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Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro
Vol. 126, No. 1, 1985 January 16, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 40-49
REQUIREMENTS AND FUNCTIONS OF VESICULAR STOMATITIS VIRUS L AND NS PROTEINS IN THE TRANSCRIPTION PROCESS --IN VITRO Bishnu
P. De and Amiya
K. Banetjee
Roche Institute of Molecular Biology Roche Research Center Nutley, New Jersey 07110 Received
November
14, 1984
The L and NS proteins of vesicular stomatitis virus were purified from and were used to study their transcribing ribonucleoprotein complex requirements and functions during reconstitution of RNA synthesis --in vitro. The requirements for L and NS proteins for optimal RNA synthesis were found to Addition of increasing be catalytic and stoichioaetric, respectively.' amounts of NS protein to N-RNA template and saturating L protein, the ratio of N-mRNA to leader RNA synthesis increased linearly. In contrast, when the concentration of L protein was increased the corresponding ratio remained constant. These results, coupled with the observation that the L protein is involved in the initiation of RNA synthesis, suggest that the NS protein is involved in the RNA chain elongation step. The NS protein possibly interacts with both the L protein and the template N-RNA and unwinds the latter to 0 1985 Academic Press. Inc. facilitate the movement of L protein on the template RNA.
The transcribing virus
is
ribonucleoprotein
a complex
(molecular
that
weight,
approximately
2,500
47,000)
In addition,
(1).
of the and
RNP complex:
400
from
molecules
the
protein
bases),
its
5'-end
0006-291X/85 Copyright All rights
negative of
there
(5).
NS (2)
of
240,000
In addition,
precedes
and lacks
poly(A)
synthesis at
its
L protein
presence
is
also
of a large
(3)
of the
four
3'-end
40
(6).
part
protein
L
calculated
and 25,000
mRNA species synthesis
are
amounts
with weight
for
NS
ribonucleoside --in vitro
of a small
in the
leader
of N nRNA and is polyphosphorylated
$1.50 Inc. reserved.
proteins
in the complex,
five there
minor
RNA
associated N (molecular
40 molecules
for
RNP, in the
stomatitie genome
tightly protein
dissociable
sequentially
which
0 1985 by Academic Press, of reproduction in any form
polarity
are approximately
weights
of vesicular
single-stranded
nucleocapsid
two other
synthesizes
of N-NS-M-G-L
(47
molecules
The transcribing
triphosphates, order
of
a linear
of phoephoprotein
molecular (4).
contains
x 106)
4
(RNP) particle
RNA at
Vol. 126, No. 1, 1985 The and
RNP is
rendered
NS proteins
conditions;
by
only
(7,8).
of
poorly
treatment
both
this
L and
NS proteins
role
The
to purify in the
initiation
recently atep
involved
studied
the
vitro
and
elongation
the
transcription
We have
in
has
in
the
extreme protein
with
mRNA and are
been
known
lability
for
in sufficient
to
the
L
strength
RNA synthesis the
the precise
--in vitro (8)
to study
is
N-RNA complex
some time,
L protein
quantity
of the
ionic
leader
process
of
removal
high
added
transcription
are
still
has made it in detail
its
process. shown
(9)
that
purified process
some post-initiation
shown
by total
complex of
of the RNA transcription
requirements have
the
L and NS proteins
phenomenon
understood.
difficult
of
inactive
reconstitution
when
Although
roles
transcriptionally
efficient
achieved
was
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
step.
of L and NS proteins that
the
NS protein
L protein
is
in vitro,
whereas
In the present
involved
studies
in the transcription is
involved
in
in the
NS protein we have process
the
-in
RNA chain
step. MATERIALS AND METHODS
bottles purified
Purification of virus. containing monolayers as described previously
VSV (Indiana serotype) of baby hamster kidney (10).
was grown in roller cells (BHK-21) and
Isolation and purification of L, NS, and N-RNA complex. The L and NS proteins were isolated from purified transcribing ribonucleoprotein (RNP) particles as described in detail recently (9). The purification of N-RNA complex from RNP has also been described (9). The protein concentrations of L, NS, and N-RNP complex were 75 Pg/ml, 120 Pglml, and 750 pg/ml, respectively. Reconstitution of RNA synthesis in vitro. contained 50 mM Tris-HCl, pH 8.0, 5 mM MgC12, 0.1 0.05 mH CTP, 1 mM ATP, CTP, and UTP, 20 pCi of 410 Cilmmole) (Amersham, Arlington Heights, IL), various concentrations of L and NS proteins experiments in each figure. Unless otherwise carried out at 30°C for 2 hr.
The reaction mixture (0.2 ml) M NaCl, 4 mN dithiothreitol, lo-32P]CTP (specific activity 2.2 Pg of N-mRNA template and as described in individual stated, the reactions were
Analysis of RNA products by polyacrylamide gel electrophoresis. For analyses of the mRNA species, the reaction products were extracted with equal volumes of phenol and the RNA in the aqueous phase was precipitated with alcohol and the pellet suspended in water. Half of the RNA was used in a reaction mixture (40 Pl) containing 20 mM Tris-HCl, pH 7.5, 10 ELM HgC12, 100 mH KCl, 0.1 mH dithiothreitol, 1 Pg oligo(dT)l2,18 (Sigma Chemicals, St. Louis, MO), 1 unit/P1 RNasin (Promega Biotec, Madison, WI), 0.5 unit RNase H (Bethesda Research Laboratories, Bethesda, MD). The reaction was incubated at 37" for 20 min, terminated by addition of sodium dodecyl sulfate (0.5%), the RNA products extracted with phenol, and the RNA removed from the aqueous layer by precipitation with ethanol. The mRNAs with poly(A) tails thus removed were 41
Vol. 126, No. 1, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
analyzed by electrophoresis in 5% polyacrylamide slab gels (32 cm x 17 cm x 0.2 cm) containing 0.16% methylene-bis-acrylamide and 7 M urea. The gels were run at 600 V for 24 hr and RNA bands were located by autoradiography on Kodak The RNA in the other half of each sample was directly electroXAR film. phoresed in 20% polyacrylamide gels containing 7 M urea (12) to determine the extent of synthesis of the leader RNA.
