Download Detection of RNA-protein complex in vaccinia virus core in vitro

Journal of General Virology (1992), 73, 1243-1249. Printed in Great Britain
1243
Detection of RNA-protein complex in vaccinia virus core in vitro
transcription system
Clarissa R. A. Dfimaso and Nissin Moussatch~*
Laborat6rio de Biologia Molecular de Virus, Instituto de Biofisica Carlos Chagas Filho, C C S - U F R J ,
21941 Rio de Janeiro, R J, Brazil
The incubation of vaccinia virus cores in appropriate
conditions promotes the release of core proteins into a
supernatant fraction. Under transcription assay conditions core mRNAs are extruded in association with
viral core proteins, however the presence of these
proteins within the core particle is not essential for
RNA synthesis and extrusion. The RNA-protein
complex is resistant to micrococcal nuclease. Five
proteins of 60K, 43K, 28K, 18K and 14.5K with RNAbinding abilities have been identified by [32p]RNA
overlay protein blot assays. These proteins are likely to
be a component of the viral ribonucleoprotein complex
since core basic proteins with similar Mrs have been
identified and at least one RNA-binding protein is
predicted in the vaccinia virus genome.
Introduction
into the supernatant fraction (Dfimaso & Moussatch6,
1992). SDS-PAGE analysis of the supernatant revealed
the presence of 17 polypeptides, four of which are
phosphorylated.
In this report we show that vaccinia virus core
mRNAs extruded during in vitro transcription are
associated with core proteins. In the presence of viral
proteins the mRNA can be protected from micrococcal
nuclease digestion and two peptides of 18K and 14.5K
are u.v. crosslinked to [32p]RNA. The identification of
the RNA-binding proteins was also performed by RNA
overlay protein blotting (Northwestern blot). This
procedure revealed the presence of five proteins with
apparent Mrs of 60K, 43K, 28K, 18K and 14-5K with
affinity for viral core mRNA. A ribonucleoprotein
(RNP) with a typical RNA-binding motif and an Mr of
14-2K was predicted within the genome of vaccinia virus
Copenhagen strain (A31R) (Goebel et al., 1990) and an
equivalent protein was found in the WR strain (SalL1 R)
with an apparent Mr of 14"9K (Smith et al., 1991). The
possibility of this protein being one of the proteins
identified in this work, and the formation of R N A protein complexes during in vitro mRNA synthesis, are
discussed.
The poxviruses are a large family that infect both
vertebrate and invertebrate hosts (for review see Moss,
1990). They are distinguished by their large size, complex
morphology and cytoplasmic site of replication. Studies
with vaccinia virus, the prototype of the family, have
contributed to our understanding of the strategies used
by these viruses to replicate and express their genome.
The infection of tissue culture cells with vaccinia virus
results in profound cytopathic effects, such as changes in
membrane permeability and inhibition of host protein,
RNA and DNA synthesis (Bablanian, 1984).
Studies on vaccinia virus transcription have been
facilitated by purification of the virus particle, and the
ability of the cores to synthesize RNA in vitro (Kates &
McAuslan, 1967; Munyon et al., 1967). These transcripts
have been characterized by sedimentation (Paoletti,
1977). R N A - D N A hybridization (Boone & Moss, 1978;
Cabrera et al., 1978) and translation in cell-free systems
(Cooper & Moss, 1978; Pelham et al., 1978).
The core transcription system was re-evaluated in
terms of RNA synthesis in the presence of Mg 2+ or Mn z+
and the effect of polyamines on the assay system
(Moussatch6, 1985). In addition, it was also verified that
some core proteins which might contribute to the
regulation of early transcription are phosphorylated
during the initiation of RNA synthesis (Moussatch6 &
Keller, 1991). Recently, we have shown that when
vaccinia virus cores are incubated in the presence of
nucleotides, several core-associated proteins are released
0001-0672 © 1992 SGM
Methods
Virus. Vacciniavirus (WR strain) was purifiedfrom infectedHeLa
cells after a 48 h infection as described previously(Moussatch6 &
Keller, 1991).The viruswas dilutedto 2 x 1011particles/ml,assuming
that 1.0 A260 unit corresponds to 1.2 × 1011 particles/ml. The 35S-
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1244
C. R. A . D&maso and N . M o u s s a t c h ~
labelled vaccinia virus was produced by a 48 h incubation of BHK-21
cells at an m.o.i, of 0-1 p.f.u./cell in the presence of 10 ~tCi/ml
[35S]methionineduring the final 24 h of infection.
