Detection of RNA-protein complex in vaccinia virus core in vitro transcription system

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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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

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.

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

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

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).

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R N A - p r o t e i n complex in vaccinia cores

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(Received 11 October 1991; Accepted 20 January 1992)