Virus Research. 25 ( 1992) 1- 13 Q 1992 Elsevier Science Publishers

VIRUS

B.V. All rights reserved

0168-1702/92/$05.00

00804

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus Kotaro Tuchiya ‘, Yoshiharu Matsuura ‘, Akihiko Kawai ‘, Akira Ishihama d and Susumu Ueda a ’ Nippon

Institute for Biological Science, Ome, Tokyo, Japan. ’ Department of Veterinary Science, Tokyo, Japan, ’ Faculty of Pharmaceutical Sciences,

National Institute of Health, Musashimurayama-shi,

Kyoto University, Kyoto, Japan and d Department of Molecular Genetics, National Institute of Genetics, Mishima, Japan (Received

31 January

1992. revision

received

20 April 1992; accepted

22 April 1992)

Summary

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera fmgiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus. Rabies virus; Glycoprotein;

Expression; Baculovirus; Fusion activity

Introduction

Rabies, an acute viral infectious disease of the central nervous system, is usually fatal in mammals once the symptoms develop, and even nowadays it is enzootic in

Correspondence to: K. Tuchiya. Nippon Tokyo 198, Japan. Fax: (81) 0428-31-6166.

Institute

for Biological

Science.

2,221-l.

Shinmachi.

Ome,

many parts of the world. Vaccines made of tissue culture-grown viruses are almost perfectly safe and effective (Wiktor et al., 1978). However, due to the low yield of the virus, tissue culture vaccines are expensive and this has prevented their broad use in developing countries. Therefore, extensive research has been performed to develop an effective and safe vaccine of low cost. Rabies virus is a member of the ~ha~do~li~jdae, genus L~ssaL,~~~s,and possesses non-segmented negative strand RNA as the genome (about 12,000 nucleotides1, which encodes 5 structural proteins designated as N, Ml, M2, G, and L (Tordo et al., 1986a,b, 1988). Bullet-shaped viral particles consist of a helical ribonucleoprotein (RNP) and the viral envelope which is made up of a lipid bilayer with external surface projections composed of a polymeric form of gly~oprot~i~ (G protein) (Dietzschold et al., 1978). The surface G protein displays both hemagglutination activity and low pH-dependent fusion activity, and plays a role in binding to the receptors on susceptible cells (Halonen et al., 1968; Mannen et al., 1982). The G protein precursors of ERA, CVS, PV, and HEP-Flury strains deduced from the respective nucIeotide sequence (~ilionis et al., 1981; Yelverton et al., 1983; Tordo et al.. 1986b: Morimoto et al., 1989) consist of 524 amino acids including a cleavable signal peptide of 19 hydrophobic amino acids (Lai and Dietzschold, 1981). The G protein is the major antigen responsible for the induction of neutralizing antibodies (Wiktor et al., 19731, and is also one of the viral components that serve as a target for virus-specific immune T-lymphocytes (Macfarlan et al, 1984; C&is et al., 1986; Kawano et al., 1990). Furthermore, isolated G protein as well as its derivatives (immunosome) confer protection on not only pre-exposed animals but also post-exposed ones (Crick and Brown, 1969; Wiktor et al., 1973; Perrin et al., 1985). Therefore, the envelope G protein has been a main target in studies for developing rabies subunit vaccines (Wunner et al., 1983). Recently, the ba~uIovirus system is being widely used to express foreign genes because of a high yield of protein products (Smith et al., 1983; Matsuura et al., 1987). In addition, expressed proteins are subjected to eukaryote-specific posttranslational modifications such as glycosylation and polymerization (Luckow and Summers, 1988). In this study, we cloned cDNA encoding the G protein of rabies virus from Nishigahara strain (subclone RC. HL) which is a standard strain for producing tissue culture-derived rabies vaccines for dogs in Japan (Ishikawa et al.. 19891, and expressed it in insect cells using a baculovirus vector. Some properties of the expressed G protein will be described.