Preparation of vaccinia virus cores for in vitro assays. The vaccinia
virus cores used in the in vitro transcription system were prepared by
incubating purified virus (2 x 1011 particles/ml) in 50 mM-Tris-HC1
pH 7.5, 10 mM-DTT,0-05% NP40 for 10 min at 37 °C and processing as
described by Moussatch6 (1985).
RNA synthesis by core-associated RNA polymerase. Vaccinia virus
cores were assayed for in vitro RNA synthesis as described (Moussatch6, 1985) with some modifications. The transcription reaction
mixtures contained 50 mM-Tris-HC1 pH 7.5, 10 mM-DTT, 5 mMMgClz, 2 mM-spermidine,20 mM-KC1,2 mM-ATP,2 mM-GTP,2 mMCTP, 0.1 mM-[3H]UTP(35 c.p.m./pmol) and 4 × 101°vaccinia virus
core particles/ml. In some experiments the [3H]UTP was replaced by
[ct-32p]UTP(250 ~tCi/ml).The 100 ktl reaction assays were incubated at
37 °C and the reaction was stopped by a 2 min centrifugation in an
Eppendorf microfuge. Each of the supernatant fractions was removed,
precipitated with 1 ml of cold 5% TCA and collected in a nitrocellulose
filter (Schleicher & Schuell, BA-85). The acid-precipitated samples
were then treated as described (Moussatch6, 1985) and the vaccinia
virus core mRNA isolated as described by Moussatch6& Keller (1991).
Nitrocellulosefilter-binding assay. To measure RNA protein interaction in solution, a nitrocellulosefilter-binding assay was used. Samples
(100 p.l)of the supernatant fraction, radioactively labelled in vitro, were
removed and directly applied to nitrocellulose filters, which had been
pre-moistened in binding buffer containing 50 mM-Tris-HCl pH 7.5,
5 mra-MgCl2,20 mM-KC1.After filtering, the nitrocellulosefilters were
washed with 2 ml of binding buffer, dried and the radioactivity
incorporated was measured in an LS-7000Beckman liquid scintillation
counter.
Micrococcal nuclease treatment of the supernatant fraction. The RNAprotein complex was analysed for resistance to micrococcal nuclease
treatment. The vaccinia virus [32p]RNA-protein complex was
separated from the cores by a 2 min centrifugation. The sample was
incubated with 0.75 mM-CaCI2 and Staphylococcus aureus nuclease
(1250 units/ml, Boehringer Mannheim). At a given time, the reaction
was stopped by the addition of 2.5 mM-EGTA and the samples were
prepared for electrophoresis in 5~ non-denaturing polyacrylamide gel
(Konarska, 1989). In another experiment, the sample was placed under
a u.v. lamp (1.2 x 107 J/m2; UVS-11 mineralight) for 20 min on ice
before micrococcalnuclease digestion. After the treatment, the samples
were prepared and analysed in a 15% polyacrylamide gel (Laemmli,
1970).
PAGE. The supernatant fraction from the core system was treated
with 5 volumesof acetone for 18 h at - 4 0 °C. After centrifugation, the
precipitate was resuspended in loading buffer containing 10~ glycerol,
100 mM-DTT,2% SDS, 0.02~ bromophenol blue and heated at 100 °C
for 5 min. The samples destined for blotting experiments were not
heated prior to loading on the gel. The polypeptides were separated
using a 12.5~ or a 15~ SDS-polyacrylamide gel as described by
Laemmli (1970). Following electrophoresis, the gel was fixed with 20~
TCA, dried and exposed to an Omat-K Kodak X-ray film or treated for
electroblotting. The laser densitometer scan was performed using an
UltraScan apparatus (model 2202, LKB).
Protein blotting. Vaccinia virus core-released proteins (20 ~tg) were
prepared for electrophoresis as described before. After electrophoresis,
the polypeptides were electrophoreticallytransferred to a nitrocellulose
membrane (Schleicher & Schuell) (4 h, 225 mA, 4 °C) in a buffer
containing 192 mM-glycine,25 mM-Trisand 20~ methanol (Towbin et
al., 1979). The membrane was incubated in a blotting solution
containing 10 mM-Tris-HClpH 7.5, 50 mM-NaC1, 1 mM-EDTA,0-02~
BSA, 0-02~ Ficoll, 0.02~ polyvinylpyrrolidone(Bowenet al., 1980)in
a sealed plastic bag for 2 h at room temperature, with gentle shaking.
