Polymerase in the Virion
Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus, II. An RNA
David Baltimore, Alice S. Huang, and Martha Stampfer
doi:10.1073/pnas.66.2.572
1970;66;572-576
PNAS
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Notes:
Proceedi11(111
of
the
National
Academy
of
Scienc68
Vol.
66,
No.2,
pp.
572-576,
June
1970
Ribonucleic
Acid
Synthesis
of
Vesicular
Stomatitis
Virus,
II.
An
RNA
Polymerase
in
the
Virion*
David
Baltimore,
Alice
S.
Huang,
and
Martha
Stampfer
DEPARTMEN'r
OF
BIOLOGY,
MASSACHUSETTS
INSTITUTE
OF
TECHNOLOGY,
CAMBRIDGE
Communicated
by
S.
E.
Luria,
March
24,
1970
Abstract.
The
virions
of
vesicular
stomatitis
virus
contain
an
enzyme
that
catalyzes
the
incorporation
of
ribonucleotides
into
RNA.
The
product
of
the
reaction
is
mainly
RNA
complementary
in
base
sequence
to
that
of
vesicular
stomatitis
virus
RNA.
Introduction.
Two
considerations
led
us
to
search
for
an
RNA
polymerase
in
the
virion
of
vesicular
stomatitis
virus,
a
single-stranded
RNA
virus.
First,
the
lack
of
infectivity
of
vesicular
stomatitis
virus
RNA,t
which
contrasts
with
the
readily
demonstrable
infectivity
of
RNA
from
arboviruses
2
or
picornaviruses,
3
could
be
explained
by
an
obligate
requirement
for
a virion
polymerase
in
order
to
initiate
a vesicular
stomatitis
virus
infection.
Second,
the
recent
finding
that
the
messenger
RNA's
that
synthesize
vesicular
stomatitis
virus
proteins
are
com-
plementary
to
the
nucleotide
sequence
of
the
virion
RNA
4
led
us
to
wonder
how
the
initial
stages
of
the
infection
proceed:
if the
virion
RNA
is
not
a messenger,
then
it
must
be
able
to
serve
as
template
for
messenger
RNA
synthesis
using
either
a polymerase
found
in
the
host
cell
or
one
that
is a
part
of
the
virion.
Two
precedents
exist
for
the
association
of
an
RNA
polymerase
with
the
virion
of
an
animal
virus.
One
is
the
finding
of
a DNA-dependent
RNA
polymerase
in
vaccinia
virions.
5
The
second
is
the
finding
of
an
RNA-dependent
RNA
poly-
merase
in
the
virions
of
reovirus
6
and
other
double-stranded
RNA
viruses.
7
Materials
and
Methods.
Purified
B
particles
8
of
vesicular
stomatitis
virus,
the
Indiana
serotype,
9
were
used
throughout
these
experiments.
Details
of
the
growth
of
high
titered
viral
stocks
in
Chinese
hamster
ovary
cells
and
purification
of
the
virions
through
sucrose
gradients
were
as
previously
described
10
except
for
some
modifications
(manuscript
in
preparation).
A
single
virus
stock
was
used
for
all
the
experiments
re-
ported
in
this
paper,
although
experiments
with
other
stocks
have
given
comparable
re-
sults.
The
stock
contained
0.5
mg
of
protein/ml
and
10
10
plaque-forming
units/ml.
Protein
was
determined
by
the
method
of
Lowry
et
al.
11
Stocks
of
purified
Newcastle
disease
virus,
type
N,
and
Sindbis
virus
were
kindly
pro-
vided
by
Dr.
Michael
A.
Bratt
and
Dr.
Boyce
W.
Burge,
respectively.
Reagents
were
obtained
from
the
following
sources:
unlabeled
nucleoside
triphosphates
from
P-L
Biochemicals;
[
8
H]GTP
from
New
England
Nuclear;
Triton
N-101,
deoxy-
ribonuclease
and
ribonuclease
from
Sigma.
Actinomycin
was
a
gift
from
Merck,
Sharpe
and
Dohme.
Rifampicin
was
supplied
by
Dr.
Harvey
Lodish.
Results.
Addition
of
intact,
purified
vesicular
stomatitis
virus
to
the
appro-
priate
reaction mixture
led
to
incorporation
of
[
3
H]GMP
into
an
acid-insoluble
form
(Table
1).
The
reaction
was
almost
totally
dependent
on
the
presence
of
a
detergent,
whose
role
was
presumably
to
strip
off
the
outer
lipid-containing
en-
572