Materials and Methods Virus and ceils Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant virus stocks were grown and assayed in monolayers of Spodoptera frugiperda (Sf) insect cells according to the method of Brown and Faulkner (1977). Sf cells were

3

maintained in Grace’s insect medium (GIBCO BRL, USA) supplemented with 10% fetal calf serum and 0.26% (w/v> Bacto tryptose broth (Difco, USA). Rabies virus, subclone RC . HL of Nishigahara strain was grown in BHK-21 cells, which were cultivated in Eagle’s minimum essential medium supplemented with 5% calf serum and 10% Tryptose phosphate broth (Difco, USA). A virus stock virtually free from defective interfering particles was prepared by three cycles of plaque isolations through BHK-21 cell cultures. The infectivity of the rabies virus was assayed by plaque formation on BHK-21 cells (Sedwick and Wiktor, 1967). RNA and enzymes

Rabies virus was purified by the method of Kawai (19771, and genome RNA was extracted from purified virions by the method of Tordo et al. (1986a). Restriction enzymes were purchased from Takara Shuzo (Japan), Toyobo (Japan), and Boehringer Mannheim (FRG). T4 DNA ligase, Escherichia coli DNA ligase, E. coli DNA polymerase I, Klenow fragment, ribonuclease H, bacterial alkaline phosphatase, RNase inhibitor, and Kilo-sequence deletion kit were purchased from Takara Shuzo (Japan). GENECLEAN, the DNA purification kit, was purchased from Bio 101 Inc. (USA). Avian myeloblastosis virus reverse transcriptase and terminal deoxynucleotidyl transferase were purchased from Midwest BioProducts (USA) and Pharmacia LKB Biotechnology (Sweden), respectively. NGlycanase, an endoglycosidase, was obtained from Genzyme (USA). Antibodies against G protein

Anti-G protein antibodies were prepared in rabbits immunized against G protein which was purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of virions (Harlow and Lane, 1988). The G protein band was excised from the gel, minced, and injected to rabbits subcutaneously twice at three-week intervals. Three weeks after the 2nd immunization, inactivated rabies virus was administered intravenously. Rabbits were bled one week after the last immunization. Cloning of G-cDNA and construction of transfer vector

Nucleic acid manipulations were carried out following the methods described by Maniatis and associates (1982). cDNA synthesis was done by the method of Tordo et al. (1986a1, which is based on the procedure of Gubler and Hoffman (1983). Primers for the cDNA synthesis of G protein gene were designed from the conserved sequences within M2 protein of ERA (Rayssiguier et al., 1986), PV (Tordo et al., 1986b), and HEP-Flury strains (Kawai, unpublished results). Two 26-mer primers (5’-TAGAATAATCAGATAATATCCCGCAA-3’ and 5’TAGAATAATCAGATTATATCCCGCAA-3’) were synthesized, which could hybridize at about 200 bases upstream of the G protein coding region (Fig. 1). Purified rabies virus RNA was annealed to the primers and reverse transcribed

’ ..TAC.GAATlC.GAGCTCGGGAAAG~GTTC EcoRi

sac,

icoRIl_

Met

C”,

-Fig. 1. Cloning of the rabies virus G-cDNA and construction of a transfer vector containing the cDNA. Rabies virus genome RNA was reverse transcribed into cDNA using specific primers and cloned into pBR322 plasmid. A fragment containing the open reading frame of G protein (G-ORFI was subcloned into pUCl8 plasmid. After removal of 5’-flanking sequence upstream from the ATG start codon of G-ORF, the resultant G-ORF cDNA was inserted into pAcYM1 transfer vector in correct orientation. P, Pstl; Hd, HindIII: E, EcoRI; K, i(pn1; B, BarnHI; L-ORF, open reading frame of L protein; PP. pol~hcdrin promoter.

