almost
all
of
the
synthesized
mRNA
was
found
to
be
of
smaller
size
(about
5
S,
data
not
shown).
No
appreciable
quantities
of
28S
mRNA
were
synthesized
in
ei-
ther
system.
Under
optimal
conditions
the
stimulation
of
protein
syn-
thesis
in
the
Krebs
II
ascites
and
wheat
embryo
extracts
in
4
a
b
o2~~~~~~~~~~~~~
2
2
3
10
20
35
hours
A
Cow
FIG.
2.
(a)
Kinetics
of
RNA
synthesis
by
VSV
ribonucleopro-
tein
cores
in
the
presence
of
a
cell-free
wheat
embryo
extract.
VSV
ribonucleoprotein
cores
were
prepared
from
3
mg
of
purified
virus
as
described
in
Materials
and
Methods
and
resuspended
in
200
1d
of
10
mM
Tris.
Twenty-five
microliters
were
added
to
a
cell-free
wheat
embryo
extract
(total
volume
50
gl)
and
incubated
in
the
presence
of
[3H]UTP
at
280.
Aliquots
(5
Ml)
were
analyzed
at
the
indicated
times
for
[H]UTP
incorporation
as
described
in
Materi-
als
and
Methods.
(b)
Stimulation
of
protein
synthesis
in
a
cell-free
extract
of
wheat
embryos
by
VSV
ribonucleoprotein
cores.
Cores
were
pre-
pared
from
3
mg
of
virus
as
described
in
Materials
and
Methods
and
resuspended
in
200
Ml
of
10
mM
Tris.HCl,
pH
7.6.
The
indicat-
ed
amounts
were
added
to
a
cell-free
wheat
embryo
extract
con-
taining
[3H]valine
as
radioactive
precursor,
and
incubated
at
28°
for
2
hr.
The
total
volume
was
100
Ml.
Aliquots
(10
Ml)
were
precipi-
tated
with
trichloroacetic
acid,
washed,
and
analyzed
for
radioac-
tivity
(18).
Complete
system
(0
0);
without
UTP
and
CTP
(0
0).
Under
identical
conditions
saturating
amounts
of
rabbit
globin
mRNA
stimulated
the
incorporation
of
14,674
cpm.
about
50%
of that
obtainable
with
rabbit
globin
mRNA.
Pro-
tein
synthesis
in
these
coupled
systems
was
dependent
on
mRNA
transcription,
as
no
stimulation
occurred
when
two
of
the four
ribonucleoside
triphosphates
(UTP
and
CTP)
were
omitted
from
the
reaction
mixture
(Fig.
2b).
Similarly,
purified
VSV
virion
RNA
did
not
stimulate
amino-acid
in-
corporation
(not
shown).
The
kinetics
of
in
vitro
protein
synthesis
in
response
to
transcribing
VSV
ribonucleoprotein
cores
showed
striking
differences
when
compared
with
the
translation
of
either
globin
mRNA
or
purified
VSV
in
vitro
mRNA
(Fig.
3).
The
rate
of
protein
synthesis
in
response
to
added
mRNA
was
usually
linear
for
30-45
min
and
then
gradually
declined.
In
contrast,
under
coupled
conditions
there
was
an
initial
lag
phase
of
15-30
min,
followed
by
linear
rates
of
protein
syn-
thesis
for
up
to
2.5
hr.
These
results
were
repeatedly
ob-
served
in
the
wheat
embryo
extract
(Fig.
Sb).
Similar
kinet-
ics
were
observed
in
the
Krebs
II
ascites
extract,
although
in
this
system
the
initial
lag
phase
was
less
pronounced
and
the
rate
of
protein
synthesis
declined
after
1-1.5
hr
(Fig.
Sa).
The
rabbit
reticulocyte
extract
was
not
used
for
the
coupled
in
vitro
transcription-translation
reaction.
An
analysis
by
gel
electrophoresis
of
the
proteins
synthe-
sized
in
vitro
in
the
coupled
transcription-translation
sys-
tems
revealed
(Fig.
4)
that
the
transcribing
ribonucleopro-
tein
cores
directed
the
synthesis
of
the
same
proteins
in
simi-
lar
relative
proportions
as
the
purified
VSV
in
vitro
mRNA
(compare
Fig.
