Virus Research, 15 (1990) 189-196

Ekevier

189

VIRUS 00564

High level expression of the two outer capsid proteins of bluetongue virus serotype 10: their relationship with the neutralization

of virus infection

Jonathon J.A. Marshall 1 and Polly Roy 1,2

’ NERC Institute of Virology and Environmental Microbiology, Mansfield Road Oxford, U.K.

and 2 University of Alabama at Birmingham, School of Public Health,

Department of Environmental Health Sciences, University Station, Birmingham, AL 35294, U.S.A.

(Accepted 24 October 1989)

DNA representing RNA segments 2 and 5 of bluetongue virus (BTV) serotype 10, corresponding to the genes that code for the outer capsid proteins VP2 and VP5, have been inserted into a baculovirus transfer vector in lieu of the coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera fmgiperda cells with wild-type AcNPV DNA in the presence of the recombinant transfer vector DNAs polyhedrin-negative recombi- nant baculoviruses were recovered. When S. frugiperda cells were infected with these recombinant viruses proteins of similar size and antigenic properties to BTV VP2 and VP5 were synthesised. The recombinant VP2, but not the recombinant VP5, was shown to be capable of inducing antibodies that neutralized the infectivity of BTV-10 in vitro.

Bluetongue virus; Baculovirus expression; Virus neutralization

Bluetongue virus (BTV) is the prototype member of the Orbiuirus genus (Family: Reoviridae) and is the causative agent of bluetongue disease in domestic ruminants

Correspondence to: P. Roy, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, U.K.

0168-1702/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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(sheep, cattle). At least 24 different serotypes have been identified by plaque reduction neutralization tests (Gorman et al., 1983). The genome consists of ten double-stranded RNA segments, each of which is monocistronic, and is located in the core of the virion. The icosahedral core contains two major (VP3 and VP7) and three minor protein species (VPl, VP4, VP6) and is surrounded by a diffuse coat of the proteins VP2 and VP5 (Verwoerd et al., 1972) which are coded for by the RNA segments L2 and M5, respectively (Mertens et al., 1984). It is known that VP2 is the main serotype-specific antigen (Huismans and Erasmus, 1981; Kahlon et al., 1983) and that solubilized VP2 induces neutralizing antibodies in sheep and is capable of protecting against viral infection (Huismans et al., 1983). A mixture of solubilized VP2 and VP5 was reported to elicit higher titers of neutralizing antibodies than solubilized VP2 alone (Huismans et al., 1983) although VP5 prepared by SDS-PAGE was unable to induce such antibodies (Huismans et al., 1987). This study was designed to determine if VP5 plays a role, on its own, in virus neutralization by expressing it in the baculovirus system.

A full-length copy of BTV-10 segment 5 was constructed from two partial length clones, pM113 and pJ90 (Purdy et al., 1986), using a unique NcoI site present in the overlapping regions and the unique EcoRV site of pBR322. The 1.6 Kb PstI fragment from the resulting vector was digested with Ba131 exonuclease (Amersham International plc) to eliminate the terminal dC-dG sequences which were introduced during the cDNA cloning process. The product DNA was repaired with the Klenow fragment of DNA polymerase and ligated into dephosphorylated pUC-4K vector which had been cut with Sal1 and the overhanging 5’ ends blunted by mung bean nuclease. The recombinant pUC-4K/lO-5 vectors were analysed by dideoxy se- quencing of the double-stranded plasmid DNA (Chen and Seeburg, 1985). One of the vectors was selected in which the 5’ end of the segment 5 sequence was intact and 24 nucleotides were missing from the 3’ end and thus contained the entire open reading frame of segment 5. The insert was released from this vector with BamHl and cloned into BamHl site of the baculovirus transfer vector pAcYM1 (Matsuura et al., 1987) and the resulting vector, pAcYMl/lO-5, isolated. Similarly a transfer vector containing the segment 2 DNA of BTV-10, pAcYMl/lO-2, was constructed. The 2.9 Kb BamHl fragment of the transfer vector pAcSI10.2, previously described (Inumaru and Roy, 1987), was inserted into the BamHl site of pAcYM1. Recombi- nant baculoviruses were obtained by transfecting S. frugiperdu cells with mixtures of infectious AcNPV DNA and pAcYMl/lO-5 or pAcYMl/lO-2 plasmid DNA. One recombinant derived from pAcYMl/lO-5 was designated YMl/lO-5 and one from pAcYMl/lO-2 was designated YMl/lO-2.