into cDNA. The double-stranded cDNA was inserted into PstI site of plasmid pBR322 by dC/dG tailing. Recombinant clones were probed with AflII-AatI and Bgf II-PsrI fragments of the cDNA of the HEP-Flury strain G protein gene (Morimoto et al., 1989). From one of the recombinant plasmids, pBR-RaGL, a Pst I-HindIII fragment containing the entire open reading frame (ORF) of G protein was excised and transferred into a plasmid pUC18 which was previously cut with PstI and HindIII. The subcloned plasmid was designated as pUC-RaG. In order to remove the 5’-non-coding flanking region of the cloned G protein cDNA (G-cDNA), pUC-RaG was cut with RamHI and iYpn1, digested with exonuclease III, and then treated with mung bean nuclease. After the end of the trimmed DNA was repaired with Klenow fragment, the DNA was self-ligated. After sequencing around the ATG start codon of the G-ORF by the dideoxy-mediated chain termination method, one of the trimmed clones, pUC-RaG1.25, was chosen for use in subsequent experiments. A fragment containing the entire G-ORF was excised from pUC-RaG1.25 by digestion with EcoRI and HiadIII. repaired with KIenow fragment, and inserted into a baculovirus transfer vector pAcYM1 (Matsuura et al., 1987) which was previously digested with BarnHI, repaired with KIenow fragment, and dephosphorylated. A recombinant plasmid

5

with correct fragments.

orientation,

pAc-RaG,

Co-transfection and selection

was obtained

ofrecombinant

after analysis of restriction

z.+uses

Sf cells were co-transfected with a mixture of purified infectious AcNPV DNA and transfer vector DNA according to the method described previously (Matsuura et al., 1986). After 4 days of incubation, the culture supernatant was harvested and subjected to plaque assay. Plaques without occlusion bodies were picked up and subjected to purification by two cycles of plaque isolation. High titer stocks (107-” PFUfml) of the recombinant viruses were obtained using Sf monolayer cultures. Imml~no~uo~escence analysis For immunofluor~scence analysis of cell surface proteins, Sf cells infected with recombinant or parent AcNPV were subjected to indirect immunofluorescence assay with rabbit anti-G protein serum and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Immunoblotting

analysis

Sf cells infected with recombinant or wild-type AcNPV at a multipIici~ of 10 PFU/cell were harvested and washed twice with PBS. Aliquots of the cells were boiled for 10 min in sample buffer (2% SDS, 5% beta-mercaptoethano1, 10% glycerol, bromophenol blue, and 62.5 mM Tris-HCl, pH 6.81, and subjected to electrophoresis in 10% polyac~lamide gels-as described by Laemmli 119701. The electrophoresed proteins were transferred onto a polyvinyliden difluoride filter (Immobiion, Millipore, USA) using a semi-dry transblot apparatus (Sartorius, Ge~any). The filter was blocked with 1% skimmed milk in PBS and incubated with rabbit anti-rabies virus G protein serum. After washing, the filter was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The protein bands reacted with the antibody were visuahzed by incubation of the filter in 0.5 mg/ml diaminobenzidine and 0.005% El&&. Cell fusion assay Cell fusion activity of the recombinant G protein was observed according to Bailey et al. (1989). In brief, the pH of the culture medium of virus-infected Sf cells was reduced from 6.2, which is an optimum pH for cell growth, to 6.0, 5.5, or 5.0. S~c~ium formation was monitored under phase-contrast microscopy. In syncytium formation inhibition experiments, Sf cell monolayers in a 3S-mm dish were infected with about 50 PFU of recombinant virus and overlaid with 1% agarose medium at pH 5.5, in which rabbit antiserum against rabies virion or G protein, or normal rabbit serum was included. After the addition of liquid medium containing the respective serum onto agarose, incubation was continued at 28°C.

h

PIaques were visualized by staining with neutral red, and the effect of antisera on syncytium formation was examined by observing the morphology of the cells in plaques.