RESULTS Optimum L and had
requirements
NS proteins
been
purified
presence
of
bound
to
small
amount
the
(20
quantity
of
optimum
the
of
RNA synthesis L protein
Addition
by approximately
concentration
synthesis
occurred
contrast,
the
significantly
in
1.4
pg/200
weight L protein
of template was
silver
or
contained
template
staining
of N-RNA
but
alone
However, resulted
depending
a
a trace
NS protein
requirement
Pl
basis)
1,
L protein
of
than
(Fig.
for
Thus,
required
higher for
optimal
was at
1)
no L
to the
addition
in
of
stimulation
on the purity
pg/200
for
optimal
of
addition
of
optimal
RNA
reaction.
In was
As shown in Fig.
at
basis)
RNA synthesis.
of the
2, at a
template
as
an NS concentration
a seven-fold
(on a molar
pl
of NS
RNA synthesis
same concentration
least
the
However,
.
0.2
optimal
42
concentration by
only
the L protein.
L and the
RNA synthesis
reaction.
of
NS protein
that of
or seventy-fold was
stimulated
at a concentration
higher
Fig.
and a saturating
significantly of
concentration
used
after
to 30-fold
lo-
hand,
was
a trace
electrophoresis,
mRNA species.
to the
retained
other
gel
in the
which
When an excess
L protein
no complete
concentrations
saturating
of
L and NS proteins
RNA synthesis
increasing
generally
on the
only
X-100
preparations.
At a fixed protein,
at 1 H NaCl,
was discernible
Triton
The
initially
The L protein,
by polyacrylamide
virtually
amounts
with
5 to 10%) of N protein.
was detectable. produced
virions
The NS protein,
in vitro.
of RNP which
by centrifugation.
was analyzed
NS protein
RNA synthesis
treatment
of purified
NS protein.
pg)
for
salt
and eluted
(approximately
template
template
by high
M NaCl followed
phosphocellulose of
of
purified
by disruption
0.4
amount
protein
were
of L and NS proteins
higher
amount
of NS protein These
results
of (on a
than
the
indicated
Vol. 126, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMOUNT OF L OJI)
Fig. 1. Rate of RNA synthesis with saturating NS protein and increasing concentrations of L protein. RNA synthesis was carried out using template saturating concentration of NS protein (1.4 pg), and increasing (2.2 pg), amounts of L protein. The RNA synthesized at each point was determined by measuring cold trichloroacetic acid insoluble radioactivity retained on nitrocellulose filters. Fig. 2. Rate of RNA synthesis with saturating L protein and increasing concentrations of NS protein. RNA synthesis was carried out using template concentration of L protein (0.2 pg), and increasing (2.2 wg), saturating amounts of NS protein as indicated. The RNA ayntheaized at each point was determined by measuring cold trichloroacetic acid insoluble radioactivity retained on nitrocellulose filters.
that
the
whereas
requirement
for
L protein
Role
of
requirement
that
oligonucleotides
understand
the role
NS or
of
such
protein saturating synthesis
leader
gels,
was
product
previously
was capable
of eynthesizing
initiated
termini
the
of
limiting
analyzed
various
kept
at was
(1.4
~1)
linearly
with
from
for
syntheais
the
ratio
the
increase
(0.24 of
poly-
and L protein. and the NS pg/200
N mRNA to
of NS protein
each
show the results
concentration
limiting
of
of individual
5% and 20% 1
a
amounts under
of NS protein
3),
43
we performed
synthesized
saturating
increased
to better
to saturating
3 and 4, and Table
a constant
(Fig.
In order
through
concentrations
RNA and mRNA
proceatr,
RNA products was
leader
such properties.
using
Figures
concentration
increased
atoichiometric
We have
RNA by electrophoresis
using
pg/200
was
elongation.
in the transcription
the
respectively.
experiments
When L protein
lacked
analyzed
reaction
and
5'
experiments
and
Each
mRNA species
the
NS protein
reconstitution
condition.
alone
of NS protein
L proteins
acrylamide
L protein
whereas
(91,
RNA synthesis
RNA chain
representing
sequences
of
in
the
for
was catalytic.
NS protein
demonstrated
series
NS protein
~1)
leader
concentration
to RNA
126, No. 1, 1965
Vol.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5678
1234
5878
-0
- LEADER
LEADER
RNA
0I
03
Fig. 3. Analysis of the RNA products eynthesiaed at saturating L protein concentration and increaeing amounts of NS protein. The mRtJAs (lanes 1 to 4) and leader RNA (lanes 5 to 8) synthesized in reconstituted reactions --in vitro containing template (2.2 pg), L protein (0.2 pg) and various amounts of NS protein were analyzed by electrophoresis, aa deecribed in Materials and Methods. Amounts of NS protein in lanes 1 and 5 ware 0.24 ng; lanes 2 and 6 were 0.48 pg; lanes 3 and 7 were 0.96 ng; and lanes 4 and 8 were 1.4 pg. The migration positions of the mRNAs for G. N, H, NS, and leader RNA are shown. 0 represents the origin of the gel. Analysis of RNA products synthesized at saturating NS proteins Fig. 4. and increasing concentrations of L protein. The mRNA products (lanes 1 to 4) and leader RNA (lanes 5 to 8) synthesized at saturating NS protein (1.4 ng), and increasing L protein amounts were analyzed by e1ectroihoresi.s as in Fig.-3. Amounts of L protein in lanes 1 and 5 were 0.02 pg; in lanes 2 and 6 were 0.04 pg; in lanes 3 and 7 were 0.08 Rg, and lanes 4 and 8 were 0.2 pg, respectively. The migration positione of the mRNAs for G, N, H, NS, and leader RNA are shown. 0 represente the origin of the gel.
(Table the
1).
In
increase
contraat, of
saturating
at
L protein
(0.2
ug/200
RNA
ratio
remaining
(Table
1).
These
results
all
rate
limiting,
hand,
when
RNA due
NS protein
(Fig.