Viral DNA was isolated from the recombinant baculoviruses as described previ- ously (Matsuura et al., 1986), digested with BumHl and subjected to Southern analysis (Southern, 1975) using segment 5 or segment 2 DNA 32P labelled by nick-translation. This confirmed the presence of DNA coding for VP5 and VP2 in the recombinant baculoviruses.

S. frugiperdu cells were infected with either recombinant baculovirus (YMl/lO-5 or YMl/lO-2), wild type AcNPV or mock infected at a multiplicity of 10 PFU/cell and incubated at 28°C for 24 h. Infection with the recombinant viruses produced

VP2 +

VP5 -e

Pal +

-180

-116

-84

-58

-48.5

-36.5

-26.6

Fig. 1. Coomasie blue stained 10% SDS-PAGE gel of mock, AcNPV, YM1/10-5 and YMl/lO-2 infected S. &g@e& c&s. The positions of the VPS, VP2 and polyhedrin (Pal) protein bands are

marked rdong with the positions of m&cular weight markers mn at the same time.

no visible nuclear inclusions. Extracts of the infected cells were subjected to electrophoresis in 10% polyacrylamide gels, as described by Laemmli (1970) and stained with Coomassie blue (Fig. 1). The YMl,/lO-5 virus directed the synthesis of a protein of the expected size for VP5 (59 kDa) and similarly for the YMf/l&2 virus a protein of the expected size for VP2 (105 kDa). Neither recombinant directed the synthesis of the polyhedrin protein seen in AcNPV infected cells. The level of expression of VP5 was not as high a level as VP2, as judged by Coomassie blue staining. This could reflect a toxicity of VP5 for S. fiugiperda cells since YMIl/lO-5 recombinant killed the cells more rapidiy than either YMfffO-2 or wild-type AcNPV (data not shown). The expression level of VP2 using the pAcYMl vector was much higher than previously reported using the pAcRP65 vector (In- umaru and Roy, 1987).

Monospecific antisera were raised to the expressed VP5 and VP2 proteins by resolving them by 10% SDS-PAGE and visualizing the protein bands by staining in 0.25 M KC1 and destaining in tap water, both at 4°C. The protein bands were excised, macerated and used to immunize either rabbits, to produce antiserum, or

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VP2

VP5

-180

-116

- 84

- 58

- 48.5

- 36.5

(A) (B) (Cl

Fig. 2. lmmunoblotting of expressed and authentic BTV proteins. Protein extracts of S. frugiperda cells infected with YMl/lO-5, YMl-10-2 or AcNPV and BHK cells mock, and BTV-10, infected were resolved

by 10% SDS-PAGE and electrophoretically blotted as described in Methods. In (A) the membrane was

probed with rabbit sera raised to BTV-10 virions. In (B) the membrane was probed with mouse ascitic

fluid raised to expressed VP5 and in (C) the membrane was probed with rabbit sera raised to expressed

VP2. The positions of the VP2 and VP5 proteins are indicated as well as those of molecular weight markers.

mice, to produce ascitic fluids, using Freunds incomplete adjuvant. The resulting sera were used, along with rabbit sera raised to purified BIT-10 virions, to characterize the expressed proteins by immunoblotting (Fig. 2). Extracts of S. frugiperdu cells infected with YMl/lO-5, YMl/lO-2 and AcNPV and of BHK cells either mock or BIT-10 infected were resolved by 10% SDS-PAGE and electro- blotted onto Durapore membrane (Millipore Corp.). Bound antibodies were de- tected using the appropriate anti IgG antiserum coupled to alkaline phosphatase (Sigma Chemical Co.) and Fast BB salt and /3-naphthyl phosphate as the substrate. Both the expressed VP5 and VP2, but not AcNPV infected cells, were recognized by antiserum to BIT-10 virions and co-migrated with the authentic proteins. In the