Results Cloning of G-cDNA and construction

of recombinant

baculorkts

The outline of cloning and insertion of the G-ORF into the AcNPV transfer vector is depicted in Fig. 1 and Materials and Methods. The recombinant transfer plasmid was co-transfected into Sf celis together with infectious AcNPV DNA. From the culture supernatant of transfected cells we isolated three independent plaques which produced no polyhedra. After successive plaque purification, stocks of putative recombinant viruses were obtained. Expression

arid glycosylation

1~~1 of rabies rirus G protein in insect cells

Surface expression of rabies virus G protein in insect cells infected with the recombinant AcNPV was examined by immunofluorescence analysis. Specific fluorescence was observed in unfixed Sf cells that had been infected with either of the three recombinant AcNPVs, but not in the parent AcNPV- or mock-infected cells (data not shown). One of the three clones, designated as AcRaG, was used for subsequent experiments. Immunoblot analysis of AcRaG-infected Sf cells demonstrated that the major recombinant proteins, reacted with monospecific anti-G protein antibodies, were about 58 and 62 kDa protein (Fig. 2A, lane 3, arrowhead) and they migrated faster than the authentic G protein (Fig. 2A, lane 1). Some minor proteins reacted with the antibody was also seen. The authors think that these smaller size proteins were probably the degradation form of G protein. To find out whether the expression of the two major species of recombinant G protein and the different mobiiities of them from the authentic one is due to a difference in glycosylation level, the glycosylation level of the recombinant G proteins was examined by digestion of oligosaccharide side chains with N-glycanase. Digestion of rabies virion G protein with N-glycanase generated three bands (Fig. 2A, Iane 2): one band migrated at the same mobility with the non-treated protein: and two bands migrated faster than the non-treated band. The fastest band, which migrated at the position of about 56 kDa, was considered to be the completely digested non-glycosylated form of the G protein because the molecular mass of the band corresponded well with that of the G proteins deduced from their amino acid sequences (56-57 kDa; Anilionis et al., 1981; Yelverton et al., 1983; Tordo et al., 1986b; Morimoto et al., 1989). Production of the intermediate band by digestion with N-glycanase probably indicates that the mature G protein in the virions of the Nishigahara strain originally carries at least two OIigosaccharide side chains. The putative N-linked glycos~~lation sites (Asn-X-Thr/Ser sequence) of the Nishiga-

N-Gly

-

+

_

+

(B) Strain l;;higahara

2

HEP-Flury Kelev

Amino acid pos?tion.of putative glycosylation 37

158

204

247

319

+

+

+ -

+ + +

+ + + +

+ + + + +

+ -

_

-

+ +

Fig. 2. Glycosylation level of G proteins. (A) Rabies virus or AcRaG-infected Spodoptera frugiperda cell lysate were incubated in the presence (2, 4) or absence (1, 3) of N-glycanase and then subjected to SDS-polyacrylamide gel electrophoresis. Proteins were blotted onto a polyvinyliden difluoride filter and were probed with anti-rabies virus G protein serum. Standard molecular size markers are indicated on the left side (kDal. (Bf Comparison of putative ~-glycosylation sites of rabies virus G proteins. Presence i+) or absence (-) of the putative N-linked glycosylation sites (Asn-X-Thr/Ser) on the ectodomain of each G protein was indicated. The sites of ERA, CVS, Pasteur (PV), HEP-Flury, and Kelev strains were predicted from the report of Anilionis et al., (1981), Yelverton et al. (19831, Tordo et al., (1986b), Morimoto et al. (19891, and Wunner et al. (19881, respectively. The sites of the Nishigahara strain was predicted from the nucleotide sequence of the cDNA used in this study (Tuchiya, unpublished results).

