Journal of General Virology (1991), 72, 359-367.

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Printed in Great Britain

Stable expression of rabies virus glycoprotein in Chinese hamster ovary cells S. R. Burger, l"f A. T. Remaley, l~ J. M. Danley, 1 J. Moore, 1 R. J. Muschel, 1 W. H. Wunner 2 and S. L. Spitainik 1. 1Department of Pathology and Laboratory Medicine, University of Pennsylvania and 2The Wistar Institute, Philadelphia, Pennsylvania 19104, U.S.A.

The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunica-

mycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial Nlinked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of Nlinked glycosylation in trafficking and antigenicity of the rabies virus G protein.

Introduction

host immune system during infection and vaccination, specifically inducing neutralizing antibodies (Cox et al., 1977; Wiktor et al., 1973) and cytotoxic T cells (Celis et al., 1988a, b). G protein cDNAs from the ERA and CVS rabies virus strains have been cloned and expressed in bacteria (Anilionis et al., 1981 ; Yelverton et al., 1983) and also in eukaryotic cells (Kieny et al., 1984; Lecocq et al., 1985; Prehaud et al., 1989). Kieny et al. (1984) used a vaccinia virus recombinant containing the cDNA for the ERA strain G protein. This membrane protein has a single transmembrane domain and the mature protein contains 505 amino acids (Anilionis et al., 1981 ; Yelverton et al., 1983). The predicted G protein amino acid sequences from both strains have three potential N-linked oligosaccharide acceptor sites of the form Asn-X-Ser/Thr, only two of which are glycosylated (Dietzschold, 1977; Wunner et al., 1985). There are no known O-linked oligosaccharides (Dietzschold, 1977; Wunner et al., 1985). At least one N-linked oligosaccharide has been

The rabies virus glycoprotein (G protein) is the only protein on the surface of the mature virus and plays a critical role in virus infection (Dietzschold et al., 1978; Sokol et al., 1971). It is the viral attachment protein and is therefore important in determining the tissue tropism of the virus (Iwasaki et al., 1973; Perrin et al., 1982; Wunner et al., 1984). Following attachment to the host cell, the G protein facilitates fusion of the viral envelope with endocytic vesicle membranes (Mifune et al., 1982; Reagan & Wunner, 1984). The G protein is also involved in budding and the release of virus particles from host cells (Mifune et al., 1982; Reagan & Wunner, 1984). Finally, the G protein is a major antigenic stimulus of the t Present address: Department of Laboratory Medicine, Washington University, St Louis, Missouri, 63110, U.S.A. :~Present address: National Heart Lung and Blood Institute, Bethesda, Maryland 20892, U.S.A. 0000-9839 © 1991 SGM

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predicted to be of the complex type by composition analysis (Dietzschold, 1977; Schlumberger et al., 1973). The importance of the N-linked oligosaccharides of the G protein is perhaps twofold. First, glycosylation may be necessary for immunogenicity. The G protein synthesized by bacteria, which fail to glycosylate proteins, is ineffective as a vaccine in animal models (Yelverton et al., 1983), whereas cloned G protein expressed and glycosylated in eukaryotic ceils and glycosylated G protein purified from rabies virions are both effective as vaccines (Kieny et al., 1984; Prehaud et al., 1989; W i k t o r et al., 1984; W u n n e r et al., 1983). H o w e v e r , the glycosylation r e q u i r e m e n t for i m m u n o g e n icity a n d a n t i g e n i c i t y is not resolved by this c o m p a r i s o n . F o r e x a m p l e , mouse m o n o c l o n a l a n t i b o d i e s w h i c h define a n t i g e n i c sites on the G p r o t e i n ( D i e t z s c h o l d et al., 1988; L a f o n et al., 1983, 1984; P r e h a u d et al., 1988; S e i f e t al., 1985) m a y n o t require glycosylation o f t h e i r t a r g e t s for o p t i m a l b i n d i n g ( W u n n e r et al., 1985). A l t e r n a t i v e l y , as for o t h e r viral g l y c o p r o t e i n s ( L e a v i t t et al., 1977; M a c h a m e r et al., 1985; N o r r i l d & P e d e r s e n , 1982; P i z e r et al., 1980; V i d a l et al., 1989), glycosylation m a y be n e c e s s a r y for the biological function, i n t r a c e l l u l a r transp o r t a n d surface e x p r e s s i o n o f the G p r o t e i n in i n f e c t e d cells, a l t h o u g h this has n o t yet b e e n d e m o n s t r a t e d . To e x a m i n e the effects o f global c h a n g e s in N - l i n k e d glycosylation o n i n t r a c e l l u l a r t r a n s p o r t , surface expression a n d a n t i g e n i c i t y o f the G p r o t e i n , we g e n e r a t e d a n d c h a r a c t e r i z e d stably t r a n s f e c t e d C h i n e s e h a m s t e r o v a r y ( C H O ) cell lines t h a t express G p r o t e i n f r o m cloned c D N A . T h e effect o f b l o c k i n g g l y c o s y l a t i o n w i t h t u n i c a m y c i n or o f altering the N - l i n k e d o l i g o s a c c h a r i d e structure using l e c t i n - r e s i s t a n t C H O cells was studied. This a p p r o a c h , c o m b i n e d w i t h site-directed m u t a g e n e s i s o f G p r o t e i n c D N A , s i m i l a r to t h a t d e s c r i b e d for the G p r o t e i n o f vesicular s t o m a t i t i s virus (VSV) ( M a c h a m e r et al., 1985), will be used in future studies to e x a m i n e these r e l a t i o n s h i p s in m o r e detail.

