VIROLOGY

191,231-236

Interaction

(1992)

of Nucleic Acids with Core-like

and Subcore-like

P. T. LOUDON**t AND PoLL’f *Laboratory

of Molecular

Particles

of Bluetongue

Virus

ROY*++’

Biophysics, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; tNERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR. United Kingdom; and *University of Alabama at Birmingham, Birmingham, Alabama 35294 Received May 19, 1992; accepted July 13, 1992

Bluetongue virus (BTV) core-like particles (CLPs) were synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus. Purified CLPs were shown to bind single-stranded RNA in three different assay systems: gel retardation, nitrocellulose binding, and sucrose gradient sedimentation. CLPs showed equal affinity for BTV-specific and non-WV RNA and also bound DNA. RNAase protection experiments demonstrated that bound RNA was accessible to immobilized ribonuclease, suggesting that the RNA was predominantly present on the outside of the CLPs. By using individually purified VP7 and VP3 in separate assays, the binding activity was shown to reside on VP3. These o 1992 results indicate further functional homologies between BlV VP3 and the rotavirus inner-core VP2 protein. Academic

Press.

Inc.

INTRODUCTION

Previous studies have demonstrated that core-like particles (CLPs) can be synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus (French and Roy, 1990). The CLPs were found to closely resemble authentic BTV cores in appearance and stoichiometry; however, they appeared to lack RNA. In the present study we have investigated the interaction between the CLP and BTV ssRNA in vitro. We demonstrate a nucleic-acid-binding activity associated with the CLP and show that the activity specifically resides on the VP3 component that also forms the subcore-like structures. The possible significance of this in the virus replication cycle and the similarities between the properties of BTV VP3 and rotavirus VP2 are discussed.

Bluetongue virus (BN) is an orbivirus in the family Reoviridae. It has a genome consisting of 10 segments of double-stranded (ds) RNA encapsidated in a doubleshelled virus particle. The outer capsid is removed soon after entry into the host cell to yield a core particle composed of two major (VP3 and VP7) and three minor proteins (VPl, VP4, and VP6; Huismans et a/., 1987). The core has a transcriptase activity which allows it to synthesize single-stranded (ss) RNA transcripts of the genomic dsRNA. The ssRNA can serve two functions. Some act as messenger RNA and are translated to form the seven structural and three nonstructural BlV proteins. However, some transcripts are encapsidated by the developing progeny virus particles and serve as the templates for the formation of the genomic dsRNA. The mechanisms by which BTV and other members of Reovidae can selectively package 10 segments of RNA are poorly understood. The recognition signals on the RNA molecules, the proteins that are involved in packaging, and the mode of selection are undefined. Two BlV proteins, core protein VP6 and nonstructural protein NS2, have been shown to bind nucleic acid and may play a role in encapsidation. The binding activity of VP6 has been shown to be nonspecific (Roy, 1990), while that of NS2 has been shown to be specific for single-stranded RNA (Huismans et al., 1987; Thomas et al., 1990).

MATERIALS AND METHODS Synthesis and purification of CLPs, VP7, and VP3 For the synthesis of CLPs, Spodoptera frugiperda (Sfl cell cultures were infected with a dual recombinant baculovirus expressing B-IV-1 7 VP3 and BTV-10 VP7. CLPs were recovered from infected cells and separated from cell debris by cesium chloride (CsCI) gradient centrifugation as described previously (French and Roy, 1990). They were then dialyzed overnight against 0.2 M Tris-HCI, pH 7.5. Purified samples of VP7 were prepared from Sfcells infected with a recombinant baculovirus expressing the BTV-10 VP7 gene as described previously (Oldfield et al., 1991; Basak et al., 1992). Subcores containing VP3 were prepared by coinfecting insect cell cultures

’ To whom reprint requests should be addressed at NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 35R, U.K. 231

0042-6822/92

$5.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction I” any form resewed.