The solution was replaced with the blotting solution containing
vaccinia virus core [32p]RNA (15 x 106 c.p.m.) and 15 ~tg/mloftRNA
as carrier. The membrane was incubated for 90 min at room
temperature in a sealed plastic bag with gentle shaking. After this
period the membrane was washed four times, for 30 min each, with 10
mM-Tris-HC1pH 7.5, 50 mM-NaC1, 1 mM-EDTA,then dried at room
temperature and exposed to an X-ray film as described before.
Results
Vaccinia core R N A - p r o t e i n c o m p l e x f o r m a t i o n
Purified v a c c i n i a virus core particles w h e n i n c u b a t e d in
the presence of the four r i b o n u c l e o t i d e s synthesize
m R N A (Kates & Beeson, 1970). T h e R N A extruded
from the core can be separated from core particles by
centrifugation. Recently, we reported that the i n c u b a tion of v a c c i n i a virus cores in the presence of nucleotides
also promotes the release of viral p r o t e i n s from the core
(D~.maso & Moussatch6, 1992). A n a l y s i s by S D S - P A G E
indicates the presence of a p p r o x i m a t e l y 17 peptides in
the s u p e r n a t a n t fraction. T h e release of core proteins
d u r i n g viral t r a n s c r i p t i o n suggests the occurrence of
R N A - p r o t e i n complexes as s h o w n i n other systems
(reviewed in Dreyfuss, 1986). T o investigate this hypothesis, we utilized the nitrocellulose filter-binding assay, in
w h i c h the protein-associated R N A is r e t a i n e d on the
filters. Protein-free R N A does n o t b i n d to the filters
u n d e r this assay c o n d i t i o n ( R i c h t e r & Smith, 1983).
Phenol-extracted v a c c i n i a virus core R N A also does not
b i n d to nitrocellulose filters (data n o t shown). Purified
cores were i n c u b a t e d in a t r a n s c r i p t i o n assay system as
described in Methods. A t a g i v e n time, fractions were
removed, the cores were pelleted by centrifugation, a n d
the a m o u n t of R N A p r e s e n t in the s u p e r n a t a n t fraction
was m e a s u r e d by acid p r e c i p i t a t i o n or by nitrocellulose
filter-binding assay. N o core c o n t a m i n a t i o n was found in
the s u p e r n a t a n t fraction after c e n t r i f u g a t i o n (data not
shown). D u r i n g the 15 m i n i n c u b a t i o n most of the
synthesized R N A extruded was associated with proteins
since n o difference i n total counts was observed using
two different procedures (Fig. 1). A f t e r this period, most
of the newly synthesized R N A was extruded free of
proteins. O n l y half of the [ 3 H ] R N A counts were retained
o n the nitrocellulose filters w h e n c o m p a r e d to the total
a m o u n t of acid-precipitated [ 3 H ] R N A .
I n c u b a t i o n of v a c c i n i a virus cores with A T P promotes
the release o f proteins into the s u p e r n a t a n t fraction
w i t h o u t R N A synthesis (Dfimaso & Moussatch6, 1992).
These proteins seem to be similar to those released
d u r i n g in vitro t r a n s c r i p t i o n , p r o m p t i n g us to verify
w h e t h e r cores recovered from A T P p r e - i n c u b a t i o n were
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RNA-protein complex in vaccinia cores
101
I
I
I
I1
/
1245
10-
/
2
X
~
X
4
~
0
5
10
Time (min)
15
0
20
Fig. 1. Retention on nitrocellulosefiltersof the RNP complex released
from vaccinia virus cores. Vaccinia virus cores were incubated in the
standard reaction mixture for transcription as described in Methods.
At the indicated times samples were removed, centrifuged and the
supernatants were precipitated with 5% TCA (O) or used in the
nitrocellulose filter-binding assay (O) as described in Methods.