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TABLE 1

Plaque reduction neutralization titers of antisera raised to expressed VP2 and VP5

Antisera BTV serotypes

10 11 13 17

Rabbit VP2 antisera >640 > 160 0 >160

Preimmune rabbit sera 0 0 0 0

Mouse VP5 ascitic fluid 0 0 0 0

Control ascitic fled 0 0 0 0

Mouse antisera to

YMl/lO-2 infected S. frugiperda cells 205k74 a (N=4)

YMl/lO-5 infected S. frugiperda cells 51k23 b - _

(N=4)

AcNPV infected S. frugiperda cells 55*40 - - (N=4)

B Significantly different from AcNPV infected S. frugiperda cells at the P = 0.05 level. b Not significantly different from AcNPV infected S. frugiperda cells at the P = 0.05 level.

case of the expressed VP5 a number of bands of lower molecular weights were also recognized, these are, presumably, degradation products of the full-sized protein.

Rabbit antisera raised to the expressed VP2 and mouse ascitic fluid raised to the expressed VP5 recognized the corresponding authentic proteins in BTV-10, but not mock, infected BHK cells whereas preimmune sera or control ascitic fluid did not. Thus it would appear that the expressed proteins have immunological properties closely related to those of the authentic BTV-10 proteins.

These sera, raised to the expressed proteins, were tested for their ability to neutralize BTV-10 in vitro (Table 1). Antiserum, diluted in PBS, was incubated with 100 PFU of BTV-10 at 4O C overnight. Then 1 x lo5 VERO cells in L15 media were added, followed by an overlay of 0.75% (w/v) carboxymethyl cellulose in L15 medium, and then incubated at 35 o C for four days. At the end of this time plaques were visualized by staining with 1.5% (w/v) crystal violet in 95% (v/v) ethanol and the plaque reduction neutralization titers were expressed as the reciprocal of the antiserum dilution that gave a 50% reduction in plaque number. VP2 antiserum neutralized the virus at a titer of greater than 640 whereas VP5 ascitic fluid, control ascitic fluid and preimmune rabbit serum did not. The VP2 antiserum was also tested for its ability to neutralize the heterologous BTV serotypes 11, 13 and 17. Serotypes 11 and 17 were neutralized to a titer of greater than 160 whereas serotype 13 was not. This pattern of cross-serotype neutralization reflects the pattern of homologies between the VP2 proteins of the serotypes (Yamaguchi et al., 1988). In order to check that the failure of the expressed VP5 protein to induce neutralizing antisera was not due to denaturation of the protein during SDS-PAGE whole infected S. frugiperdu cells were used to raise mouse antisera. Groups of four mice each received two inoculations of 3 X lo6 infected cells, intraperitoneally and the resulting sera were tested for their ability to neutralize. Titers were 205, 51 and 55 for cells infected with YMl/lO-2, YMl/lO-5 and AcNPV, respectively. Thus even in

194

the absence of denaturation by SDS-PAGE VP5 was unable to elicit neutralizing antibodies even though VP2 could. The reason for the relatively high background neutralizing titer of antisera to AcNPV infected cells is unknown.

The data presented support the conclusion that the outer capsid protein VP2 plays a direct role in neutralization of BTV whilst VP5 does not. A previous study by Huismans and associates (1987) demonstrated that VP5 and VP2 together were capable of inducing higher neutralizing titers than VP2 alone. It may be that interactions between the two proteins can potentiate the ability of VP2 to induce neutralizing antibodies or indeed confer this ability on VP5 More extensive studies are in progress to determine if such interactions have a role in the neutralization of BTV infection.

Acknowledgement

We would like to acknowledge C.D. Hatton for photographic work and Miss S.J. Pinniger for typing. This work was supported by EEC contract BAP-0120 UK and by Public Health Service Grant AI26879 from the National Institutes of Health, U.S.A.

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(Received 7 August 1989; revision received 24 October 1989)