232

LOUDON

with two recombinant baculoviruses (both at an m.o.i. of 3 PFU/cell)-one expressing BTV-17 VP3 and the other expressing a chimeric form of BTV-10 VP7 carrying a short foreign protein epitope (15 residues of SIV gag protein) at the N terminus (unpublished data). The infected cells were harvested after 3 days, lysed, and sedimented on a discontinuous sucrose gradient as described previously (French and Roy, 1990). The purified fraction, which consisted entirely of VP3 subcores, was dialyzed overnight against 0.2 MTris-HCI, pH 7.5. The protein contents of all the purified preparations were determined by gel electrophoresis. For the preparation of 3H-labeled protein samples, monolayers of 1 X lo* Sfcells were infected with the VP3AIP7 dual recombinant baculovirus at an m.o.i. of 5 PFU/cell as described previously (Inumaru and Roy, 1987). After 24 hr incubation, the medium was replaced with lysine-free TCl 00 medium containing 10% FCS (Gibco, Ltd., U.K.) previously dialyzed overnight against water. After 8 hr, 100 &i of [3H]lysine (Amersham, Ltd., U.K.) was added in a final volume of 1 ml of lysine-free TC 100. Following a further 24 hr incubation, infected cells were harvested and CLPs were purified as described above.

AND ROY

EDTA). For competition assays, yeast tRNA (Sigma Chemical Co., Ltd., U.K.) was added to the binding reaction to a final concentration of 15 pg/pl. Following incubation of 1 hr on ice, the samples were resolved on a 0.8% agarose gel in TBE buffer according to the protocols described in Maniatis et a/. (1982). After 1 hr of electrophoresis, the gel was fixed in 10% methanol, 10% glacial acetic acid and either stained with Coomassie brilliant blue or dried and autoradiographed.

Nitrocellulose

binding assays

The procedure employed for the nitrocellulose binding assay was adapted from the methods described by Kingsbury and Jones (1987) with some minor modifications. Reaction mixtures of 40 ~1 were set up containing 30 ng of 32P-labeled BTV-specific ssRNA and 8 fig of protein in binding buffer (see above). After 30 min at room temperature, the samples were filtered through 0.45-pm nitrocellulose filters (Millipore, Inc., U.S.A.) and washed with 10 ml binding buffer to remove the unbound radioactive material. The amount of 32P-labeled probe retained on the filters was measured by scintillation counting.

Nucleic acid probes Two BTV-specific RNA probes were used. One was a 2.9-kb transcript consisting of the coding region of BTV-10 segment 2. It was generated by SP6 transcription of plasmid pSPT19.1 OBTV2 previously linearized with HindIll, as described by Liu and Roy (1992). The second was a full-length 1.2-kb transcript of segment 7 of BlV-1 (Australia) and was an exact copy of BTV mRNA. It was generated byT7 transcription of pBTVEx + 7 previously linearized with SnaBI, as described by Wade-Evans et al., (1992). Both of the BTV-specific probes were synthesized in the presence of 10 &i [32P]UTP and were subsequently separated from free nucleoside triphosphates by Sephadex G-50 bead chromatography. A nonviral RNA species was also prepared by T7 transcription of pGEM 3zf (+) (Promega, Inc., U.S.A.) linearized with Dral, which cuts at a site 1.2 kbp downstream from the T7 promoter. To prepare a dsDNA probe, a cDNA clone representing the coding sequence of BTV segment 7 BTV-10 was excised from pAcYM1 BTV10.7 (Oldfield et al., 1991). It was then purified by electrophoresis on a 1% agarose gel and nick translated in the presence of [32PldATP using an Amersham kit (Amersham plc, U.K.). Gel retardation

experiments

Sixteen micrograms of CLP was mixed with either 0.4 or 4 ng of 32P-labeled ssRNA in a final volume of 1 ~1 of binding buffer (50 mNI Tris-HCI, pH 7.5, 2.5 rnM

Sucrose gradient

centrifugation

The buoyant density of CLP/RNA complexes was determined by sucrose gradient centrifugation. Approximately 30 ng of 32P-labeled BTV ssRNA probe were mixed with approximately 6000 cpm of purified 3H-labeled CLP in binding buffer. After 30 min at room temperature, the samples were sedimented on a linear 1O-50% w/v sucrose gradient (in 0.2 /1/1Tris-HCI, pH 7.5) at 26,000 rpm, for 3 hr at 4’ in SW41 tubes (Beckman, Inc., U.S.A.). The gradient was harvested into 0.5-ml fractions and the 3H and 32P cpm of each fraction were determined by scintillation counting. For RNAase protection experiments, 30 ng of the 32P-labeled BTV ssRNA were incubated with approximately 4 pg of unlabeled CLPs as described above. After 30 min, 1 mg of RNAase A immobilized on polyacrylamide beads was added to the reaction mixture. The reaction was incubated for a further 15 min with mixing, the beads removed and the products sedimented on a sucrose gradient as described above. The distribution of radioactivity in the gradient was determined. DNA binding assays were performed by replacing the ssRNA probe with an equal number of cpm of a 32P-labeled dsDNA species. The CLP/DNA complexes were then sedimented through a sucrose gradient and fractions were collected and counted as described above.