Methods Cell lines and tissue culture. Wild-type CHO cells (CHO-K1D) were obtained from Dr Stomato, Wistar Institute. Wild-type (Pro-5) and lectin-resistant CHO cell lines (Lec 1, Lec 2 and Lec 8) (Stanley et al., 1975) were obtained from the American Type Culture Collection. The CHO-K1D cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum (FCS), 2 mMglutamine, 100 international units (IU)/ml of penicillin, 100 ~tg/ml of streptomycin and 0.25 Ixg/ml of amphotericin B. All other cell lines were grown in alpha-MEM supplemented as above. Transfected cell lines were maintained in complete media containing 0-5 mg/ml of active G418 (Gibco). For some experiments, cells were cultured overnight in complete medium containing the glycosylation inhibitor tunicamycin (5 Ixg/ml). Antibodies. Rabies virus G protein-specific monoclonal antibodies 101-1 (antigenic site II) and 509-6 (antigenic site I) from the Wistar

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Fig. 1. Construction of plasmid pSG5rabgp. Plasmid pSG5rabgp was produced by excision of the cDNA encoding the G protein from ptg155pro using BgllI and ligation of the purified fragment into the BgllI site of pSG5. In pSG5, A represents the simian virus 40 (SV40) early promoter, B represents rabbit fl-globin intron II, C represents the bacteriophage T7 promoter, D represents the SV40 polyadenylation signal, AmpR represents the ampicillin resistance gene and ori indicates the origin of replication. The hatched regions in ptg155pro and pSG5rabgp represent the G protein cDNA.

Institute were described previously (Dietzschold et al., 1988; Flamand et al., 1980; Lafon et al., 1983); monoclonal antibodies 62-36-7, 62-80-6, 62-15-2, 62-11-6 and 62-114-6 were generously provided by J. Smith, Centers for Disease Control, Atlanta, Ga., U.S.A. (Smith et al., 1984). Hybridoma cell lines producing IgG mouse monoclonal antibodies OKT11 and anti-Lewis a (151-6-a7-9)were obtained from the American Type Tissue Culture Collection. Hybridoma culture supernatant was concentrated 20-fold by ammonium sulphate precipitation and subsequent dialysis. Polyclonal antibody to wild-type CHO cells was produced by three sequential weekly intraperitoneal immunizations of eight BALB/c mice with 5 x 106 CHO-K1D cells. Immune serum was obtained 1 week after the last immunization, pooled and stored in aliquots at - 20 °C.

Plasmid construction. The full-length cDNA of the G protein (ERA strain) was excised from the ptg155pro plasmid (Kieny et al., 1984) with BgllI and ligated into the unique BgllI site of the pSG5 eukaryotic expression vector (Stratagene) (Green et al., 1988) to produce pSG5rabgp (Fig. 1), Plasmid pSG5rabgp was used to transform Escherichia coli HB 101 and constructs containing the insert in the sense and antisense orientations were isolated, characterized and amplified using standard techniques (Sambrook et aL, 1989). Transfection. CHO cells were cotransfected with 25 ~tgof pSG5rabgp and 1 ktgof pSV2neo (Southern & Berg, 1982)by the calcium phosphate precipitation method (Sambrook et al., 1989) with a 2 rain glycerol shock at 4 h (Sambrook et al., 1989). After 48 h the medium was replaced with fresh complete medium containing 1 mg/ml of active G418 and thereafter the medium was replaced approximately every 2 days with fresh complete medium containing G418. After approximately 14 days G418-resistant colonies were isolated, amplified and screened for expression of rabies virus G protein by indirect immunofluorescence. Clonal cell lines were isolated by repetitive subcloning by limiting dilution.