1),
namely
the
viral
NS,
N,
and
M
proteins,
small
amounts
of
the
putative
G
protein
precursor
(Fig.
4,
slot
7),
and
a
very
similar
pattern
of
proteins
not
correspond-
ing
in
size
to
the
major
viral
proteins.
Moreover,
the
ratio
of
the
different
newly
synthesized
viral
proteins
was
identical
after
short
incubation
times
(30
min,
Fig.
4,
slot
5)
and
long-
er
incubation
times
(60-150
min,
Fig.
4,
slot
6),
showing
that.
response
to
transcribing
VSV
ribonucleoprotein
cores
was
Biochemistry:
Breindl
and
Holland
the
transcription
of
at
least
three,
and
possibly
four,
of
the
2548
Biochemistry:
Breindl
and
Holland
0
2
0
b.
2
g
c
Eo
I
Z
3
2
3
hours
hours
4
FIG.
3.
(a)
Kinetics
of
cell-free
protein
synthesis
in
a
Krebs
II
ascites
extract,
without
added
mRNA
(0
O),
with
2
,g
of
VSV
in
vitro
mRNA
(X
X),
or
with
40
gl
of
VSV
ribonucleoprotein
cores
containing
about
8
,ug
of
VSV
template
RNA
(*
*)
in
a
total
volume
of
100
,l.
VSV
in
vitro
mRNA
and
ribonucleoprotein
cores
were
prepared
as
described
in
Materials
and
Methods.
At
the
indicated
times
5
gl
aliquots
were
analyzed
for
[35S]methionine
incorporation
into
hot
trichloroacetic-acid-precipitable
proteins.
The
high
endogenous
protein
synthesis
in
this
experiment
was
due
to
a
contamination
of
the
rabbit
reticulocyte
initiation
factors
with
mRNP.
In
similar
experiments
with
rat
liver
initiation
factors
no
endogenous
protein
synthesis
was
observed
(not
shown).
(b)
Kinetics
of
cell-free
protein
synthesis
in
a
wheat
embryo
ex-
tract,
without
added
mRNA
(o
o),
with
4
Mg
of
rabbit
globin
mRNA
(X
X),
or
with
40
Ml
of
VSV
ribonucleoprotein
cores
(-
0)
in
a
total
volume
of
100
Ml.
For
details
see
above.
Globin
mRNA
translation
was
done
in
an
independent
experiment
with
[3H]valine
rather
than
[35Sjmethionine
as
radioactive
precursor
and
included
in
this
figure
for
the
purpose
of
comparison.
Back-
ground
subtractions
(zero
time
samples)
were
1.1
X
104
35S
cpm
and
60
3H
cpm,
respectively.
The
high
[P5Sjmethionine
back-
ground
was
due
to
binding
of
acid-soluble
methionine
to
the
filter
paper.
It
can
be
substantially
reduced
by
addition
of
reducing
agents
to
the
10%
trichloroacetic
acid
wash.
five
viral
mRNAs
initiates
early
in
the
reaction.
Even
after
very
long
periods
of
linear
mRNA
and
protein
synthesis
in
the
wheat
embryo
extract
(compare
Figs.
2
and
3)
no
detect-
able
L
protein
was
synthesized
(Fig.
4,
slot
7).
DISCUSSION
The
results
presented
in
this
paper
show
that
VSV
ribonucle-
oprotein
cores
when
added
directly
to
protein
synthesizing
systems
of
eukaryotic
origin
are
an
excellent
generating
source
of
functional
viral
mRNA
which
is
effectively
trans-
lated
into
proteins
(Figs.
2
and
3).
It
has
been
shown
(7)
that
under
in
vitro
conditions
the
transcription
of
the
entire
VSV
genome
is
complete
after
50-60
min.
In
our
experiments
mRNA
synthesis
was
linear
for
at
least
3
hr
(Fig.
2a).
This
apparently
complete
transcription
of
the
virus
genome
has
been
verified
by
hybridization
studies
(Table
1)
showing
that
the
entire
VSV
genome
was
transcribed
under
the
con-
ditions
employed
in
these
experiments.
Since
protein
synthe-
sis
in
the
coupled
systems
was
linear
for
at
least
1
hr
and
as
long
as
2.5
hr
(Fig.