able to synthesize a n d extrude m R N A . I n this experim e n t , cores were p r e - i n c u b a t e d in the presence of A T P
for 20 m i n , recovered by c e n t r i f u g a t i o n a n d r e s u s p e n d e d
in the reaction m i x t u r e for t r a n s c r i p t i o n , as described in
Methods. As s h o w n i n Fig. 2, p r e - i n c u b a t e d cores were
i n fact c a p a b l e of synthesizing a n d e x t r u d i n g R N A from
the particle w h e n t r a n s c r i p t i o n was m e a s u r e d by acid
precipitation. Most of the transcripts were extruded free
of p r o t e i n since a small a m o u n t of R N A was able to b i n d
to the nitrocellulose filter. W e t h e n d e t e r m i n e d w h e t h e r
this R N A could reassociate with the proteins previously
released d u r i n g p r e - i n c u b a t i o n , by a d d i n g the p r o t e i n
s u p e r n a t a n t fraction (10 ~tg/ml) to the R N A synthesized
from p r e - i n c u b a t e d cores. T h e R N A - p r o t e i n complex
was reconstituted, since the r e t a i n i n g capacity of the
nitrocellulose filters was restored (Fig. 2).
4
0
t
10
15
Time (min)
5
T
20
Fig. 2. Retention on nitrocellulose filters of the RNP complex reformed in vitro.Vaccinia virus cores were pre-incubated in the presence
of ATP for 20 min. After this period the mixture was centrifuged and
the supernatant removed. Pre-incubated vaccinia virus cores were
incubated in the standard reaction mixture for transcription as
described in Methods. At indicated times samples were removed,
centrifuged and the supernatants were precipitated with 5% TCA (O),
or used in nitrocellulosefilter-binding assay in the absence (A) or in the
presence (O) of the supernatant fraction obtained during the preincubation period.
Table 1. Effect of different treatments on the reconstitution
of the RNA-protein complex
Complex fraction
Treatment*
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
RNA
None
None
50 mM-EDTA
500 mM-NaC1
30 mg/ml Heparin
120mg/ml Heparin
500 mg/ml Heparin
10 mg/ml BSA
500 mg/ml BSA
30 mg/ml tRNA
30 mg/ml Globin mRNA
2.8 mg/ml Core RNA
+
+
+
+
+
+
supernatant
supernatant
supernatant
supernatant
supernatant
supernatant
+ supernatant
+ supernatant
+ supernatant
Radioactivity
(% of control)
20
100
45
42
46
38
22
28
29
40
50
50
RNA-protein complex analysis
T o study the f o r m a t i o n of the R N A - p r o t e i n complex, we
tested the effect of different t r e a t m e n t s in r e c o n s t i t u t i n g
the R N P complex. F o r this assay we utilized 2.8 ~tg of
[ 3 H ] R N A / m l c o r r e s p o n d i n g to the total a m o u n t synthesized d u r i n g a 20 m i n i n c u b a t i o n . R N A was c o m b i n e d
with the total proteins released d u r i n g p r e - i n c u b a t i o n
(10p, g/ml). T a b l e 1 shows that 20% of the R N A
* Vaccinia virus cores were pre-incubated in the presence of ATP for
20 min. The supernatant fraction was removed by centrifugation and
the core fraction was resuspended in the transcription reaction mixture
in the presence of [3H]UTP for 20 min. After this time RNA extruded
from the cores was removed by centrifugation and assayed for binding
to nitrocellulose filters in the absence or in the presence of the core
supernatant fraction obtained during the pre-incubation period as
described in Methods. The treatment was performed by mixing the
agent with the supernatant fraction before [3H]RNA addition.
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C. R. A. Dgtmaso and N. Moussatch~
1246
(a)
(e)
(b)
1
2
3
4
5
I
1
I I[
II
[
-66K
--45K
-36K
4--29K
-24K
-20.1K
.~
4r--14.2K
Mobility
Fig. 3. Micrococcal nuclease treatment of the [32p]RNA-protein complex. Supernatant fraction from the vaccinia virus core
transcription system and purified vaccinia virus core RNA were isolated, non-irradiated (a) or u.v.-irradiated (b), digested with
micrococcal nuclease and analysed by PAGE as described in Methods. (a) 5~ Non-denaturing gel: lane 1, RNA; lane 2, RNA after
5 min of micrococcal nuclease digestion; lane 3, supernatant fraction; lanes 4 and 5, supernatant fraction after 5 and 20 min of
micrococcalnucleasedigestion. (b) 15~ Denaturing gel: lane 1, supernatant fraction; lanes 2 to 5, supernatant fraction after 1, 5, 10 and
20 min of micrococcal nuclease digestion. The arrows indicate two major (14.5K and 18K) and one minor (28K) complexesobserved
after digestion. (c) Laser densitometer tracing of the autoradiogram shown in (b), lane 5.
synthesized from pre-incubated cores was still retained
on the nitrocellulose filters. The addition of 50 mME D T A or 500mM-NaC1 during reconstitution of the
complex reduced the binding capacity to 45 and 4 2 ~
respectively.