Rabies virus glycoprotein expression

Southern blotting. Genomic DNA with a high Mr was extracted from cultured cells by standard methods (Sambrook et al., 1989), digested with EcoRI, separated by electrophoresis on a 0.8~ agarose gel, and blotted onto Zeta-Probe nylon sheets (Bio-Rad). EcoRI was used because it is active in cutting genomic DNA and because both pSV2neo and pSG5rabgp have unique EcoRI restriction sites. Appropriate plasmid restriction fragments were purified twice by agarose gel electrophoresis and extracted from the gel with Gene-Clean (Bio 101). The purified fragments were then labelled with [32p]dCTP by nick translation following the manufacturer's instructions (BRL). The blot was treated with the labelled probe overnight at 65 °C in 0.5 MNaHPO4, 1 mM-EDTA pH 7.2 containing 7~ SDS, washed (two washes with 40 mM-NaHPO4, 1 mM-EDTA pH 7-2 containing 1~ SDS and two washes with 40 mM-NaHPO4, 1 mM-EDTA pH 7.2 containing 5 ~ SDS; 60 rain per wash at 65 °C), and the probe that hybridized to the DNA was detected by exposure to Kodak XAR-5 X-ray film (Eastman Kodak). Indirect immunofluoreseence. Indirect immunofluorescence was performed on cells grown in eight-well tissue culture chamber slides (Nunc). For surface fluorescence, live unfixed cells were incubated for 45 rain at 4 °C with primary antibody diluted in DMEM medium containing 30 mg/ml bovine serum albumin (DMEM-BSA). For cytoplasmic staining, cells were first permeabilized with acetone for 5 min at 4 °C and then air-dried before incubation with primary antibody. After three washes with DMEM-BSA, the cells were incubated at 4 °C for 45 rain with the fluoresceinated F(ab')2 fragment of goat anti-mouse lgG (H + L) (Tago) diluted 1 : 50 in DMEM-BSA. The slides were washed as above and then covered with 50 ~ glycerol in phosphate-buffered saline (pH 7.4) and a coverslip, and examined with a Leitz Orthoplan epifluorescence microscope (Wild Leitz USA). Flow cytometry. Flow cytometry was performed on a BectonDickinson FACS Analyzer. Cells were removed from culture dishes with a dilute trypsin-EDTA solution (0.002~ trypsin and 0.004~ EDTA for 30 min at 37 °C), centrifuged and resuspended in medium for 2 h at 37 °C before staining. This method was used to minimize proteolysis of cell surface glycoproteins. Live cells were then treated in the same manner as described above for indirect immunofluorescence. Immunopreeipitation. Logarithmically growing cells were metabolically labelled for 48 h with 0.25 ~tCi/ml of [14C]leucine in leucine-free DMEM (ICN) supplemented with 10~ FCS, 2mM-glutamine, 100 IU/ml of penicillin, 100 Ixg/mlof streptomycin, and 0.25 ~tg/ml of amphotericin B. G protein was solubilized from transfected cells with lysis buffer (50 mM-Tris-HC1, 150 mM-NaCI, 5 mM-EDTA pH 7-4 with 0.5~ NP40 and 200 lag/ml PMSF) as previously described (Wunner et al., 1980). Aliquots of solubilized protein containing approximately 200000 c.p.m, in a total volume of 200 ~tlwere incubated at 4 °C for 1 h with ascites fluid containing anti-G protein antibodies (1:100 final dilution) or with the indicated control antibodies. Fixed Staphylococcus aureus (Sigma) was then added (25 p.l of a 10~ suspension) and incubated at 4 °C for 1 h before centrifugation of the immune complexes. Immune complexes were washed five times in 15 mM-TrisHCI, 0.5 M-NaC1, 5 mM-EDTA pH 7-4 with 5 ~ sucrose and 1~ NP40, electrophoresed on a 5 to 15~ gradient SDS-polyacrylamide gel and analysed by fluorography with Amplify (Amersham).