4)
resulted
to
limiting in
that
rapidly
at
excess,
the
over
44
thin
whereas
limiting
NS protein,
Rg/200
corresponding
saturating
of rate
(0.02
the
initiated,
concentration
of
limiting in
constant
indicated were
concentration from
virtually
chains
wae
saturating
concentration ~1)
leader
protein,
a constant
to
N mRNA
to
concentration
range
concentrations
of
chain
elongation
NS protein. step
ul)
On was
the
the initiation
L
was other
Vol. 126, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
Radioactivity incorporated (cpr) Leader RNA
Additions Expt
N-RNA pg/200
.
A
B
L reaction
ul
NS
2.2
0.2
0.24
2.2
0.2
0.48
2.2
0.2
0.96
1
Ratio N-mRNA N-mRNA
791
Leader
RNA
652
0.8
1111
1937
1.7
1398
6139
4.4
2.2
0.02
1.4
176
642
2.2
0.00
1.4
269
1044
3.9
2.2
0.2
1.4
373
1253
3.4
3.6
Reconstitution reactions were carried out using indicated amounts of N-RNA complex, L, and NS proteins. The mRNAs and leader RRAs eynthesized in Expt. A and B were analyzed by polyacrylamide gel electrophoresis as described in Fig. 3 and Fig. 4, respectively. The labeled bands representing the N mRNA and the leader RNA under each condition were excised from the gel, and radioactivity was quantitated by Cerenkov counting.
of
RNA synthesis
extended
by L protein.
and completed
In order
to
further
step,
heat
NS proteins
were
heated for
products L
were
protein
at
completed 12, leader
13).
the
completed
results
chains.
by polyacrylamide
various
temperatures
In
contrast,
origin
were
mRNA chains
heated
and the
contrast
less being
6)
and
with
the the
its effect
on its
thermolabile, 45
of
of
to elongate capacity failed
but
11,
migrating
synthesis
of
of heating.
temperature
that
both
to synthesize
RNA species
interpretation
capability
able
RNA
purified
RNA (lanes
8, 9, and lo),
increase
and the
synthesis leader
L or
at saturating
Heating
were
preterminated
RNA (lanes
with
eliminated
L,
electrophoresis.
and
the RNA chain
experiments
NS preparations
leader
sharply
had
5,
quickly
Purified
2 min and used
abolished
of small,
consistent
but
A.
in
performed.
reconstitution gel
were
of NS protein.
were
virtually
as a series
effectively
In
in
chains
of NS protein
and 80°C for
RNA synthesis
lanes
RNA
of excess
experiments
at AO", 60",
mRNA decreased
NS protein large
inactivation
5,
initiated
the role
analyzed
RNA as well
between
These
investigate
mRNA (Fig.
and
all
due to the presence
elongation
concentrations
Thus,
heat
treatment
and complete
to complete to initiate
small
of the RNA
RNA chains
Vol. 126, No. 1, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1234587
3 91011121314
-LE
Fig. 5. Effect of temperature of the L and NS proteins on mRNA and leader RNA synthesis in in vitro reconstitution reactions. The mRNAs (lanes 1 to 7) and leader RNA (lanes 8 to 14) were synthesized using heated L (0.2 pg) or NS proteins (1.4 pg) and template (2.2 pg) and analyzed as described in Fig. 3. NS protein was heated at 40°C for 2 min (lanes 1 and 8); 60' for 2 min (lanes 2 and 9); 80' for 2 min (lanes 3 and IO). L protein was heated at 40°C for 2 min (lanes 4 and 11); 60" for 2 min (lanes 5 and 12); 80' for 2 q in (lanes 6 and 13). Lanes 7 and 14 represent control reactions at 30°C for 2 hr. The migration positions of mRNAs for G, N, M, NS, and leader RNA are shown. 0 represents the origin of the gel.
after
being
heated;
NS protein
consequently,
no RNA was synthesized
even though
excess
was present. DISCUSSION
In protein
this for
communication, an optimal
a weight
basis
protein.
This
a 1:l
molar
the
the
L
be due
--in
the
of
the
L and
in
the
studies.
different
that
discrepancy previous
strains
reported of
one (8)
reported
(8)
basis)
earlier
which
the
extreme
may
have
higher than
for
on the
where optimal lability
complicated
of thermolability
snd our preparations these
of NS
(8.11)
to be required
in the degree
VSV used in 46
7-fold
on a molar
may be due to report
the requirement
approximately
was found
The difference
of the
that
higher
from
NS proteins
This
is
70-fold
is different
vitro.
L proteins
demonstrated
of transcription
approximately
used
protein
to
(or
ratio
stoichiometry
between
rate
conclusion
RNA synthesis of
we have
studies.
may
also
In a similar
L
Vol. 126, No. 1, 1985 reconstitution disease
experiment,
virus
(NDV),
requirement for
the
for
the
and
of
ratio
molar
ratio
1:40:400
to
concentration in
2,
corrected
system,
the
molar
nucleocapsid
ratio
facilitate
continued
synthesis
but
the
is packaged
process
within
template,
was unable
incomplete
reactions alone
whereas
the
elongation when
of
(Table
1).
can
Thus,
1).
2,500
in
than of
molar
replication with
the
loo-fold)
than
NS protein
might
RNP in the
a suboptimal
is the
high
associated
(greater
It
NS in vitro,
present
NS protein
infected
level
of
contrast, excess
[o-32P)ATP
cells,
of NS protein
and
the ability
was ratio
it
seems
that
RNA chains
but
failed
L protein
e.g.
increased,
ratio
complete 47
when present
to N-RNA
However, was
using
shown
that
L
AC and AACA, etc.,
RNA chains.
demonstrated
concentration
L protein, to
it
A chain
by the fact
in the presence
of N mRNA/leader
the corresponding
and
CTP,
to initiate
is directly
the
when added
mRNAs (9).