Heparin is a polyanion that has been used to block the
formation of nucleic acid-protein complex (Dynan &
Burgess, 1979). Our results show that increasing concentrations of heparin gradually prevented reconstitution of
the R N A - p r o t e i n complex. When a very high concentration of heparin (500 gg/ml) was used, reconstitution of
the complex was prevented. The core protein fraction
could not be replaced by high concentrations of BSA. In
competition experiments, addition of the same amount
of unlabelled vaccinia virus R N A (2.8 I_tg/ml) reduced
the binding capacity to 50~o. However, t R N A and globin
m R N A must be present in at least a 10-fold excess over
32p-labelled vaccinia virus R N A to lower the amount of
radioactivity retained on the filter by 40 and 5 0 ~
respectively.
Micrococcal nuclease digestion of the RNA-protein
complex
To examine the R N A - p r o t e i n complex formed in vitro
we used the u.v. crosslinking approach described by
Dreyfuss (1986). The supernatant fraction from the
vaccinia virus core transcription system was isolated as
described in Methods. The R N A - p r o t e i n complex and
purified vaccinia virus core R N A were digested with
micrococcal nuclease and analysed by P A G E as described. Fig. 3(a) shows that purified vaccinia virus
R N A was completely digested by the micrococcal
nuclease after a 5 min treatment. However, the R N A protein complex was partially resistant to 20 rain of
micrococcal nuclease treatment. Alternatively, the protein was u.v. crosslinked to the newly synthesized core
[32p]RNA and was also analysed by SDS P A G E after
nuclease digestion as described in Methods. As shown in
Fig. 3(b) viral peptides crosslinked to [32p]RNA were
radioactively labelled after micrococcal nuclease treatment. Fig. 3 (c) shows the laser densitometer scanning of
the autoradiogram shown in Fig. 3 (b, lane 5). Two major
complexes of [32p]RNA-protein with Mrs of 14-5K and
18K and a minor peak of 28K were observed after 20
mins of nuclease digestion.
Detection of vaccinia virus core RNA-binding protein
As previously described, 17 polypeptides were released
when the cores were pre-incubated in the presence or
absence of factors enabling viral transcription. To
analyse which of these proteins could associate with the
viral R N A s , the proteins were separated by S D S - P A G E
and electroblotted onto nitrocellulose filters as described
in Methods. The resulting blots were assayed for the
binding of [3 2p]RN A free of proteins prepared from preincubated cores. Fig. 4 shows the identification of five
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R N A - p r o t e i n complex in vaccinia cores
(a)
1
2
3
(b)
(c)
1247
was scanned in a laser densitometer and the p e a k areas
corresponding to the R N A - p r o t e i n complexes were
determined in arbitrary units. Table 2 shows that 99% of
the radioactivity was linked to proteins with Mrs of 18K
and 14.5K whereas the other three protein bands
represented 1% of the total radioactivity.
Discussion
Fig. 4. Detection of vaccinia virus core nucleic acid-binding proteins
by the RNA overlay protein blot assay. Vaccinia virus core proteins
released after pre-incubation with ATP were separated by 12.5% SDSPAGE and electroblotted onto nitrocellulose membranes. The membranes were incubated with [32p]RNA synthesized by pre-incubated
cores in vitro as described in Methods. (a) Lane 1, [35S]methioninelabelled vaccinia virus; lane 2, vaccinia virus cores, lane 3, supernatant
fraction. (b) Autoradiogram of the membrane after exposure for 40 h
with an intensifying screen as described in Methods. (c) Autoradiogram of the membrane after exposurefor 24 h as described in Methods.
The arrows indicate the 18K and 14.5K bands.
Table 2. Hybridization o f the vaccinia virus R N P complex
Mr
Arbitrary
units*
Percentage of
total
60K
43K
28K
18K
14.5K
3.1
1.0
9-5
567.8
764-6
0.23
0.07
0.71
42-18
56-81
* The autoradiogram shown in Fig. 4 was subjected to densitometry
in an UltraScan laser densitometer (LKB). The values presented as
units were obtained by cutting and weighing the absorbance peaks
recorded during the scanning of each band of the autoradiograph.
proteins with apparent Mrs of 60K, 43K, 28K, 18K and
14.5K with viral R N A binding capability (Fig. 4a, lane
2). In a different gel exposure the two bands of 18K and
14.5K were more distinguishable. The autoradiogram
Incubation of vaccinia virus core with the four ribonucleoside triphosphates provides a useful in vitro system in
which to study transcription. In this assay, since the
particle is not disrupted the endogenous D N A is used
and the synthesized m R N A probably resembles the
transcripts made in vivo early in infection (Cooper &
Moss, 1978; Kates & Beeson, 1970; Pelham et al., 1978).