Results Isolation and characterization o f cell lines expressing G protein R e c o m b i n a n t p l a s m i d s c o n t a i n i n g the G p r o t e i n in e i t h e r the sense or a n t i s e n s e o r i e n t a t i o n were isolated,

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c h a r a c t e r i z e d a n d purified. T h e s e were t h e n e a c h i n d i v i d u a l l y t r a n s f e c t e d into w i l d - t y p e C H O cells along with pSV2neo. F o l l o w i n g selection w i t h G418, i n d i v i d u a l colonies a n d p o l y c l o n a l p o p u l a t i o n s o f s t a b l y t r a n s f e c t e d r e s i s t a n t cells were o b t a i n e d . To confirm the i n t e g r a t i o n o f G p r o t e i n c D N A i n t o cellular g e n o m i c D N A , a S o u t h e r n blot w a s p e r f o r m e d w i t h D N A f r o m polyclonal G 4 1 8 - r e s i s t a n t p o p u l a t i o n s o f C H O - K 1 D cells t r a n s f e c t e d w i t h e i t h e r the sense o r a n t i s e n s e versions o f p S G 5 r a b g p (Fig. 2). P r o b i n g the S o u t h e r n blot o f EeoRI-cleaved cellular g e n o m i c D N A w i t h a 32p-labelled p S G 5 r a b g p BglII f r a g m e n t c o n t a i n ing the G p r o t e i n c D N A r e v e a l e d the p r e s e n c e o f a b r o a d b a n d c e n t r e d at 8 k b for the a n t i s e n s e - t r a n s f e c t e d C H O K 1 D cells (lane 7) a n d two r e l a t i v e l y discrete b a n d s o f 9 k b a n d 5 k b for the s e n s e - t r a n s f e c t e d C H O - K 1 D cells (lane 8). P l a s m i d p S G 5 r a b g p has a single EcoRI r e s t r i c t i o n site w h i c h is o u t s i d e the r e g i o n o f the r a b i o l a b e l l e d BglII f r a g m e n t p r o b e , t h e r e f o r e i f no r e a r r a n g e m e n t o f the p l a s m i d D N A o c c u r r e d following i n t e g r a t i o n e a c h c o p y o f i n t e g r a t e d D N A should yield a single b a n d on the S o u t h e r n blot. O u r results s h o w i n g a few b a n d s o f v a r i o u s sizes (lanes 7 a n d 8) t h e r e f o r e i n d i c a t e r a n d o m i n t e g r a t i o n o f r e l a t i v e l y few copies o f G p r o t e i n c D N A into the C H O cell g e n o m i c D N A (Southern & Berg, 1982). I n c o n t r a s t , n o b a n d s were d e t e c t e d using D N A from u n t r a n s f e c t e d C H O - K 1 D cells (lane 5) or f r o m cells t r a n s f e c t e d w i t h o n l y p S V 2 n e o , the p l a s m i d c o n t a i n i n g the selectable m a r k e r (lane 6). W h e n the first p r o b e was s t r i p p e d f r o m the blot a n d t h e blot r e - p r o b e d w i t h a 32p-labelled B g I I I - B a m H I restriction f r a g m e n t o f p S V 2 n e o c o n t a i n i n g n e o m y c i n resist a n c e (neo R) c o d i n g sequences, h y b r i d i z a t i o n b a n d s were specifically d e t e c t e d for b o t h the sense- a n d a n t i s e n s e t r a n s f e c t e d cells, as well as for the cells t r a n s f e c t e d w i t h p S V 2 n e o alone (lanes 14 to 16). T h i s d e m o n s t r a t e s the specificity o f the p r o b e s used in this e x p e r i m e n t a n d t h a t p l a s m i d D N A c o n t a i n i n g the selectable m a r k e r was also i n t e g r a t e d into the cellular g e n o m i c D N A o f t h e a p p r o p r i a t e cell lines. T h e p o l y c l o n a l p o p u l a t i o n o f C H O - K 1 D cells transfected w i t h the sense version o f p S G 5 r a b g p was e x a m i n e d for e x p r e s s i o n o f the G p r o t e i n b y i m m u n o fluorescence (Fig. 3). W h e n live, i n t a c t cells were s t a i n e d w i t h m o n o c l o n a l a n t i b o d y 101-1 (b), a p p r o x i m a t e l y 2 0 ~ o f the cells were positive, a n d 10~o were h i g h l y positive. T h e fluorescence p a t t e r n o f the p o s i t i v e l y s t a i n e d t r a n s f e c t e d cells was u n i f o r m a n d diffuse a n d was q u a l i t a t i v e l y s i m i l a r to t h a t seen w h e n the cells were s t a i n e d w i t h a n t i - C H O cell p o l y c l o n a l a n t i s e r u m (c) a n d (d). I n contrast, 101-1 d i d n o t b i n d to u n t r a n s f e c t e d C H O - K 1D cells (a) o r to cells t r a n s f e c t e d w i t h G p r o t e i n c D N A in the a n t i s e n s e o r i e n t a t i o n ( d a t a n o t shown). N e i t h e r o f the i s o t y p e - m a t c h e d n e g a t i v e c o n t r o l anti-