RNA BTV-CORE 1

2

3

4

FIG. 1. Gel retardation of single-stranded (ss) RNA by BTV CLPs. 32P-labeled BTV ssRNA transcripts were incubated with purified BTV CLPs. Samples were resolved by 0.8% agarose gels in TBE buffer as described by Maniatis eta/. (1982). The gel was fixed and autoradiographed as described in the text. Lane 1, negative control: RNA with no added protein, Lane 2, 4 ng of 32P-labeled RNA incubated with 16 pg of CLPs. Lane 3, 4 ng of labeled RNA incubated with 16 pg of CLP in the presence of 400 pg of yeast tRNA. Lane 4, 0.4 ng of labeled RNA incubated with 16 rg of CLP.

RESULTS Ability

of BTV CLPs to bind RNA

CLPs were purified from Sfcells infected with a dual recombinant baculovirus expressing BTV VP3 and VP7 (French and Roy, 1990). To determine whether they could interact with BTV-specific RNA they were incubated with a 32P-labeled transcript of the BTV-10 L2 gene. A control reaction containing the RNA probe without CLPs was also set up. The formation of CLPRNA complexes was then assayed by gel retardation. As shown in Fig. 1 (lane 1) 4 ng of the 2.9-kbp L2 transcript from sample lacking CLPs migrated into the gel. Addition of 16 pg of CLPs to the binding reaction resulted in retardation of most of the probe, with the retarded RNA remaining in the loading wells (Fig. 1, lane 2). Staining the gel with Coomassie blue demonstrated that protein was also located in the loading wells (result not shown). When 0.4 ng of RNA was incubated with 16 pg of CLPs, virtually all the RNA bound to the CLPs and was retarded (Fig. 1, lane 4). To determine whether the formation of RNA-CLP complexes was dependent on BTV RNA sequences, an excess of yeast tRNA (15 pg) was included in the binding reaction. As shown in Fig. 1 (lane 3) the tRNA effectively displaced all the labeled BTV RNA from the CLPs, indicating that the CLPs do not possess an absolute specificity for BTV ssRNA.

INTERACTIONS

RNA binding activity

233

of CLP resides on VP3 protein

The CLP consists of two proteins, trimers of VP7 (Basak et a/., 1992) laid down on a subcore of VP3 (Loudon and Roy, 1991). To determine which of these proteins was responsible for RNA binding a nitrocellulose binding assay was used. VP7 was purified from CLPs derived from Sfcell cultures infected with the recombinant baculovirus expressing the BTV S7 gene. VP3, in the form of subcores, was purified from Sf cultures coinfected with two recombinant baculoviruses, one expressing VP3 and the other a mutant form of VP7 carrying a foreign epitope (see Materials and Methods). The purity of the preparations was confirmed by SDS-PAGE and Coomassie blue staining (data not shown). The abilities of VP3, VP7, and the CLP to bind RNA were then compared. An equal amount of each protein was incubated with a 32P-labeled BTV-specific ssRNA probe, then filtered through nitrocellulose. The amount of radioactivity retained on the filter was then measured. RNA transcripts without added protein did not bind to the filter (Fig. 2A, lane 1). When CLPs were employed radioactivity was retained on the filter, indicating the formation of CLP-RNA complexes (Fig. 2A, lane 2). When VP7 was employed the amount of radioactivity retained on the filter was equivalent to the background level obtained with RNA alone (Fig. 2, lane 4). In contrast, the treatment with VP3 subcores yielded high counts of membrane-bound radioactivity (Fig. 2, lane 3) with a given mass of VP3 able to bind approximately twice as much RNA as an equal mass of CLPs. Similar results were obtained using gel shift assay (data not shown). Interaction between CLP and ssRNA is nonspecific and is inhibited by the addition of salt The specificity of RNA binding to the CLPs was determined by measuring the ability of an unlabeled, nonspecific ssRNA species to compete for binding with an exact, full-length copy of BTV-1 RNA segment 7 (Wade-Evans et al., 1992). In these reactions 8 pg of CLPs were incubated with 30 ng of 32P-labeled BTV-1 S7 transcript in the presence of 0, 30, 90, 150, or 480 ng of unlabeled nonviral RNA. After 30 min the samples were filtered through nitrocellulose membranes and the amount of bound BTV-specific RNA measured by scintillation counting. Addition of the nonspecific competitor species reduced the amount of radioactivity retained on the filters (Fig. 2B). Titration of the competitor against BTV-specifit RNA demonstrated that the CLP exhibited an equal affinity for the two species, with the addition of a 16-fold excess of competitor reducing the amount of bound radioactivity to approximately background levels.