hara strain were predicted from nucleotide sequence of the G-ORF (Tuchiya, unpublished results) and are shown in comparison with other known rabies virus G proteins Fig. 2B). Our data show that the G protein of the Nishigahara strain possesses three putative glycosylation sites, two of which may be glycosylated in the G protein of rabies virion. The G protein expressed in insect cells was also treated similarly (Fig. 2A, lane 4). The upper 62 kDa band became faint, but the lower band remained at the position of 58 kDa. which was slightly larger than that of the completely digested G protein of the virion (about 56 kDa). In order to examine whether the 58 kDa recombinant protein had completely lost oiigosaccharide side chains or not, we tried to detect oligosaccharide side chains in the band by using carbohydrate detection kit, G.P. SENSOR (Honen, Japan). Carbohydrates were detected in the N-glycanase-treated 58 kDa component of recombinant G proteins, upper two

bands of authentic G protein but not in the 56 kDa band of the authentic G protein (data not shown). This indicates that the recombinant G protein in insect cells is glycosylated differently from the -abies virion-associated G protein.

a

b

e

‘Fig. 3. Syncytium formation of Spodopferu frugiperda cells by the recombinant G protein. Spmfoptem cells were infected with AcRaG (a-g) or AcNPV (h). Immediately after infection, pH of the medium was adjusted to 5.0 (a), 5.5 (b, e-h), 6.0 (c), or 6.2 (d). In syncytium formation-inhibition experiments (e-g), rabbit anti-rabies virus antiserum (e), rabbit anti-G protein monospecific antiserum (f), or normal rabbit serum (g) were included in the medium at pH 5.5. Syncytium formation was monitored at 72 h postillf~ction.

frugiperda

Y

Fusion activity

qf the recombinant G protein

To examine whether the recombinant G protein has biological activities as does authentic G protein, we investigated the fusogenic properties of recombinant G protein. In the rabies virus-infected cell cultures, this activity is observed when pH of the culture medium is decreased (Mannen et al., 1982). When the pH of the AcRaG-infected insect cell culture was decreased from 6.2 to 6.0-5.0, syncytium formation was observed at pH below 5.5 (Fig. 3a-d). The syncytium formation was not observed in AcNPV-infected cells (Fig. 3h). Moreover the syncytium formation was inhibited by immune rabbit sera against the rabies virion and G protein, but not by non-immunized rabbit serum (Fig. 3e-g). In spite of the difference in the glycosylation level, the recombinant G protein was thus found to carry the fusion activity as the authentic G protein associated with rabies virus-infected cells.

Discussion

In this study, we established an insect cell/baculovirus vector system for expressing a high level of the rabies virus G protein of the Nishigahara strain and characterized the recombinant protein. At least a portion of the recombinant G proteins produced in the insect cells was expressed on the cell surface and shown to display the fusogenic activity. The cell fusion activity was induced by the expressed G protein, because syncytium formation was completely inhibited by antisera against rabies virions or isolated G protein. G proteins on the rabies virion and on the surface of the virus-infected cells are thought to form a polymeric structure (Dietzschold et al., 1978) and recently, Gaudin et al. (1991) reported that when the G protein showed fusogenic activity at low pH, the G protein underwent a reversible conformational change which could be detected by monoclonal antibodies. Therefore, our results that the G protein expressed by insect cells has fusogenic activity suggest that the recombinant protein forms correct higher-order structure such as native polymerization which can further undergo conformational change to show the activity at low pH. Unlike the authentic virion G protein, two glycosylated species of recombinant G protein were detected by SDS-PAGE; both migrated faster than the authentic G protein (Fig. 2A). Similar phenomena (different electrophoretic mobility from the authentic G protein and production of heterogeneous species of G protein) were also reported in the case of recombinant baculovirus-mediated expression of G protein of the CVS strain of rabies virus (Prehaud et al., 1989) and of another rhabdovirus, vesicular stomatitis virus (Bailey et al., 1989). The authentic G protein had three putative carbohydrate chain acceptor sites, two of which were glycosylated. The presence of the two G proteins of different electrophoretic mobilities might reflect the difference in glycosylation, or incomplete glycosylation at either or both sites of the protein. After the digestion of the recombinant G proteins with N-glycanase. it is not clear whether the completely de-glycosylated G protein was present, because the location of the protein (56 kDa) was covered with the 58-kDa