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Fig. 2. Analysis of wild-type and transfected CHO-K1D cell lines by Southern blotting. Genomic D N A was isolated, digested with EcoRl, separated by agarose gel electrophoresis and blotted onto Zeta-Probe, as described in Methods. The blot was probed with either 32p-labelled G protein cDNA (a) or with a 32p.labelled segment of the neo s gene (b). The sizes of the labelled lambda D N A standards (kb; lanes I and 9) are indicated on the left. Lanes 2 to 4 and 10 to 12 contain 50, 20 and 10 pg of EcoRI-digested pSG5rabgp (sense orientation) plasmid DNA, respectively. The remaining lanes contain cellular genomic DNA. Lanes 5 and 13, untransfected CHO-K1D cells; lanes 6 and 14, CHOK 1D cells transfected with pSV2neo alone; lanes 7 and 15, CHO-K 1D cells cotransfected with pSG5rabgp (antisense orientation) and pSV2neo; lanes 8 and 16, CHO-K1D cells cotransfected with pSG5rabgp (sense orientation) and pSV2neo.

bodies, OKT 11 and anti-Lewisa, bound to any of the cells tested (data not shown). The polyclonal population of pSG5rabgp (sense orientation)-transfected cells was further examined with six additional G protein-specific monoclonal antibodies. Similar to the results found with

Fig. 3. Indirect immunofluorescence of wild-type- and pSG5rabgp (sense orientation)-transfected CHO-K 1D cells. Wild-type- and sensetransfected cells were grown on eight-well slides, stained unfixed with the indicated primary antibody (G protein-specific mouse monoclonal antibody 101-1 or mouse anti-CHO cell polyclonal antibody) followed by fluorescein-labelled anti-mouse IgG secondary antibody, and examined by fluorescence microscopy as described in Methods. (a) Untransfeeted cells and monoclonal antibody 101-1; (b) transfected cells and monoclonal antibody 101-1; (c) untransfected ceils and antiCHO polyclonal antibody; (d) transfected cells and anti-CHO polyclonal antibody. Bar marker represents 25 Ixm.

monoclonal antibody 101-1, approximately 20~ of the polyclonal population of transfected cells was positive with each of the G protein-specific monoclonal antibodies tested (data not shown). Thus, the G protein epitopes recognized by these antibodies were appropriately expressed at the cell surface. Similar results were obtained with a clonal derivative of wild-type CHO cells, Pro-5 (Stanley et al., 1975), transfected with pSG5rabgp (data not shown). The polyclonal population of ceils transfected with pSG5rabgp (sense orientation) was subcloned by repetitive limiting dilution and screened for expression of G protein by indirect immunofluorescence. Clone T3.30.11, which expressed a high level of G protein when examined by indirect immunofluorescence with all

Rabies virus glycoprotein expression of the anti-G protein antibodies, was further analysed by flow cytometry. A uniform population of brightly fluorescent cells stained positively with both the 101-1 and 509-6 monoclonal antibodies (Fig. 4). No staining was seen when clone T3.30.11 was incubated with OKT11, an isotype-matched control antibody of irrelevant specificity. CHO cells transfected with G protein cDNA in the antisense orientation were negative with all antibodies tested except for the anti-CHO polyclonal serum (Fig. 4). The T3.30.11 clone and three other carefully studied clones were found to express the G protein stably; no diminution in expression of the protein was noted either by indirect immunofluorescence or by flow cytometry after more than 6 months of continuous culture. Transfected CHO cells were metabolically labelled with [x~C]leucine and detergent-soluble cellular proteins were immunoprecipitated with G protein-specific monoclonal antibodies (Fig. 5). A band at 67K was observed for three independently isolated sense-transfected clonal cell lines (lanes 6 to 8). This band comigrates with authentic 67K G protein specifically immunoprecipitated from rabies virus-infected baby hamster kidney (BHK) cells (lanes 1 and 2) and also with the G protein from purified rabies virions (lane 4). No corresponding band was seen following immunoprecipitation of the non-transfected cells (lane 5) or when proteins from infected cells were immunoprecipitated with an isotypematched negative control antibody (lane 3). Similarly, no proteins were specifically immunoprecipitated from antisense-transfected cells (data not shown). These results suggest that the 67K protein immunoprecipitated from the transfected cells is identical to fully glycosylated authentic G protein.