LOUDON

234

It was previously demonstrated that CLPs prepared by CsCl gradient centrifugation lacked detectable nucleic acids. Since the results described above indicate that CLPs bind RNA, an investigation was undertaken to determine whether salt had any effect on the formation of CLP-RNA complexes. Binding reactions were therefore performed in the presence of various concentrations of CsCI. As shown in Fig. 2C, salt addition had a strong inhibitory effect on binding. A final concentration of 20% CsCl in the reaction mixture effectively prevented labeled RNA from binding to the CLPs and reduced the radioactivity retained on the nitrocellulose membrane to background levels.

CPM x103

50

A

40 3WssRNA Protein

1 Various CPM x103

3 preparation

32

B 24

32P-ssRNA Protein

Sedimentation

-

16

1

2

3

Concentration CPM

4

2 protein

AND ROY

4

5

6

of RNA competitors

32 C

x103

24l---

-4

=P-SSRNA Protein 16

1

2 Concentration

4

3

5

of CsCl

FIG. 2. Nitrocellulose binding assay of the interaction between BTV proteins and BTV-specific RNA. Purified CLPs were incubated with 32P-labeled BTV-specific ssRNA, then filtered through nitrocellulose as described in the text. The amount of RNA retained on the filters was determined by scintillation counting. (A) Comparison of the RNA-binding ability of CLPs, VP3, and VP7. Graph shows RNA with no added protein (1) and RNA mixed with 8 rg CLP (Z), 8 rg VP3 (3) or 8 pg VP7 (4). (B) Specificity of the binding between the CLP and BTVspecific ssRNA. The interaction between CLPs and BTVspecific RNA was carried out in the presence of various concentrations of an unlabeled, nonspecific competitor RNA species. Graph shows negative control of 30 ng BTV-specific 3zP-labeled RNA withssRNA out added protein (1) 30 ng of 3zP-labeled BTVspecific mixed with CLP without competitor RNA (2) interaction between 30 ng of 3zP-labeled BTV-specific ssRNA in the presence of 30 ng (3) 90 ng (4) 150 ng (5) or 480 ng (6) of unlabeled nonviral RNA. (C) The effect of cesium chloride concentration on binding. Graph shows RNA with no added protein (1) RNA bound to CLPs (2) and RNA transcripts bound to CLPs in the presence of 10% w/v CsCl(3), 20% w/v CSCI (4), 40% w/v CSCI (5).

coefficient

of CLP-RNA

complexes

To determine the sedimentation coefficient of CLPRNA complexes, 3H-labeled CLPs were incubated with 32P-labeled BTV-specific RNA then sedimented through a linear sucrose gradient. RNA transcripts alone, incubated without added protein, remained at the top of the gradient (Fig. 3C). RNA transcripts bound to CLPs were recovered as a sharp peak of radioactivity in fraction 8 (Fig. 3A), corresponding to a sedimentation coefficient of approximately 440 S. The distribution of 3H-labeled CLPs was essentially similar to that of the bound 32P-labeled probe, demonstrating the formation of RNA-CLP complexes. Similar profiles were obtained when the ssRNA probe was replaced by dsDNA (Fig. 3B). To determine the location of bound RNA on the CLP, an RNAase protection experiment was undertaken. Complexes between the CLP and the exact copy transcript of BTV S7 RNA were treated with RNAase A covalently linked to polyacrylamide beads, then sedimented on a sucrose gradient. Using immobilized RNAase it was assumed that RNA bound inside the CLP would be protected from digestion, while that bound to the outside would be RNAase-sensitive. RNAase treatment led to the disappearance of the 440 S peak of 32P (Fig. 3C), indicating that the bound RNA was accessable to the ribonuclease. DISCUSSION The ability of CLPs to bind ssRNA was demonstrated using three different assay systems. The migration of BTV-specific ssRNA on agarose gels was retarded by the addition of CLPs. All the retarded RNA was retained within the loading wells of the gel. The binding activity was not specific for Bn/ RNA since the addition of an excess of yeast tRNA prevented binding of the BlV RNA. The CLP/RNA complex sedimented in a sharp peak using linear gradients of 1 O-5096 w/v sucrose. The observed sedimentation coefficient of 440 S is comparable with that of 470 S observed for virus-

RNA BTV-CORE 2,000

T

1,800 1,600 1,400 CPM 1,200

.?