In this situation, core m R N A is made and extruded from
the particles. Proteins from the cores can also be released
when incubated in the presence of A T P (Ben-Hamida et
al., 1983). Recently, we have shown that during in vitro
m R N A synthesis, core proteins are also released from
the particle (Damaso & Moussatch6, 1992). The role of
these proteins is still unknown and can probably be
related to m R N A extrusion. In the present paper, we
demonstrate that vaccinia virus core m R N A s are
extruded in association with core proteins (Fig. 1). In
addition, core R N A extruded free of proteins can reform the ribonucleoprotein complex with the corereleased proteins (Fig. 2, Table 1). The R N A - p r o t e i n
complex is resistant to micrococcal nuclease treatment
(Fig. 3) and five proteins have R N A - b i n d i n g ability (Fig.
4). However, two polypeptides with apparent MrS of 18K
and 14.5K have higher affinities for the viral R N A than
the 60K, 43K and 28K peptides.
The data supporting the presence of non-ribosomal
R N P s in eukaryotic cells has been reviewed by Dreyfuss
(1986). In growing H e L a cells eight R N P s , ranging from
34K to 120K were identified as being associated with
heterogeneous nuclear R N A ( h n R N A ) and also had
affinity for s s D N A (Dreyfuss, 1986). Cytoplasmic nonribosomal R N P complexes ( m R N P ) have also been
isolated and characterized. These complexes are resistant to high concentrations of NaC1 (0-5 M) and are not
associated with cellular polyribosomes (Dreyfuss, 1986).
Several proteins of similar M r have been found associated with m R N A in different cell lines (Greenberg,
1977; K u m a r & Pederson, 1975; Morel et al., 1971).
R N P complexes were also described as being formed
with viral transcripts. In cells infected with adenovirus 2
it was found that h n R N A complexes formed with both
viral and cellular proteins that were probably involved in
the processing and transport of the viral m R N A to the
cytoplasm (Van Eekelen et al., 1981). An R N P complex
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1248
C. R. A. D~maso and N. Moussatch~
was found in vesicular stomatitis virus (VSV)-infected
cells formed by the N protein, viral mRNAs and host
mRNPs (Adam et al., 1986). The function of the VSV N
protein-mRNA interaction is not known but it was
demonstrated that this complex inhibited protein synthesis in rabbit reticulocyte lysates at the level of initiation
(Rosen et al., 1984). The presence of mRNP not
associated with the polyribosomes early in vaccinia
virus-infected cells was demonstrated by Metz et al.
(1975). These authors suggested that this structure could
be a precursor of the virus mRNA found in polyribosomes. Recently, the complete sequence of the genome of
vaccinia virus was determined and an RNP with an
RNA-binding signature (A31R) and an M r of 14-2K was
predicted but not identified (Goebel et al., 1990). It is
possible that the 14.5K protein identified in this
communication is the same as the one identified as being
encoded by the DNA of vaccinia virus.
The presence of nucleic acid-binding proteins associated with vaccinia virus has been already demonstrated. Most of these proteins were reported to bind to
both ss- and dsDNA molecules (Ichihashi et al., 1984;
Kao et al., 1981; Soloski et al., 1978; Yang & Bauer,
1988). Several other basic proteins associated with
vaccinia virus core particles have been identified but
they were not referred to as nucleic acid-binding proteins
(Oie & Ichihashi, 1981). Proteins with MrS ranging from
18.5K to 11K were first identified and grouped as VP9,
VPi0 and VP11 and correspond to 30% of total vaccinia
virus proteins (Sarov & Joklik, 1972). Some of these
proteins were phosphorylated and could be associated
with cellular ribosomes (Sagot & Beaud, 1979). Ichihashi
et al. (1984) identified four DNA-binding proteins with
MrS of 57K, 27K, 13"8K and 13K. All four proteins were
associated with the core/lateral body fraction and were
not associated with the DNA structures; this is an area
which needs further investigation as the role of lateral
bodies in the biology of vaccinia virus is still unknown. It
is possible that some of these basic proteins could
associate with viral RNA and play a role in the transport
of RNA to the ribosomes or in the translation of
transcripts. The latter role has already been proposed to
occur in a viral system. It has been suggested that during
human immunodeficiency virus infection a viral protein
(tat gene) linked to the 5" end of the viral mRNAs acts as
a positive translational control (Rosen et al., 1986). The
role of vaccinia virus RNA-binding proteins in viral
transcription and translation is currently under investigation in our laboratory.