3)
conditions
seemed
adequate
for
com-
plete
transcription
and
translation
of
the
entire
genetic
in-
formation
of
the
virus.
Nevertheless,
the
translation
product
consisted
mainly
of
three
of
the
five
viral
proteins
(the
NS,
N.
and
M
proteins)
and
possibly
small
amounts
of
a
fourth
2 3
4
5
6
7
I.
0
06
2
0
I
-a
30
FIG.
4.
Analysis
of
proteins
synthesized
in
the
coupled
tran-
scription-translation
reaction.
VSV
ribonucleoprotein
cores
were
prepared
and
added
to
cell-free
protein
synthesizing
systems
pre-
pared
from
wheat
embryos
and
Krebs
II
ascites
cells
as
described
in
Materials
and
Methods.
Aliquots
(10
1d)
were
analyzed
by
sodi-
um
dodecyl
sulfate-polyacrylamide
slab
gel
electrophoresis
(24)
and
autoradiography.
The
results
of
different
experiments
are
summarized
in
this
figure.
The
slots
contain
[14C]valine-labeled
VSV
proteins
extracted
from
infected
cells
(1),
proteins
synthe-
sized
in
the
wheat
embryo
system
without
(2)
and
with
(3)
addi-
tion
of
VSV
ribonucleoprotein
cores,
proteins
synthesized
in
the
Krebs
II
ascites
system
without
(4)
and
with
addition
of
VSV
ri-
bonucleoprotein
cores
after
30
min
(5)
and
150
min
(6)
of
coupled
mRNA
and
protein
synthesis.
The
autoradiograms
shown
in
slots
1-6
were
exposed
for
2
days.
Slot-
7
shows
the
product
after
3
hr
of
coupled
in
vitro
mRNA
and
protein
synthesis
in
the
wheat
embryo
system
(compare
Fig.
3b).
This
autoradiogram
was
exposed
for
10
days
to
show
the
presence
of
the
putative
G
protein
precursor
(dotted
arrows)
and
the
absence
of
detectable
amounts
of
the
L
protein.
viral
protein,
the
unglycosylated
precursor
of
the
viral
G
protein
(Fig.
4).
The
same
result
was
obtained
when
VSV
mRNA
was
transcribed
in
vitro
for
three
hr
and
translated
after
purification
(Fig.
1).
The
G,
NS,
N,
and
M
proteins
to-
gether
represent
about
50%
of
the
genetic
information
of
the
virus,
the
rest
being
required
to
code
for
the
L
protein
(mo-
lecular
weight
1.5
to
1.9
X
105,
refs.
5
and
25).
Since
tran-
scripts
representing
100%
of
the
viral
genome
were
present
in
the
in
vitro
mRNA
(Table
1),
the
absence
of
the
L
protein
in
the
in
vitro
protein
product
(Figs.
1
and
4)
indicates
that
the
L
protein
mRNA
was
transcribed
but
not
translated
in
the
cell-free
systems.
This
is
most
likely
due
to
a
degradation
of
the
L
protein
mRNA,
since
no
detectable
quantities
of
full-size
28S
mRNA
were
present
in
the
in
vitro
transcrip-
tion
product
(compare
above).
It
cannot
be
ruled
out,
how-
ever,
that
small
amounts
of
full-size
28S
mRNA
were
syn-
thesized
in
the
in
vitro
systems,
but
not
translated
as
effi-
ciently
as
the
other
viral
mRNA
species,
thus
reflecting
a
possible
translational
control
of
VSV-specific
protein
synthe-
sis.
Consistent
with
this
possibility
is
the
fact
that
the
viral
L
protein
is
present
in
infected
cells
in
a
much
lower
molar
concentration
than
the
other
viral
proteins
(refs.
4
and
27,
and
compare
the
marker
proteins
in
Figs.
1
and
4,
slots
1),
although
all
viral
mRNAs
seem
to
be
present
in
equimolar
amounts
(28).
The
underrepresentation
of
the
viral
G
pro-
tein
precursor
in
the
translation
products
might
also
be
due
to
mRNA
degradation.
Another
possibility
is
that
specific
conditions
are
necessary
for
the
translation
of
the
G
protein
mRNA
which
are
not
met
by
the
in
vitro
systems.