1,000 800 600 400

.:

200

L-l

0

0 2,000

4

8

12

__

--

20

16

B

1,800 1,600 1,400 CPM 1,200 1,000 800 600 400 200

-

OI 0

4

8

12

s--q,

, ; 16

,

, 20

Fraction numbers FIG. 3. Sucrose gradient sedimentation of CLP-nucleic acid complexes CLPs were mixed with 3*P-labeled nucleic acid probes, then sedimented on 1O-50% w/v linear gradients of sucrose. Fractions were collected from the bottom and the distribution of radioactivity was determined by scintillation counting. (A) 3*P-labeled BTWspecific ssRNA bound to 3H-labeled CLPs. 3H (---), 32P (A); 32P-labeled dsDNA bound to unlabeled CLPs. DNA alone sedimented on the gradient without added protein (M), DNA-CLP complexes (X). (B) Treatment of CLP-RNA complexes prior to sedimentation with RNAase A linked to polyacrylamide beads (see text for details). (Cl) [3’P]RNA sedimented on gradient without added protein, (A) [32P]RNA-CLP complexes, (---) RNAase-treated [32P]RNA-CLP comolexes.

derived cores (Huismans eta/., 1987). The CLPs exhibited a nonspecific ability to bind nucleic acid. In nitrocellulose binding assays an equal affinity for BTV-specific and nonviral ssRNA was observed. Binding of dsDNA to the CLP was also demonstrated using sucrose gradient sedimentation. The ability of CLPs to bind nucleic acids is in contrast to earlier reports in which freshly purified CLP samples

INTERACTIONS

235

were found to be devoid of nucleic acid (French and Roy, 1990). However, the present study demonstrated that the concentrations of cesium chloride used to purify CLPs prevented binding. Hence nucleic acid bound to the CLP in viva may be lost during subsequent purification. Using individually purified VP7 and VP3 in the form of subcores, the ability of the CLP to bind nucleic acids was shown to reside on VP3. In previous studies we have demonstrated that the VP7 component of CLPs can be removed by dialysis against low salt to yield a population of subcores composed of VP3 alone (Loudon and Roy, 1991). In the present study we used a new method of subcore purification. VP3 was coexpressed with a chimeric form of VP7 carrying a foreign protein epitope (manuscript in preparation). This modified VP7 appeared to destabilize the interactions between VP7 and VP3, so that the fraction purified from sucrose gradients consisted entirely of VP3 subcores. In nitrocellulose binding assays it was determined that a given mass of VP3 was able to retain approximately twice as much RNA as the same mass of CLP, while VP7 samples exhibited no detectable binding activity. The binding activity found on BTV VP3 has strong similarities with that of rotavirus VP2. Both bind ssRNA in a non-sequence-specific manner, and both also bind dsDNA (Boyle and Holmes, 1986). Rotavirus VP2 and BTV VP3 have other similarities. Both of these proteins are approximately 103 kDa in size and are predicted to have low negative charge at neutral pH. Both form a subviral particle, designated the core in rotavirus (Labbe et al., 1991) and the subcore in BTV (Loudon and Roy, 1991). These structures are approximately the same size and have a similar appearance on uranyl acetate staining. Despite these functional similarities, a comparison of amino acid sequences reveals no significant homology, and the predicted leucine zippers and hydrophilic N-terminal region of rotavirus VP2 (Kumar et a/., 1989) do not appear to be present on BTV VP3. The function of an RNA binding domain on BTV VP3 is unclear; it may be involved in assembly. Studies with other members of the Reoviridae have shown that during assembly positive-sense ssRNA interacts with subviral particles (Olkkonen et a/., 1990; Gottlieb et al., 1991). In rotaviruses, RNAase-sensitive ssRNA molecules have been found extending from the surface of the subviral replicative intermediates (Patton and Gallego, 1990). The RNA subsequently moves into the particle and is replicated while the remaining structural proteins assemble to form the single-shelled particle. The ability of BTV CLPs to bind external nucleic acid may have a role at an early stage in morphogenesis, during the incorporation of ssRNA into a replicative intermediate, or during assembly of VP3 onto a preformed RNA-protein complex. Alternatively, BTV VP3