RNA chains,
NS protein 3),
alone,
full-length
NS protein
(Fig.
in
L protein
initiate
lacked for
In sharp
Table
that
be approximately
for
concentration
that
synthesize
efficiently
L protein
was
initiated
to
NS protein,
NS protein
25,000
the optimum
in a cell-free
higher
high
previously
concentration
excess
Moreover,
maturation,
containing
function
the
that
the virion.
We have demonstrated
protein
than
of L and NS).
also
of RNA from progeny
of virus
to
weights
synthesized
the
the
The corresponding
requirements
to be considerably Perhaps,
that
(3).
is assumed
reported
mRNA are
(15.16). newly
(17).
during
its
it
be 1:40:2800.
been
the higher
of 240,000
of genome RNA (2),
would has
and
of
and if
the molecular
like
cells
was found
L protein
for
NS protein
infected
here
observed
--in vitro.
per molecule
virion
that,
also
of Newcastle
of NS in NDV) was higher
are used,
determined
purified
have
of L, NS, and N proteins
are bound
note
the
(14)
analogue
respectively,
the
of
amounts
the
weights
of RNA:L:NS
interesting
al.
RNA synthesis
optimal
N protein
(ref.
--et
P, and the NP-RNA complex
for
(4),
in
L,
(the
47,000
molar
Hamaguchi
molecular
molecules
using
P protein
L protein
If (4),
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
increased
remained
of an linearly
constant
was increased in excess,
them due to lack
that
when
(Fig.
4,
may have
of NS protein
Vol. 126, No. 1, 1985 which
was the
the
ratio
all
rate
of
increase
completion
to
leader
showed
which
the than
Since efficient progress
polymerase,
been
ref.
interacts
prepares
the
order
interesting
results
them
with
the
to
study
full
moving
replace
might site any
of other
for
that
a8 with
genome.
the
new
and the
the L protein
initiate
anal1
the
N protein it.
synthesis
in vitro
by host
cell
components
the
putative
host
factors
Perhaps
NS
This
explains
possibly
the region
It
should cellular
Stimulation (18).
the
molecule,
with
example,
reported
facilitate
and
from
--in vitro.
has been
RNA
the N protein
RNA synthesis. for
is
The NS protein
to interact
agent,
RNA that
the L protein
may move along
the NS function
may
48
polypeptide
demonstrating
to
displaces
(L or NS)
a large
mRNA sizes.
be required
protein,
whether
capacity
for little
to L. For each L protein
synthetic
to be seen
the
to the
of NS relative
whether
conclusion
which
required
proteins
presented
as well
access
species.
candidate
the
length
protein
to gain
the
to
two
of only
the L protein
70 NS molecules
preceding
has
L protein
such that
requirement of
interaction
aborted
(8,10,13),
being
proper
has been
support which
an RNA unwinding
molar
N-RNA
by the
extend
L protein
to be the
evidence
synthesized
template
genome RNA for
on the
seems
L protein,
mRNA but
are
vitro the
5) clearly
as numerous
the message
of
brought of N-mRNA
(Fig.
L and NS proteins
which
in excess,
full-length
RNA as well
in --
the
ratio
studies
complex
The
RNA polymerase
possibly
N-RNA
with
rapidly
a constant
than
both
elucidate
no direct
cannot
is
the
3),
The above
the but
to
leader
that
were
to complete
smaller
RNA polymerase.
mRNAs are
N-RNA template.
of
made
although
high
RNA, but
of
daltone,
protein
leader
transcription
the
probably
to complete
of
was present
inactivation
capability
increase
RNA occurred
L protein
maintaining
heat
a linear
to leader
by the
thus
ite
discovery
constitutes (240,000
lost
the original
has
chains
initiated
The
capacity
the
respect
Thus,
When NS protein
NS protein,
NS protein
RNA8 larger
in the reaction. with
had been
RNA synthesis.
retained
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concentration.
by the
that
viral
step
mRNA synthesis
of NS protein
to
the
limiting
the
RNA chains
still
BIOCHEMICAL
be or
of RNA It
NS function
remaina or can
Vol. 126, No. 1, 1985 replace firmly
it
--in
establiah
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
vitro.
Future
the functions
studies
along
this
of L and NS in the
line
would
transcription
certainly
help
process.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Wagner, R.R. (1975) in, Compreheneive Virology (H. Fraenkel-Conrat and R.R. Wagner, eds.), pp. l-80, Plenum Press, New York. Bishop, D.H.L., and Roy, P. (1972) J. Virol. 10, 234-243. Schubert, H., Harmison, G.G., and Meier, E. (1984) J. Virol. 51, 505-514. Gallione, C.J., Greene, J.R., Iverson, L.E., and Rose, J.K. (1981) J. Virol. 39, 529-535. Banerjee, A.K., Abraham, G., and Colonno, R.J. (1977) J. Gen. .Virol. 34, 108. Colonno, R., and Banerjee, A.K. (1978) Nucl. Acids Res. 5, 4165-4176. Abraham, G., and Banerjee, A.K. (1976) Virology 71, 230-241. Emerson, S.U., and Yu, Y.H. (1975) J. Virol. 15, 1438-1356. De, B.P., and Banerjee, A.K. (1984) J. Virol. 51, 628-634. Banerjee, A.K., Moyer, S.A., and Rhodee, D.P. (1974) Virology 61, 527-558. Naito, S., and Ishihama, A. (1976) J. Biol. Chem. 251, 4307-4314. Thornton, G.B., De, B.P., and Banerjee, A.K. (1984) J. Gen. Virol. 65, 663-668. Emerson, S'.U., and Wagner, R.R. (1972) J. Virol. 10, 297. Hamaguchi, H., Yoshida, T., Nishikawa, K., Naruse, H., and Nagai, Y. (1983) Virology 128, 105-117. Hsu, C., Kingebury, D.W., and Murti, K.G. (1979) J. Virol. 32, 304-313. Villarreal, L.P., Briendl, H., and Holland, J.J. (1976) Biochemistry 15, 1663-1667. Patton, J.T., Davis, N.L., and Wertz, G.W. (1983) J. Virol. 45, 155-164 Rose, J.K., Lodieh, H.F., and Brock, M.L. (1977) J. Virol. 21, 683-693.