protein. But these observations suggest that both the number and structure of carbohydrate side chains on the recombinant G protein are different from those on the viral G protein. Nevertheless, the recombinant G protein was shown to display certain biologicai activities similar to that of viral G protein. This indicates that the recombinant G protein is expressed on the cell surface through the maturation pathway common to membrane proteins and that the expressed G protein keeps similar. if not identical, three-dimensional structure. Prehaud et al. (1989) also reported the expression of G protein of rabies virus CVS strain in insect cells. In this case, the expressed G protein reacted with monoclonal antibodies against the conformational epitope II of G protein (Prehaud et al., 1988). Data from Wiktor et al. t 1984) suggested that correct folding was a prerequisite for the expression of the G protein on the cell surface and for the subsequent representation of biological activities. It is reported that vesicular stomatitis virus G protein did not require glycosylation for its surface expression and representation of its activity. Further examinations are needed to make the participation clear in fusion activities of the rabies virus G protein. In general the level of expression of recombinant genes on baculovirus vector is usually high in insect cells, but in most cases the mode and level of glycosylation are different from the authentic proteins in their respective hosts. For instance, oligosaccharide side chains of the influenza virus hemagglutinin expressed in Sf cells are smaller than those of virions grown in vertebrate hosts (Kuroda et al.. 19901. This may account for the size difference between the authentic and recombinant G proteins Further examinations are also needed for the detailed structure of oligosaccharide side chains on the expressed G protein. In spite of having different oligosaccharide side chains, the recombinant G proteins were found to be active in cell fusion and in inducing protective immunity against lethal challenge by rabies virus (Prehaud et al., 1989; Tuchiya, unpublished results). For the development of live rabies vaccines, recombinant vaccinia viruses carrying the G protein gene have been extensively investigated (Kieny et al., 1984; Wiktor et al., 1984; Blancou et al., 19861. Up to now, however, few practicable ways have been developed for the production of a subunit vaccine. In this report as well as in Prehaud et al. (19891, it is suggested that the high yields of the immunologically active G protein are supplied by the recombinant baculovirus/insect cell system, which will promise to establish a new system for producing rabies subunit component vaccines. Quite recently, Fu et al. (1991) reported that rabies virus N protein was produced abundantly using a bacuIovirus vector and that priming of animals with the recombinant N protein. instead of the authentic RNP, prior to inoculation with inactivated rabies vaccine also resulted in high 1eveI production of virus-neutralizing antibodies, leading to protection of animals after only a Low dose of the inactivated vaccine. So, there may be a possibility for developing more effective and economic subunit rabies vaccines by a combination of the G and N proteins. Difficulty to perform large-scale cultures of insect ceils. which has been a serious problem for the large-scale production of recombinant proteins by using a baculovirus expression system, is now being overcome by using airlift bioreactors (Maiorella et al., 1988: Murhammer and Goochee, 1988). Along

11

this line, the next breakthrough should be to develop purification of the recombinant G protein in large scale.

a simple method

for

We thank Dr. Kinjiro Morimoto (Kyoto Univ.) for helpful discussion, and Dr. Yoshihisa Ishikawa, Messrs. Masao Usui and Yoshiaki Nakamura for help in the animal experiments. Some of the results in this paper were presented at the 24th Joint Viral Diseases Panel Meeting of U.S.-Japan Cooperative Medical Science Program, held on September 27-29, 1990, in Sendai.