236

LOUDON

may function as a nucleocapsid protein, as suggested for rotavirus VP2 (Kumar et a/,, 1989) and hold the genomic dsRNA in the correct conformation to allow it to be packaged. ACKNOWLEDGMENTS We gratefully acknowledge the support of the National Institute of Health, USA (Grant NIH Al 26879) and the Agriculture and Food Research Council, U.K. (Grant LRG 43535) for enabling this research to take place. We thank Dr. A. Wade-Evans of the Institute of Animal Health, Pirbright, for the gift of plasmid pETVEx + 7 and Dr. A. Belyaev for the gift of BTV VP7 SIV recombinant baculovirus.

REFERENCES BASAK, A. K., STUART, D. I., and ROY, P. (1992). Preliminary crystallographic study of bluetongue virus capsid protein VP7. J. Mol. Viol., (in press.) BELYAEV, A. S.. BOOTH, T. F., and ROY, P. (1992). Presentation of Hepatitis B virus preS, epitope on bluetongue virus core-like particles. \/irology, (in press). BOYLE, J. F., and HOLMES, K. V. (1986). RNA-binding proteins of bovine rotavirus. J. Viral. 58, 561-568. FRENCH,T. J., and ROY, P. (1990). Synthesis of bluetongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. /. Viral. 64, 1530-l 536. FUJIMURA,T., and WICKNER, R. B. (1988). Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain. Cell 55, 663-67 1. GOTTLIEB, P., STRASSMAN, J. FRUCHT, A. QIAO, X., and MINDICH, L. (1991). In vitro packaging of the bacteriophage 06 ssRNA genomic precursors. Virology 181, 589-594. HUISMANS, H., VAN DIJK, A. A., and ELS, H. J. (1987). Uncoating of parental bluetongue virus to core and subcore particles in infected cells. Virology 157, 180-l 88. INUMARU, S., and ROY, P. (1987). Production and characterization of

AND ROY the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector. Virology 157, 472-479. KUMAR, A., CHARPILIENNE,A., and COHEN, J. (1989). Nucleotide sequence of the gene encoding for the RNA binding protein (VP”) of RF bovine rotavirus. Nucleic Acids Res. 17, 2126. LABBE, M., CHARPILIONNE,A., CRAWFORD, S., ESTES, M. K., and CoHEN, J. (1991). Expression of rotavirus VP2 produces empty corelike particles. 1. Viral. 65, 2946-2952. LIU, H. M., BOOTH, T. F., and ROY, P. (1991). Interactions of in vitro translated core and capsid proteins of bluetongue virus. 1. Gen. Viral., in press. LOUDON, P. T., and ROY, P. (199 1). Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: Inclusion of the largest protein VP1 in the core and virus-like particles. Virology 160,798-802. MANIATIS, J., FRITSCHE, E. F., and SAMBROOK, 1. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. OLDFIELD, S., HIRASAWA,T., and ROY, P. (1991). Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature from the outer capsid protein VP2. J. Gen. Viral.

72,449-451. PATTON, 1. T., and GALLEGO,C. 0. (1990). Rotavirus RNA replication: Single-stranded RNA extends from the replicase particle. J. Gen. Viral. 71, 1087-l 094. PRASAD, B. V. V., YAMAGUCHI, S., and ROY, P. (Supervisor) (1992). Three-dimensional structure of single-shelled BTV. /. viral. 66, 2135-2142. ROY, P. (1990). Use of baculovirus expression vectors: Development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus. In “FEMS Microbiology Immunology,” Vol. 64, pp. 223-224. Elsevier Biomedical, Amsterdam. THOMAS, C. P., BOOTH, T. F., and ROY, P. (1990). Synthesis of bluetongue viral-coded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: It binds the singlestranded RNA species. /. Gen. Viral. 71, 2073-2083. WADE-EVANS, A. M., MERTENS, P. P. C., and BOSTOCK,C. J. (1992). In v&o transcription of exact RNA copies of bluetongue virus genome segments from full length cDNA copies. Nucleic Acids Rest, submitted.

Interaction of nucleic acids with core-like and subcore-like particles of bluetongue virus.

Bluetongue virus (BTV) core-like particles (CLPs) were synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus. Purified CLPs ...
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