Role of glycosylation in intracellular transport of G protein To assess the role of the two N-linked oligosaccharides in intracellular transport of the G protein, the polyclonal population of stably transfected CHO cells was treated with tunicamycin; this compound specifically and completely blocks N-linked glycosylation of proteins (see Fig. 6). Abundant surface staining of G protein was observed by indirect immunofluorescence in both nonpermeabilized and permeabilized cells that were not treated with tunicamycin (Fig. 7a and b). However, when the transfected cells were grown in the presence of 5 ~tg/ml of tunicamycin for 16 h, immunofluorescence was observed only if the cells were first permeabilized (c and d). Similar results were obtained with each of the four transfected clonal cell lines expressing G protein which were examined (data not shown). These findings indicate that G protein lacking both N-linked oligosac-

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101 102 103 Green fluorescence Fig. 4. Analysis of transfected cell lines by flow cytometry. (a) T3.30.11, a clonal CHO-KID cell line which was cotransfected with pSG5rabgp (sense orientation) and pSV2neo, and (b) T3. AS, a CHOK1D cell line which was cotransfected with pSG5rabgp (antisense orientation) and pSV2neo, were analysed by flow cytometry as described in Methods. G protein-specific mouse monoclonal antibodies 101-1 (-.-) and 509-6 (--), an isotype-matched negative control mouse monoclonal antibody OKT11 (-) and a positive control mouse anti-CHO cell polyclonal antibody (aCHO) (-.-) were used.

charides is not transported or is inefficiently transported to the plasma membrane and instead accumulates intracellularly. Previous studies of the carbohydrate composition of purified G protein suggested that at least one of the two N-linked oligosaccharides is of the complex variety and contains sialic acid (Dietzschold, 1977; Schlumberger et al., 1973). To examine the importance of the type of oligosaccharide present on the G protein for proper intraceUular transport, the cDNA was transfected into three lectin-resistant mutants of Pro-5 CHO cells (Lec 1, Lec 2 and Lec 8; see Fig. 6). These cells allow inital Nlinked glycosylation to occur, but later steps in processing of the oligosaccharides are blocked. Lec 2 ceils are markedly defective in the sialylation of oligosaccharides; complex type oligosaccharides of G protein expressed in these cells are therefore expected to lack terminal sialic acid. In contrast, the oligosaccharides synthesized on G protein produced in Lec 1 cells are expected to be limited to the high mannose type (Stanley et al., 1975), whereas those synthesized in Lec 8 cells could be of either the high mannose or hybrid variety (Stanley et al., 1975). Following transfection of pSG5rabgp into each of the lectin-resistant cell lines, the G418-resistant cells were

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Fig. 5. Immunoprecipitation of rabies virus G protein. The left panel represents results obtained with,BI-IKcells infected with rabies virus ERA strain and metabolicallylabelled with [35S]methionineas described (Wunner et aL, 1985). Detergent lysateswere prepared as described in Methods (lanes 1 to 3); intact rabies virions were prepared as described (Clark et aL, 1981). CHO-K1D cells were metabolically labelled with [14C]leucine and detergent-soluble proteins were immunoprecipitated, separated by SDS-PAGE and autoradiographed as described in Methods. Lanes 1 to 3 and 5 to 8 represent immunoprecipitated proteins. Lane 4 represents all the labelled proteins found in intact rabies virions; viral proteins L, G, N, NS and M are present and labelled. The location of the G protein is indicated. The migration positions of Mr standards are noted on the left of each gel. The two gels were run on separate occasions. Rabies virus-infectedBHK cells were immunoprecipitated with antibody 509-6(lane 1), antibody 101-1 (lane 2) or an isotype-matched negative control antibody (lane 3). Proteins were immunoprecipitated with monoclonal antibody 509-6 from non-transfected CHOK 1D cells (lane 5) or three distinct clonal cell lines, T3.14.14 (lane 6), T3.30.11 (lane 7) and T3.45.3.16 (lane 8), isolated from CHOKID cells cotransfected with pSG5rabgp (sense orientation) and pSV2neo.

isolated and examined by indirect immunofluorescence with G protein-specific antibodies. In each case, the lectin-resistant cell lines were found to express the transfected G protein on their plasma m e m b r a n e (data not shown). The degree of surface expression of the G protein for each of the three lectin-resistant cell lines was comparable to that observed above for the transfected wild-type C H O ceils. For each lectin-resistant transfected cell line, tunicamycin blocked surface expression of G protein (data not shown). These results indicate that although some type of N-linked glycosylation is critical for intracellular transport and surface expression of G protein, the specific structures of the N-linked oligosaccharides on this glycoprotein are less important in this process.