References Anilionis, A., Wunner. W.H. and Curtis, P.J. (1981) Structure of the glycoprotein gene in rabies virus. Nature 294. 275-278. Bailey, M.J., McLeod, D.A., Kang, C.-Y. and Bishop, D.H.L. (1989) Glycosyiation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells. Virology 169, 323-331. Blancou, J., Kieny, M.P.. Lathe, R., Lecocq, J.P., Pastoret, P.P., Soulebot, J.P. and Desmettre, P. (1986) Oral vaccination of the fox against rabies using a live recombinant vaccinia virus. Nature 322, 373-375. Brown. M. and Faulkner, P. (1977) A plaque assay for nuclear polyhedrosis viruses using a solid overlay. J. Gen. Viral. 36. 361-364. Celis. E.. Miller, R.W.. Wiktor, T.J., Dietzschold, B. and Koprowski, H. (1986) Isolation and characterization of human T-cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-y. J. Immunol. 136. 692-697. Crick, J. and Brown, F. (1969) Viral subunits for rabies vaccination. Nature 222, 92. Dietzschold. B., Cox, J.H., Schneider. LG., Wiktor. T.J. and Koprowski, H. (1978) Isolation and purification of a polymeric form of the glycoprotein of rabies virus. J. Gen. Viroi. 40, 131-139. Fu, Z.F.. Dietzschold, B., Schumacher, C.L., Wunner, W.H.. Ertl, H.C.J. and Koprowski, H. (1991) Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine. Proc. Nati. Acad. Sci. USA 88, 2001-2005. Gaudin, Y., Tuffereau, C.. Segretain. D., Knossow, M. and Flamand. A. (1991) Reversible conformational changes and fusion activity of rabies virus glycoprotein. J. Virol. 65, 4853-4859. Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25. 263-269. Halonen, P.E.. Murphy. F-A., Fields, B.N. and Reese; D.R. (1968) Hemagglutination of rabies and some other bullet-shaped viruses. Proc. Sot. Exp. Biol. Med. 127, 1037-1042. Harlow. E. and Lane, D. 11988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory. New York. Ishikawa, Y., Samejima, T.. Nunoya, T., Motohashi, T. and Nomura, Y. (1989) Biological properties of the cell culture-adapted RC.HL strain of rabies virus as a candidate strain for an inactivated vaccine. J. Jpn. Vet. Med. Assoc. 42, 437-643. Kawai, A. (1977) Transcriptase activity associated with rabies virion. J. Virol. 24, 826-835. Kawano, H.. Mifune, K., Ohuchi, M., Mannen, K., Cho. S., Hiramatsu, K. and Shichijo, A. (199tJ) Protection against rabies in mice by a cytotnxic T-ceil clone recognizing the glycoprotein of rabies virus. J. Gen. Viral. 71, 381-287. Kieny, M.P., Lathe, R., Drillien. R.. Spehner. D., Skory. S., Schmitt, D.. Wiktor, T., Koprowski, H. and Lecocq, J.P. (1984) Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 312. 163-166.