This
latter
possibility
is
supported
by
the
findings
that
mRNA
(14)
or
total
cytoplasmic
extracts
(29)
from
VSV-infected
cells
also
direct
the
synthesis
of
very
little,
if
any,
G
protein
in
vitro
Proc.
Nat.
Acad.
Sci.
USA
72
(1975)
M.
M.
M
--i-
4ma
Boom
II
-
ikLi..
"
on,
Proc.
Nat.
Acad.
Sci.
USA
72
(1975)
2549
under
conditions
suitable
for
the
VSV
L,
NS,
N,
and
M
pro,
tein
mRNA
translation.
The
three
different
cell-free
systems
reproducibly
synthe-
sized
a
nearly
identical
pattern
of
discrete
proteins
not
cor-
responding
in
size
to
the
major
viral
proteins
(Fig.
1,
slots
3,
5,
and
7).
These
proteins
were
also
produced
in
the
coupled
transcription-translation
reaction
(Fig.
4,
slots
3
and
6),
and
might
be
the
products
of
premature
termination
during
the
transcription
and/or
the
translation
process.
An
intriguing
possibility
is
that
these
polypeptides
are
viral
proteins
which
are
modified
or
prematurely
terminated
at
defined
signals
on
the
mRNA
molecules,
having
a
function
in
the
virus
life
cycle.
This
possibility
is
supported
by
the
observation
that
polypeptides
with
the
same
electrophoretic
mobility
are
present
in
extracts
of
VSV-infected
cells,
although
in
lesser
amounts
than
are
produced
in
vitro
(M.
Breindl
and
J.
J.
Holland,
unpublished).
An
interesting
result
is
the
finding
that
the
protein
prod-
ucts
after
short
(30
min,
i.e.,
shortly
after
the
end
of
the
lag
period,
Fig.
3)
and
long (150
min)
periods
of
coupled
tran-
scription
and
translation
in
the
Krebs
II
ascites
extract
are
identical
(Fig.
4,
slots
5
and
6).
Similar
results
were
obtained
with
the
wheat
embryo
extract
(not
shown).
This
shows
that
the
transcription
of
at
least
three,
and
possibly
four,
of
the
five
viral
mRNAs
initiates
early
in
the
reaction.
However,
more
knowledge
about
the
nature
of
the
initial
lag
period
(see
below)
as
well
as
other
parameters
of
the
reaction
is
nec-
essary
to
definitely
decide
whether
there
is
synchronous
or
sequential
transcription
of
the
individual
viral
mRNA
mole-
cules.
A
major
characteristic
of
the
coupled
transcription-trans-
lation
reaction
is
the
15-
to
30-min
lag
phase
in
protein
syn-
thesis
(Fig.
3),
which
is
not
seen
for
mRNA
transcription
(Fig.
2a).
The
cause
for
this
lag
period
is
not
known.
How-
ever,
it
can
be
calculated
from
the
incorporation
of
radioac-
tive
ribonucleoside
triphosphates
of
known
specific
activity
into
the
in
vitro
product
RNA
that
the
VSV
polymerase
achieves
1-
to
2-fold
net
RNA
synthesis
in
1
hr
(compare
Fig.
2a).
Since
most
of
the
particles
of
a
virus
preparation
are
active
in
RNA
transcription
(13),
30-60
min
are
re-
quired
to
synthesize
one
genome
equivalent
of
the
virus
(7),
and
it
seems
possible
that
the
major
portion
of
the
lag
period
reflects
the
time
necessary
to
complete
and
release
the
indi-
vidual
mRNA
molecules.
The
coupled
in
vitro
transcription-
translation
system
characterized
in
this
publication
provides
a
most
useful
tool
to
study
this
and
other
problems
of
VSV-
specific
protein
synthesis.
We
thank
Estelle
Bussey
for
excellent
technical
assistance
and
Dr.
J.
Perrault
for
the
gift
of
[3H]uridine-labeled
VSV
virion
RNA
and
for
helpful
discussion.
This
work
was
supported
by
Grant
no.
10802
from
the
National
Cancer
Institute
and
by
a
research
fellow-
ship
from
the
Deutsche
Forschungsgemeinschaft.
1.
Huang,
A.
S.
&
Wagner,
R.
R.
(1966)
J.
Mol.
Biol.
22,
381-
384.
2.
Repik,
P.
&
Bishop,
D.
H.
L.