The authors wish to thank Dr S. J. Keller for valuable support,
Ademilson N. Bizerra and Paulo Sergio Lopes for technical assistance
and the Radiology Department of the UFRJ Hospital. This research
was supported by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnolbgico (CNPq), Financiadora de Estudos e
Projetos (Finep) and Conselho de Ensino para Graduados da UFRJ
(CEPG).
References
ADAM, S. A., CHOI, Y. D. & DREYFUSS,G. (1986). The interaction of
mRNA with proteins in VSV infected cells. Journal of Virology 57,
614-622.
BABLANIAN,R. (1984). Poxvirus cytopathogenicity: effects on cellular
macromolecular synthesis. In Comprehensive Virology, vol. 19, pp.
391-427. Edited by R. R. Wagner & H. Fraenkel-Conrat. New
York: Plenum Press.
BEN-HAMIDA,F., PERSON,A. & BEAUD,G. (1983). Solubilization of a
protein synthesis inhibitor from vaccinia virions. Journalof Virology
45, 452-455.
BOONE, R. F. & MOSS, B. (1978). Sequence complexity and relative
abundance of vaccinia virus mRNAs synthesized in vivo and in vitro.
Journal of Virology 26, 554-569.
BOWEN, B., STEINBERG,J., LAEMMLI,U. K. & WEINTRAUB,H. (1980).
The detection of DNA-binding proteins by protein blotting. Nucleic
Acids Research $, 1-20.
CABRERA,C. V., ESTEBAN,M., McCARRON,R., MCALLISTER,W. T. &
HOLOWCZAK,J. (1978). Vaccinia virus transcription: hybridization
of mRNA to restriction fragments of vaccinia DNA. Virology 86,
102-114.
COOPER, J. A. • MOSS, B. (1978). Transcription of vaccinia virus
mRNA coupled to translation in vitro. Virology 88, 149-165.
D.L~IASO,C. R. A. & MOUSSATCHI~,N. (1992). Proteins released from
vaccinia cores are probably not involved in protein synthesis
inhibition in vitro. Brazilian Journal of Medical and Biological
Research (in press).
DREYFUSS, G. (1986). Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annual Review of Cell Biology 2,
459-498.
DYNAN, W. S. & BURGESS,R. R. (1979). In vitro transcription by wheat
germ ribonucleic acid polymerase II. Effect of heparin and the role of
templete integrity. Biochemistry 18, 4581-4588.
GOEBEL,S. J., JOHNSON,G. P., PERKUS,M. E., DAVIS,S. W., WINSLOW,
J. P. & PAOLETTI,E. (1990). The complete DNA sequence of
vaccinia virus. Virology 179, 247-266.
GREENBERG,J. (1977). Isolation of messenger ribonucleoproteins in
cesium sulfate density gradient: evidence that polyadenylate and
non-polyadenylate messenger RNAs are associated with protein.
Journal of Molecular Biology 108, 403-416.
ICHIHASHI,Y., OIE, M. & TSURtmA~, T. (1984). Location of DNAbinding proteins and disulfide-linked proteins in vaccinia virus
structural elements. Journal of Virology 50, 929-938.
KAO, S.-Y., RESSNER, E., KATES, J. & BAUER, W. R. (1981).
Purification and characterization of a superhelix binding protein
from vaccinia virus. Virology 111, 500-508.
KATES, J. R. & BEESON, J. (1970). Ribonucleic acid synthesis in
vaccinia virus. I. The mechanism of synthesis and release of RNA in
vaccinia cores. Journal of Molecular Biology 50, 1-18.
KATES, J. R. & McAUSLAN, B. R. (1967). Poxvirus DNA-dependent
RNA polymerase. Proceedings of the National Academy of Sciences,
U.S.A. 58, 134-141.
KONARSKA, M. M. (1989). Analysis of splicing complexes and small
nuclear ribonucleoprotein particles by native gel electrophoresis.