12 Kuroda. K., Geyer, H.. Geyer, R., Doerfler, W. and Klenk. H.-D. (IYYO) The oligosaccharides of influenza virus hemagglutinin expressed in insect cells hy a haculovirus vector, Virology 173, 418-429. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227. 680-685. Lai, C.Y. and Dietzschold. B. (1981) Amino acid composition and terminal sequence analysis of the rabies virus glycoprotein: identification of the reading frame on the cDNA sequence, Biochem. Biophys. Res. Commun. 103. 536-542. Luckow, V.A. and Summers, M.D. (1988) Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47-55. Macfarlan, RI.. Dietzschold, B.. Wiktor. T.J., Kiel, M.. Houghten, R., Lerner. R.A.. Sutclifee, J.G. and Koprowski. H. (1984) T-cell responses to cleaved rabies virus glycoprotein and to synthetic peptides. J. lmmunol. 133. 3748-2752. Maiorella, B.. Inlow, D., Shauger, A. and Harano. D. (1988) Large-scale insect cell-culture for recombinant protein production. Bio/Technology 6, 1306-1410. Maniatis, T.. Fritsch, E.F. and Sambrook. J. (19X2) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Mannen. K.. Ohuchi, M. and Mifune, K. (1982) pH-dependent hemolysis and cell fusion of rhabdovirus. Microbial. Immunol. 26, 1035-1042. Matsuura, Y.. Possee. R.D. and Bishop. D.H.L. i 1986) Expression of the S-coded genes of lymphocytic choriomeningitis arenavirus using a baculovirus vector. J. Gen. Viral. 67. 1515 1529. Matsuura, Y.. Possee, R.D.. Overton. H.A. and Bishop. D.H.L. (1987) Baculovirus expression vectors: the requirements for high level expression of proteins. including glycoproteins. J. Gen. Virol. 68, 1233-1250. Morimoto, K.. Ohkuho. A. and Kawai. A. (1989) Structure and transcription of the glycoprotein gene of attenuated HEP-Flury strain of rabies virus. Virology 173. 465-477. Murhammer, D.W. and Goochee, C.F. (1988) Scale-up of insect cell cultures: protective effect of Pluronic F-68. Bio/Technology h, 141l-1418. Perrin. P.. Thibodeau, L. and Sureau. P. tlYX5) Rabies immunosome (subunit vaccine) structure and immunogenicity. Pre- and post-exposure protection studies. Vaccine 3. 325-332. Prehaud, C.. Coulon. P., Lafay, F., Thiers. C. and Flamand. A (1988) Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. J. Viral. 62. 1-7. Prehaud, C., Takehara, K.. Flamand, A. and Bishop. D.H.L. (1989) Immunogenic and protective properties of rabies virus giycoproteiI1 expressed by baculovirus vectors. Virology 173, 390-399. Rayssiguier, C.. Coie, L.. Withers. E., Wunner. W.H. and Curtis. P.J. (19%) Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence. Virus Res. 5, 177-1YO. Sedwick, W.D. and Wiktor, T.J. (1967) Reproducible plaquing system for rabies. lymphocytic choriomeningitis, and other ribonucleic acid viruses in BHK-21/!3S agarose suspensions. J. Virol. 1. 1234-1226. Smith, G.E., Summers, M.D. and Fraser. M.J. (1983) Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3, 215667165. Tordo, N., Poch, O., Ermine. A. and Keith. G. (1986a) Primary structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VSV. Nut. Acids Res. 14. 2671-2683. Tordo. N., Poch. O., Ermine, A., Keith, G. and Rougeon. F. tlY86b) Walking along the rabies genome: is the large G-L intergenic region a remnant gene’? Proc. Natl. Acad. Sci. USA 83. 3914-3918. Tordo, N.. Poch. 0.. Ermine, A.. Keith, G. and Rougeon, F. (1988) Completi~~n of the rabies virus genome sequence determination: highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses. Virology 165. 565-576. Wiktor. T.J.. Gyorgy, E.. Schlumberger, H.D.. Sokol, F. and Koprowski. H. (1973) Antigenic properties of rabies virus components. J. Immunol. 1IO, X9-276. Wiktor, T.J., Plokin. S.A. and Koprowski, H. (1978) Development and clinical trials of the new human rabies vaccine of tissue culture (human diploid cell) origin. Dev. Biol. Stand. 40, 3-Y.

13 Wiktor, T.J., Macfarlan, R.I., Reagan, K.J.. Dietzschold, B., Curtis, P.J., Wunner, W.H., Kieny, M.-P., Lathe, R., Lecocq, J.-P., Mackett, M.. Moss, B. and Koprowski, H. (19841 Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proc. Natl. Acad. Sci. USA 81, 7194-7198. Wunner, W.H., Dietzschold, B.. Curtis, P.J. and Wiktor, T.J. (1983) Rabies subunit vaccines. J. Gen. Virol. 64, 1649-1656. Wunner. W.H., Larson. J.K., Dietzschold, B. and Smith, C.L. (1988) The molecular biology of rabies viruses. Rev. Infect. Dis. 10 (Suppl 4), S771-5784. Yelverton, E., Norton, S., Obijeski. J.F. and Goeddel, D.V. (1983) Rabies virus giycoprotein analogs: biosynthesis in Escherichia coli. Science 219. 614-620.