(1973)
J.
Virol.
12,969-983.
3.
Kang,
C.
J.
&
Prevec,
L.
(1969)
J.
Virol.
3,404-413.
4.
Mudd,
J.
A.
&
Summers,
D.
F.
(1970)
Virology
42,328-340.
5.
Wagner,
R.
R.,
Prevec,
L.,
Brown,
F.,
Summers,
D.
F.,
Sokol,
F.
&
McLeod,
R.
(1972)
J.
Virol.
10,
1228-1230.
6.
Baltimore,
D.,
Huang,
A.
&
Stampfer,
M.
(1970)
Proc.
Nat.
Acad.
Sci.
USA
66,572-576.
7.
Bishop,
D.
H.
L.
(1971)
J.
Virol.
7,486-490.
8.
Roy,
P.
&
Bishop,
D.
H.
L.
(1971)
J.
Mol.
Biol.
57,513-527.
9.
Villarreal,
L.
P.
&
Holland,
J. J.
(1973)
Nature
New
Biol.
246,
17-19.
10.
Banerjee,
A.
K.
&
Rhodes,
D.
(1973)
Proc.
Nat.
Acad.
Sci.
USA
70,3566-3570.
11.
Huang,
A.
S.,
Baltimore,
D.
&
Stampfer,
M.
(1970)
Virology
42,946-957.
12.
Mudd,
J.
A.
&
Summers,
D.
F.
(1970)
Virology
42,958-968.
13.
Roy,
P.
&
Bishop,
D.
H.
L.
(1971)
J.
Mol.
Biol.
58,799-814.
14.
Morrison,
T.,
Stampfer,
M.,
Baltimore,
D.
&
Lodish,
H.
F.
(1974)
J.
Virol.
13,
62-72.
15.
Both,
G.
W.,
Moyer,
S.
A.
&
Banerjee,
A.
K.
(1975)
Proc.
Nat.
Acad.
Sci.
USA
72,274-278.
16.
Reichmann,
M.
E.,
Villarreal,
L.
P.,
Kohne,
D.,
Lesnaw,
J.
&
Holland,
J.
J.
(1974)
Virology
58,
240-249.
17.
Stewart,
A.
G.,
Gander,
E.
S.,
Morel,
C.,
Luppis,
B.
&
Scher-
rer,
K.
(1973)
Eur.
J.
Biochem.
34,205-212.
18.
Breindl,
M.
&
Gallwitz,
D.
(1974)
Eur.
J.
Biochem.
45,
91-97.
19.
Johnston,
F.
B.
&
Stern,
H.
(1957)
Nature
179,
160-163.
20.
Shih,
D.
S.
&
Kaesberg,
P.
(1973)
Proc.
Nat.
Acad.
Sci.
USA
70,
1799-1803.
21.
Roberts,
B.
E.
&
Patterson,
B.
M.
(1973)
Proc.
Nat.
Acad.
Sci.
USA
70,2330-2334.
22.
Schreier,
M.
M.
&
Staehlin,
T.
(1973)
J.
Mol.
Biol.
73,
329-
349.
23.
Blatti,
S.
P.,
Ingles,
C.
J.,
Lindell,
T.
J.,
Morris,
P.
W.,
Weaver,
R.
F.,
Weinberg,
F.
&
Rutter,
W.
J.
(1970)
Cold
Spring
Har-
bor
Symp.
Quant.
Biol.
35,
649-657.
24.
Laemmli,
U.
K.
(1970)
Nature
227,680-685.
25.
Oblijeski,
J.
F.,
Marchenko,
A.
T.,
Bishop,
D.
H.
L.,
Cann,
B.
W.
&
Murphy,
F.
A.
(1974)
J.
Gen.
Virol.
22,21-33.
26.
Both,
G.
W.,
Moyer,
S.
A.
&
Banerjee,
A.
K.
(1975)
J.
Virol.
15,
1012-1019.
27.
Kang,
C.
J.
&
Prevec,
L.
(1971)
Virology
46,678-690.
28.
Stamminger,
G.
&
Lazzarini,
A.
(1974)
Cell
3,
85-93.
29.
Grubmann,
M.
J.
&
Summers,
D.
F.
(1973)
J.
Virol.
12,
265-
274.
Biochemistry:
Breindl
and
Holland