49
to
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 40-49
REQUIREMENTS AND FUNCTIONS OF VESICULAR STOMATITIS VIRUS L AND NS PROTEINS IN THE TRANSCRIPTION PROCESS --IN VITRO Bishnu
P. De and Amiya
K. Banetjee
Roche Institute of Molecular Biology Roche Research Center Nutley, New Jersey 07110 Received
November
14, 1984
The L and NS proteins of vesicular stomatitis virus were purified from and were used to study their transcribing ribonucleoprotein complex requirements and functions during reconstitution of RNA synthesis --in vitro. The requirements for L and NS proteins for optimal RNA synthesis were found to Addition of increasing be catalytic and stoichioaetric, respectively.' amounts of NS protein to N-RNA template and saturating L protein, the ratio of N-mRNA to leader RNA synthesis increased linearly. In contrast, when the concentration of L protein was increased the corresponding ratio remained constant. These results, coupled with the observation that the L protein is involved in the initiation of RNA synthesis, suggest that the NS protein is involved in the RNA chain elongation step. The NS protein possibly interacts with both the L protein and the template N-RNA and unwinds the latter to 0 1985 Academic Press. Inc. facilitate the movement of L protein on the template RNA.
The transcribing virus
is
ribonucleoprotein
a complex
(molecular
that
weight,
approximately
2,500
47,000)
In addition,
(1).
of the and
RNP complex:
400
from
molecules
the
protein
bases),
its
5'-end
0006-291X/85 Copyright All rights
negative of
there
(5).
NS (2)
of
240,000
In addition,
precedes
and lacks
poly(A)
synthesis at
its
L protein
presence
is
also
of a large
(3)
of the
four
3'-end
40
(6).
part
protein
L
calculated
and 25,000
mRNA species synthesis
are
amounts
with weight
for
NS
ribonucleoside --in vitro
of a small
in the
leader
of N nRNA and is polyphosphorylated
$1.50 Inc. reserved.
proteins
in the complex,
five there
minor
RNA
associated N (molecular
40 molecules
for
RNP, in the
stomatitie genome
tightly protein
dissociable
sequentially
which
0 1985 by Academic Press, of reproduction in any form
polarity
are approximately
weights
of vesicular
single-stranded
nucleocapsid
two other
synthesizes
of N-NS-M-G-L
(47
molecules
The transcribing
triphosphates, order
of
a linear
of phoephoprotein
molecular (4).
contains
x 106)
4
(RNP) particle
RNA at
Vol. 126, No. 1, 1985 The and
RNP is
rendered
NS proteins
conditions;
by
only
(7,8).
of
poorly
treatment
both
this
L and
NS proteins
role
The
to purify in the
initiation
recently atep
involved
studied
the
vitro
and
elongation
the
transcription
We have
in
has
in
the
extreme protein
with
mRNA and are
been
known
lability
for
in sufficient
to
the
L
strength
RNA synthesis the
the precise
--in vitro (8)
to study
is
N-RNA complex
some time,
L protein
quantity
of the
ionic
leader
process
of
removal
high
added
transcription
are
still
has made it in detail
its
process. shown
(9)
that
purified process
some post-initiation
shown
by total
complex of
of the RNA transcription
requirements have
the
L and NS proteins
phenomenon
understood.
difficult
of
inactive
reconstitution
when
Although
roles
transcriptionally
efficient
achieved
was
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
step.
of L and NS proteins that
the
NS protein
L protein
is
in vitro,
whereas
In the present
involved
studies
in the transcription is
involved
in
in the
NS protein we have process
the
-in
RNA chain
step. MATERIALS AND METHODS
bottles purified
Purification of virus. containing monolayers as described previously
VSV (Indiana serotype) of baby hamster kidney (10).
was grown in roller cells (BHK-21) and
Isolation and purification of L, NS, and N-RNA complex. The L and NS proteins were isolated from purified transcribing ribonucleoprotein (RNP) particles as described in detail recently (9). The purification of N-RNA complex from RNP has also been described (9). The protein concentrations of L, NS, and N-RNP complex were 75 Pg/ml, 120 Pglml, and 750 pg/ml, respectively. Reconstitution of RNA synthesis in vitro. contained 50 mM Tris-HCl, pH 8.0, 5 mM MgC12, 0.1 0.05 mH CTP, 1 mM ATP, CTP, and UTP, 20 pCi of 410 Cilmmole) (Amersham, Arlington Heights, IL), various concentrations of L and NS proteins experiments in each figure. Unless otherwise carried out at 30°C for 2 hr.
The reaction mixture (0.2 ml) M NaCl, 4 mN dithiothreitol, lo-32P]CTP (specific activity 2.2 Pg of N-mRNA template and as described in individual stated, the reactions were
Analysis of RNA products by polyacrylamide gel electrophoresis. For analyses of the mRNA species, the reaction products were extracted with equal volumes of phenol and the RNA in the aqueous phase was precipitated with alcohol and the pellet suspended in water. Half of the RNA was used in a reaction mixture (40 Pl) containing 20 mM Tris-HCl, pH 7.5, 10 ELM HgC12, 100 mH KCl, 0.1 mH dithiothreitol, 1 Pg oligo(dT)l2,18 (Sigma Chemicals, St. Louis, MO), 1 unit/P1 RNasin (Promega Biotec, Madison, WI), 0.5 unit RNase H (Bethesda Research Laboratories, Bethesda, MD). The reaction was incubated at 37" for 20 min, terminated by addition of sodium dodecyl sulfate (0.5%), the RNA products extracted with phenol, and the RNA removed from the aqueous layer by precipitation with ethanol. The mRNAs with poly(A) tails thus removed were 41
Vol. 126, No. 1, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
analyzed by electrophoresis in 5% polyacrylamide slab gels (32 cm x 17 cm x 0.2 cm) containing 0.16% methylene-bis-acrylamide and 7 M urea. The gels were run at 600 V for 24 hr and RNA bands were located by autoradiography on Kodak The RNA in the other half of each sample was directly electroXAR film. phoresed in 20% polyacrylamide gels containing 7 M urea (12) to determine the extent of synthesis of the leader RNA.