Discussion Both wild-type and lectin-resistant C H O cell lines transfected with G protein c D N A express this protein in a stable manner without any apparent cytotoxic effect. The transfected wild-type C H O cells specifically produce a protein of 67K (Fig. 5), which agrees with the Mr of the G protein isolated from infected cells or purified virions

(Wiktor et al., 1973; Wunner et al., 1985). Since previous studies demonstrated that the unglycosylated protein migrates faster than the 67K fully glycosylated viral protein (Wunner et al., 1985), the present result suggests that the protein produced by the transfected cells is appropriately glycosylated. Using indirect immunofluorescence and flow cytometry, the expressed protein was detected on the plasma m e m b r a n e of the transfected cells (Fig. 3 and 4) in a manner similar to that found with virus-infected ceils (Lodmell & Ewalt, 1987; Wiktor & Koprowski, 1978; Wiktor et al., 1984). The configuration of the transfected protein appears to be similar to that of the virus-produced protein, since all of the G protein-specific monoclonal antibodies tested were able to recognize the protein on the surface of transfected cells. Glycosylation of several viral G proteins is important for their proper folding, expression, transport and function (Basak & Compans, 1983; Gibson et al., 1978; Leavitt et al., 1977; Norrild & Pedersen, 1982; Pizer et al., 1980; Sodora et al., 1989; Vidal et al., 1989). We examined the role of N-linked glycosylation in the intracellular transport of G protein by using both the glycosylation inhibitor tunicamycin and three lectinresistant C H O cell lines with well characterized glycosy-

365

Rabies virus glycoprotein expression

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lation defects. Tunicamycin treatment of transfected CHO cells blocked surface expression of G protein, indicating that glycosylation is essential for its proper transport to the plasma membrane (Fig. 7). Treatment of rabies virus-infected cells with tunicamycin prevents glycosylation of the G protein (Wunner et al., 1985), but the effect of tunicamycin on transport of this glycoprotein to the plasma membrane of infected cells or on production of infectious virions has not been studied. Our results suggest that unglycosylated G protein is not efficiently transported to the cell surface and therefore tunicamycin would greatly inhibit production of infectious virions by virus-infected cells. Similar results have been reported for the G protein of VSV-infected cells (Gibson et al., 1978; Kotwal et al., 1986; Leavitt et al., 1977). Deletion of both N-linked acceptor sites on VSV G protein by site-directed mutagenesis also blocked surface expression of the protein, whereas deletion of either site alone had little effect on translocation (Machamer et al., 1985; Machamer & Rose, 1988 a; Rose

Fig. 7. Indirect immunofluorescence of G protein cDNA-transfected CHO-KID cells treated with tunicamycin. Cells cotransfected with pSG5rabgp (sense orientation) and pSV2neo were grown on eight-well slides and incubated for 16 h in the presence or absence of 5 Ixg/ml of tunicamycin. Live or acetone-fixed (permeabilized) cells were then stained with G protein-specific mouse monoclonal antibody 101-1 and fluorescein-labelled secondary antibody and examined by fluorescence microscopy as described in Methods. (a) Live and (b) fixed cells incubated without tunicamycin; (c) live and (d) fixed ceils incubated with tunicamycin. Bar marker represents 25 ~tm.

& Bergmann, 1982). The N-linked oligosaccharides on VSV G protein have been proposed to promote translocation by allowing proper protein folding and by preventing formation of aberrant intermolecular disulphide bonds (Machamer & Rose, 1988a, b). The Nlinked oligosaccharides on the rabies virus G protein may play a similar role; analogous studies with the G protein are currently under way using site-directed mutagenesis and stable transfection of the mutated G protein cDNAs. Lectin-resistant CHO cell lines were used to examine whether certain types of N-linked oligosaccharides present on the G protein would affect surface expression of this glycoprotein. Whether the oligosaccharides were restricted to be of the high mannose type (Lec 1 cells),