Methods in Enzymology 180, 422-453.
KUMAR, A. & PEDERSON,T. (i975). Comparison of proteins bound to
heterogeneous nuclear RNA and messenger R N A in HeLa cells.
Journal of Molecular Biology 9, 353-365.
LAEMMLI, O. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, London 227, 680685.
METZ, D. H., ESTEBAN,M & DANmLESCU,G. (1975). The formation of
virus polyribosomes in L cells infected with vaccinia virus. Journalof
General Virology 27, 181-195.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:27:54
R N A - p r o t e i n complex in vaccinia cores
MOREL, C., KAYIBANDA,B. & SCHERRER,K. (1971). Proteins associated
with globin messenger RNA in avian erythroblasts: isolation and
comparison with proteins bound to nuclear messenger-like RNA.
FEBS Letters 18, 84-88.
Moss, B. (1990). Poxviridae and their replication. In Virology, 2nd edn.,
pp. 2079-2111. Edited by B. N. Fields & D. M. Knipe. New York:
Raven Press.
MOUssATCHI~,N. (1985). Polyamines stimulate DNA-dependent RNA
synthesis catalysed by vaccinia virus. Biochimica et biophysica acta
826, 113-120.
MOUssATCHI~, N. & KELLER, S. J. (1991). Phosphorylation of vaccinia
virus proteins during transcription in vitro. Journal of Virology 65,
2555-2561.
MUNYON, W. E., PAOLETTI, E. & GRACE, J. T., JR (1967). RNA
polymerase activity in purified infectious vaccinia virus. Proceedings
of the National Academy of Sciences, U.S.A. 58, 2280-2288.
OIE, M. & ICHIHASHI, Y. (1981). Characterization of vaccinia
polypeptides. Virology 113, 263-276.
PAOLETTI, E. (1977). In vitro synthesis of a high molecular weight
virion-associated RNA by vaccinia. Journal of Biological Chemistry
252, 856-861.
PELHAM, H. B., SYKES,J. i . i . & HUNT, T. (1978). Characteristics of a
coupled cell-free transcription and translation system directed by
vaccinia cores. European Journal of Biochemistry 82, 199-209.
RIcI-rrER, J. D. & SMITH. L. D. (1983). Developmentally regulated
RNA binding proteins during oogenesis in Xenopus laevis. Journal of
Biological Chemistry 258, 4864-4869.
ROSEN, C. A., SIEKIERKA, J., ENNXS, H. L. & COHEN, P. S. (1984).
Inhibition of protein synthesis in vesicular stomatitis virus infected
1249
Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein
particle. Biochemistry 23, 2407-2411.
ROSEN, C. m., SODROSKI,J. G., CHUN-GoH, W. C., DAYTON, A. I.,
LIPPKE, J. & HASELTINE, W. A. (1986). Post-transcriptional
regulation accounts for the transactivation of the human Tlymphotropic virus type III. Nature, London 319, 555-559.
SAGOT, J. & BEAUD, G. (1979). Phosphorylation in vivo of a vaccinia
virus structural protein found associated with the ribosomes from
infected cells. European Journal of Biochemistry 98, 131-140.
SAROV, I. & JOKLIK, W. K. (1972). Studies on the nature of the capsid
polypeptides of vaccinia virions. Virology 50, 579-592.
SMITH, G. S., CHAN, Y. S. & HOWARD, S. T. (1991). Nucleotide
sequence of 42 kbp of vaccinia virus strain WR from near the right
inverted terminal repeat. Journal of General Virology 72, 1349-1376.
SOLOSKI, M. J., ESTEBAN, M. & HOLOWCZAK, J. A. (1978). DNA
binding proteins in the cytoplasm of vaccinia virus-infected mouse L
cells. Journal of Virology 25, 263 273.
TOWmN, H., STAEHELIN, T. & GORDON, J. (1979). Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proceedingsof National Academy of
Sciences, U.S.A. 76, 4350-4354.
VAN EEKELEN, C. A. G., MARIMAN,E. C. M., REINDERS, R. J. & VAN
VENROOIJ, W. J. (1981). Adenoviral heterogeneous nuclear RNA is
associated with. host cell proteins. European Journal of Biochemistry
119, 461-473.
YANa, W.-P. & BAUER,W. R. (1988). Purification and characterization
of vaccinia virus structural protein VP8. Virology 167, 578-584.
(Received 11 October 1991; Accepted 20 January 1992)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:27:54