RESULTS Optimum L and had
requirements
NS proteins
been
purified
presence
of
bound
to
small
amount
the
(20
quantity
of
optimum
the
of
RNA synthesis L protein
Addition
by approximately
concentration
synthesis
occurred
contrast,
the
significantly
in
1.4
pg/200
weight L protein
of template was
silver
or
contained
template
staining
of N-RNA
but
alone
However, resulted
depending
a
a trace
NS protein
requirement
Pl
basis)
1,
L protein
of
than
(Fig.
for
Thus,
required
higher for
optimal
was at
1)
no L
to the
addition
in
of
stimulation
on the purity
pg/200
for
optimal
of
addition
of
optimal
RNA
reaction.
In was
As shown in Fig.
at
basis)
RNA synthesis.
of the
2, at a
template
as
an NS concentration
a seven-fold
(on a molar
pl
of NS
RNA synthesis
same concentration
least
the
However,
.
0.2
optimal
42
concentration by
only
the L protein.
L and the
RNA synthesis
reaction.
of
NS protein
that of
or seventy-fold was
stimulated
at a concentration
higher
Fig.
and a saturating
significantly of
concentration
used
after
to 30-fold
lo-
hand,
was
a trace
electrophoresis,
mRNA species.
to the
retained
other
gel
in the
which
When an excess
L protein
no complete
concentrations
saturating
of
L and NS proteins
RNA synthesis
increasing
generally
on the
only
X-100
preparations.
At a fixed protein,
at 1 H NaCl,
was discernible
Triton
The
initially
The L protein,
by polyacrylamide
virtually
amounts
with
5 to 10%) of N protein.
was detectable. produced
virions
The NS protein,
in vitro.
of RNP which
by centrifugation.
was analyzed
NS protein
RNA synthesis
treatment
of purified
NS protein.
pg)
for
salt
and eluted
(approximately
template
template
by high
M NaCl followed
phosphocellulose of
of
purified
by disruption
0.4
amount
protein
were
of L and NS proteins
higher
amount
of NS protein These
results
of (on a
than
the
indicated
Vol. 126, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMOUNT OF L OJI)
Fig. 1. Rate of RNA synthesis with saturating NS protein and increasing concentrations of L protein. RNA synthesis was carried out using template saturating concentration of NS protein (1.4 pg), and increasing (2.2 pg), amounts of L protein. The RNA synthesized at each point was determined by measuring cold trichloroacetic acid insoluble radioactivity retained on nitrocellulose filters. Fig. 2. Rate of RNA synthesis with saturating L protein and increasing concentrations of NS protein. RNA synthesis was carried out using template concentration of L protein (0.2 pg), and increasing (2.2 wg), saturating amounts of NS protein as indicated. The RNA ayntheaized at each point was determined by measuring cold trichloroacetic acid insoluble radioactivity retained on nitrocellulose filters.
that
the
whereas
requirement
for
L protein
Role
of
requirement
that
oligonucleotides
understand
the role
NS or
of
such
protein saturating synthesis
leader
gels,
was
product
previously
was capable
of eynthesizing
initiated
termini
the
of
limiting
analyzed
various
kept
at was
(1.4
~1)
linearly
with
from
for
syntheais
the
ratio
the
increase
(0.24 of
poly-
and L protein. and the NS pg/200
N mRNA to
of NS protein
each
show the results
concentration
limiting
of
of individual
5% and 20% 1
a
amounts under
of NS protein
3),
43
we performed
synthesized
saturating
increased
to better
to saturating
3 and 4, and Table
a constant
(Fig.
In order
through
concentrations
RNA and mRNA
proceatr,
RNA products was
leader
such properties.
using
Figures
concentration
increased
atoichiometric
We have
RNA by electrophoresis
using
pg/200
was
elongation.
in the transcription
the
respectively.
experiments
When L protein
lacked
analyzed
reaction
and
5'
experiments
and
Each
mRNA species
the
NS protein
reconstitution
condition.
alone
of NS protein
L proteins
acrylamide
L protein
whereas
(91,
RNA synthesis
RNA chain
representing
sequences
of
in
the
for
was catalytic.
NS protein
demonstrated
series
NS protein
~1)
leader
concentration
to RNA
126, No. 1, 1965
Vol.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5678
1234
5878
-0
- LEADER
LEADER
RNA
0I
03
Fig. 3. Analysis of the RNA products eynthesiaed at saturating L protein concentration and increaeing amounts of NS protein. The mRtJAs (lanes 1 to 4) and leader RNA (lanes 5 to 8) synthesized in reconstituted reactions --in vitro containing template (2.2 pg), L protein (0.2 pg) and various amounts of NS protein were analyzed by electrophoresis, aa deecribed in Materials and Methods. Amounts of NS protein in lanes 1 and 5 ware 0.24 ng; lanes 2 and 6 were 0.48 pg; lanes 3 and 7 were 0.96 ng; and lanes 4 and 8 were 1.4 pg. The migration positions of the mRNAs for G. N, H, NS, and leader RNA are shown. 0 represents the origin of the gel. Analysis of RNA products synthesized at saturating NS proteins Fig. 4. and increasing concentrations of L protein. The mRNA products (lanes 1 to 4) and leader RNA (lanes 5 to 8) synthesized at saturating NS protein (1.4 ng), and increasing L protein amounts were analyzed by e1ectroihoresi.s as in Fig.-3. Amounts of L protein in lanes 1 and 5 were 0.02 pg; in lanes 2 and 6 were 0.04 pg; in lanes 3 and 7 were 0.08 Rg, and lanes 4 and 8 were 0.2 pg, respectively. The migration positione of the mRNAs for G, N, H, NS, and leader RNA are shown. 0 represente the origin of the gel.
(Table the
1).
In
increase
contraat, of
saturating
at
L protein
(0.2
ug/200
RNA
ratio
remaining
(Table
1).
These
results
all
rate
limiting,
hand,
when
RNA due
NS protein
(Fig.
4)
resulted
to
limiting in
that
rapidly
at
excess,
the
over
44
thin
whereas
limiting
NS protein,
Rg/200
corresponding
saturating
of rate
(0.02
the
initiated,
concentration
of
limiting in
constant
indicated were
concentration from
virtually
chains
wae
saturating
concentration ~1)
leader
protein,
a constant
to
N mRNA
to
concentration
range
concentrations
of
chain
elongation
NS protein. step
ul)
On was
the
the initiation
L
was other
Vol. 126, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
Radioactivity incorporated (cpr) Leader RNA
Additions Expt
N-RNA pg/200
.
A
B
L reaction
ul
NS
2.2
0.2
0.24
2.2
0.2
0.48
2.2
0.2
0.96
1
Ratio N-mRNA N-mRNA
791
Leader
RNA
652
0.8
1111
1937
1.7
1398
6139
4.4
2.2
0.02
1.4
176
642
2.2
0.00
1.4
269
1044
3.9
2.2
0.2
1.4
373
1253
3.4
3.6
Reconstitution reactions were carried out using indicated amounts of N-RNA complex, L, and NS proteins. The mRNAs and leader RRAs eynthesized in Expt. A and B were analyzed by polyacrylamide gel electrophoresis as described in Fig. 3 and Fig. 4, respectively. The labeled bands representing the N mRNA and the leader RNA under each condition were excised from the gel, and radioactivity was quantitated by Cerenkov counting.
of
RNA synthesis
extended
by L protein.
and completed
In order
to
further
step,
heat
NS proteins
were
heated for
products L
were
protein
at
completed 12, leader
13).
the
completed
results
chains.
by polyacrylamide
various
temperatures
In
contrast,
origin
were
mRNA chains
heated
and the
contrast
less being
6)
and
with
the the
its effect
on its
thermolabile, 45
of
of
to elongate capacity failed
but
11,
migrating
synthesis
of
of heating.
temperature
that
both
to synthesize
RNA species
interpretation
capability
able
RNA
purified
RNA (lanes
8, 9, and lo),
increase
and the
synthesis leader
L or
at saturating
Heating
were
preterminated
RNA (lanes
with
eliminated
L,
electrophoresis.
and
the RNA chain
experiments
NS preparations
leader
sharply
had
5,
quickly
Purified
2 min and used
abolished
of small,
consistent
but
A.
in
performed.
reconstitution gel
were
of NS protein.
were
virtually
as a series
effectively
In
in
chains
of NS protein
and 80°C for
RNA synthesis
lanes
RNA
of excess
experiments
at AO", 60",
mRNA decreased
NS protein large
inactivation
5,
initiated
the role
analyzed
RNA as well
between
These
investigate
mRNA (Fig.
and
all
due to the presence
elongation
concentrations
Thus,
heat
treatment
and complete
to complete to initiate
small
of the RNA
RNA chains
Vol. 126, No. 1, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1234587
3 91011121314
-LE
Fig. 5. Effect of temperature of the L and NS proteins on mRNA and leader RNA synthesis in in vitro reconstitution reactions. The mRNAs (lanes 1 to 7) and leader RNA (lanes 8 to 14) were synthesized using heated L (0.2 pg) or NS proteins (1.4 pg) and template (2.2 pg) and analyzed as described in Fig. 3. NS protein was heated at 40°C for 2 min (lanes 1 and 8); 60' for 2 min (lanes 2 and 9); 80' for 2 min (lanes 3 and IO). L protein was heated at 40°C for 2 min (lanes 4 and 11); 60" for 2 min (lanes 5 and 12); 80' for 2 q in (lanes 6 and 13). Lanes 7 and 14 represent control reactions at 30°C for 2 hr. The migration positions of mRNAs for G, N, M, NS, and leader RNA are shown. 0 represents the origin of the gel.
after
being
heated;
NS protein
consequently,
no RNA was synthesized
even though
excess
was present. DISCUSSION
In protein
this for
communication, an optimal
a weight
basis
protein.
This
a 1:l
molar
the
the
L
be due
--in
the
of
the
L and
in
the
studies.
different
that
discrepancy previous
strains
reported of
one (8)
reported
(8)
basis)
earlier
which
the
extreme
may
have
higher than
for
on the
where optimal lability
complicated
of thermolability
snd our preparations these
of NS
(8.11)
to be required
in the degree
VSV used in 46
7-fold
on a molar
may be due to report
the requirement
approximately
was found
The difference
of the
that
higher
from
NS proteins
This
is
70-fold
is different
vitro.
L proteins
demonstrated
of transcription
approximately
used
protein
to
(or
ratio
stoichiometry
between
rate
conclusion
RNA synthesis of
we have
studies.
may
also
In a similar
L
Vol. 126, No. 1, 1985 reconstitution disease
experiment,
virus
(NDV),
requirement for
the
for
the
and
of
ratio
molar
ratio
1:40:400
to
concentration in
2,
corrected
system,
the
molar
nucleocapsid
ratio
facilitate
continued
synthesis
but
the
is packaged
process
within
template,
was unable
incomplete
reactions alone
whereas
the
elongation when
of
(Table
1).
can
Thus,
1).
2,500
in
than of
molar
replication with
the
loo-fold)
than
NS protein
might
RNP in the
a suboptimal
is the
high
associated
(greater
It
NS in vitro,
present
NS protein
infected
level
of
contrast, excess
[o-32P)ATP
cells,
of NS protein
and
the ability
was ratio
it
seems
that
RNA chains
but
failed
L protein
e.g.
increased,
ratio
complete 47
when present
to N-RNA
However, was
using
shown
that
L
AC and AACA, etc.,
RNA chains.
demonstrated
concentration
L protein, to
it
A chain
by the fact
in the presence
of N mRNA/leader
the corresponding
and
CTP,
to initiate
is directly
the
when added
mRNAs (9).
RNA chains,
NS protein 3),
alone,
full-length
NS protein
(Fig.
in
L protein
initiate
lacked for
In sharp
Table
that
be approximately
for
concentration
that
synthesize
efficiently
L protein
was
initiated
to
NS protein,
NS protein
25,000
the optimum
in a cell-free
higher
high
previously
concentration
excess
Moreover,
maturation,
containing
function
the
that
the virion.
We have demonstrated
protein
than
of L and NS).
also
of RNA from progeny
of virus
to
weights
synthesized
the
the
The corresponding
requirements
to be considerably Perhaps,
that
(3).
is assumed
reported
mRNA are
(15.16). newly
(17).
during
its
it
be 1:40:2800.
been
the higher
of 240,000
of genome RNA (2),
would has
and
of
and if
the molecular
like
cells
was found
L protein
for
NS protein
infected
here
observed
--in vitro.
per molecule
virion
that,
also
of Newcastle
of NS in NDV) was higher
are used,
determined
purified
have
of L, NS, and N proteins
are bound
note
the
(14)
analogue
respectively,
the
of
amounts
the
weights
of RNA:L:NS
interesting
al.
RNA synthesis
optimal
N protein
(ref.
--et
P, and the NP-RNA complex
for
(4),
in
L,
(the
47,000
molar
Hamaguchi
molecular
molecules
using
P protein
L protein
If (4),
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
increased
remained
of an linearly
constant
was increased in excess,
them due to lack
that
when
(Fig.
4,
may have
of NS protein
Vol. 126, No. 1, 1985 which
was the
the
ratio
all
rate
of
increase
completion
to
leader
showed
which
the than
Since efficient progress
polymerase,
been
ref.
interacts
prepares
the
order
interesting
results
them
with
the
to
study
full
moving
replace
might site any
of other
for
that
a8 with
genome.
the
new
and the
the L protein
initiate
anal1
the
N protein it.
synthesis
in vitro
by host
cell
components
the
putative
host
factors
Perhaps
NS
This
explains
possibly
the region
It
should cellular
Stimulation (18).
the
molecule,
with
example,
reported
facilitate
and
from
--in vitro.
has been
RNA
the N protein
RNA synthesis. for
is
The NS protein
to interact
agent,
RNA that
the L protein
may move along
the NS function
may
48
polypeptide
demonstrating
to
displaces
(L or NS)
a large
mRNA sizes.
be required
protein,
whether
capacity
for little
to L. For each L protein
synthetic
to be seen
the
to the
of NS relative
whether
conclusion
which
required
proteins
presented
as well
access
species.
candidate
the
length
protein
to gain
the
to
two
of only
the L protein
70 NS molecules
preceding
has
L protein
such that
requirement of
interaction
aborted
(8,10,13),
being
proper
has been
support which
an RNA unwinding
molar
N-RNA
by the
extend
L protein
to be the
evidence
synthesized
template
genome RNA for
on the
seems
L protein,
mRNA but
are
vitro the
5) clearly
as numerous
the message
of
brought of N-mRNA
(Fig.
L and NS proteins
which
in excess,
full-length
RNA as well
in --
the
ratio
studies
complex
The
RNA polymerase
possibly
N-RNA
with
rapidly
a constant
than
both
elucidate
no direct
cannot
is
the
3),
The above
the but
to
leader
that
were
to complete
smaller
RNA polymerase.
mRNAs are
N-RNA template.
of
made
although
high
RNA, but
of
daltone,
protein
leader
transcription
the
probably
to complete
of
was present
inactivation
capability
increase
RNA occurred
L protein
maintaining
heat
a linear
to leader
by the
thus
ite
discovery
constitutes (240,000
lost
the original
has
chains
initiated
The
capacity
the
respect
Thus,
When NS protein
NS protein,
NS protein
RNA8 larger
in the reaction. with
had been
RNA synthesis.
retained
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concentration.
by the
that
viral
step
mRNA synthesis
of NS protein
to
the
limiting
the
RNA chains
still
BIOCHEMICAL
be or
of RNA It
NS function
remaina or can
Vol. 126, No. 1, 1985 replace firmly
it
--in
establiah
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
vitro.
Future
the functions
studies
along
this
of L and NS in the
line
would
transcription
certainly
help
process.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Wagner, R.R. (1975) in, Compreheneive Virology (H. Fraenkel-Conrat and R.R. Wagner, eds.), pp. l-80, Plenum Press, New York. Bishop, D.H.L., and Roy, P. (1972) J. Virol. 10, 234-243. Schubert, H., Harmison, G.G., and Meier, E. (1984) J. Virol. 51, 505-514. Gallione, C.J., Greene, J.R., Iverson, L.E., and Rose, J.K. (1981) J. Virol. 39, 529-535. Banerjee, A.K., Abraham, G., and Colonno, R.J. (1977) J. Gen. .Virol. 34, 108. Colonno, R., and Banerjee, A.K. (1978) Nucl. Acids Res. 5, 4165-4176. Abraham, G., and Banerjee, A.K. (1976) Virology 71, 230-241. Emerson, S.U., and Yu, Y.H. (1975) J. Virol. 15, 1438-1356. De, B.P., and Banerjee, A.K. (1984) J. Virol. 51, 628-634. Banerjee, A.K., Moyer, S.A., and Rhodee, D.P. (1974) Virology 61, 527-558. Naito, S., and Ishihama, A. (1976) J. Biol. Chem. 251, 4307-4314. Thornton, G.B., De, B.P., and Banerjee, A.K. (1984) J. Gen. Virol. 65, 663-668. Emerson, S'.U., and Wagner, R.R. (1972) J. Virol. 10, 297. Hamaguchi, H., Yoshida, T., Nishikawa, K., Naruse, H., and Nagai, Y. (1983) Virology 128, 105-117. Hsu, C., Kingebury, D.W., and Murti, K.G. (1979) J. Virol. 32, 304-313. Villarreal, L.P., Briendl, H., and Holland, J.J. (1976) Biochemistry 15, 1663-1667. Patton, J.T., Davis, N.L., and Wertz, G.W. (1983) J. Virol. 45, 155-164 Rose, J.K., Lodieh, H.F., and Brock, M.L. (1977) J. Virol. 21, 683-693.
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