366

S. R. Burger and others

hybrid type (Lec 8 cells) or complex type lacking the terminal sialic acid (Lec 2 cells), G protein was expressed on the surface of transfected cells in comparable amounts. These studies, however, do not exclude the possibility of different rates of intracellular transport of the variably glycosylated protein. Previous studies have shown that several different lectin-resistant cell lines infected with VSV all produce infectious virions, indicating that, as for the rabies virus G protein, the specific type of oligosaccharide present on VSV G protein is not critical for surface expression (Robertson et al., 1978). Eukaryotic cell lines which stably express G protein, such as those described in this report and by Lecocq et al. (1985), offer several advantages over other available means to study this protein. Although intact rabies virus infects a broad range of tissue culture cells in vitro (Wunner, 1987), it is inconvenient to work with infected cells owing to the risk of human infection from live virus and because of the cytopathic effect of the virus on the host cells. Cells infected with recombinant vaccinia virus containing G protein cDNA can be used to study the protein without risk of human infection (Kieny et al., 1984), but the vaccinia virus vector also has a cytopathic effect on host cells. Finally, G protein has been expressed in E. coli, but the expressed protein does not undergo normal post-translational modification, including glycosylation; the unmodified protein produced by these cells has been found to be ineffective as a vaccine in animal models (Yelverton et al., 1983). In contrast, the approach described in this report offers some unique advantages. The ability to alter the glycosylation of G protein by expressing it in lectin-resistant cell lines will allow studies exploring the functional and antigenic significance of the oligosaccharides on this protein. In addition, site-directed mutagenesis of G protein can be used to delete and create N-linked oligosaccharide acceptor sites, permitting further study of its structure-function relationships. Since antigenic epitopes on the G protein are preserved in transfected ceils, this suggests that comparison of wild-type and mutant G proteins in transfected mammalian cell lines will be useful for studying both humoral and cellular host immune responses to rabies virus (Bunschoten et al., 1989; Celis et al., 1988a; Eager et al., 1989; Morgeaux et al., 1989). In summary, a model system for studying the rabies virus G protein is described which should be useful in a wide range of studies.

We thank M. Kelsten for expert guidance in preliminary flow cytometry studies. This work was supported in parts by grants from the National Institutes of Health (CA45690, AI18883) and the American Cancer Society (JFRA-193). S.L.S. is a Mallinckrodt Scholar. A.T.R. was supported by post-doctoral fellowship award 5T32CA09140-15

from the National Institutes of Health. This work was presented in part at the Xth International Conference of Glycoconjugates in Jerusalem, Israel, 1989.

References ANILIONI$,A., WOtCNER,W. H. & CURTIS,P. J. (1981). Struture of the glycoprotein gene in rabies virus. Nature, London 294, 275-278. BASAK,S. & COMI'ANS,R. W. (1983). Studies on the role of glycosylation in the functions and antigenic properties of influenza virus glycoproteins. Virology 128, 77-91. BUNSCHOTEN,H., KLAPMLrrS,R. J., CLAAgSEN,I. J. T. M., REYNEVELD, S. D., OSTERHAUS,A. D. M. E. & UYTDEHAAG,F. G. C. M. (1989). Rabies virus-specific human T cell clones provide help for an in vitro antibody response against neutralizing antibody-inducing determinants of the viral glycoprotein. Journal of General Virology70, 15131521. CELIS, E., KARR, R. W., DIETZSCHOLD, B., WUNNER, W. H. & KOPROWSKI, H. (1988a). Genetic restriction and fine specificity of human T cell clones reactive with rabies virus. Journal of Immunology 141, 2721-2728. CELIS, E., Ou, D., DIETZSCHOLD, B. & KOPROWSKI H. (1988b). Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients. Journal of Virology 62, 3128-3134. CLARK, H F., PARKS, N. F. & WLrI~ER, W. H. (1981). Defective interfering particles of fixed rabies viruses: lack of correlation with attenuation or auto-interference in mice. Journal of General Virology 52, 245-258. Cox, J. H., DIETT_,SCHOLD,B. & SCHNEIDER,L. G. (1977). Rabies virus glycoprotein. II. Biological and serological characterization. Infection and Immunity 16, 754-759. DIETZSCHOLD,B. (I 977). Oligosaccharides of the glycoprotein of rabies virus. Journal of Virology 23, 286-293. DIET~OLD, B., COX, J. H., SCHNEIDER,L. G., WIKTOR, T. J. & KOPROWSKI, H. (1978). Isolation and purification of a polymeric form of the glycoprotein of rabies virus. Journal of General Virology 40, 131-139. DIETZ,SCHOLD,B., RUPPRECHT,C. E., TOLLIS,M., LAFON,M., MATTEI, J., WIKTOR,T. J. & KOPROWSKI,H. (1988). Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Reviews of Infectious Diseases 10, 5785-5798. EAGER, K. B., HACKETT, C. J., GERHARD, W. O., BENN1NK, J., EISENLOHR,L. C., YEWDELL,J. & RICX2IARDI,R. P. (1989). Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes. Journal of Immunology 143, 2328-2335. FLAMAND, A., WIKTOR, T. J. & KOPROWSKI, H. (1980). Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rubies-related virus proteins. II. The glycoprotein. Journal of General Virology 48, 105-109. GIBSON, R., LEAVITr, R., KORNFELD, S. & SCHLESINGER,S. (1978). Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein. Cell 13, 671-679. GREEN, S., ISSEMANN,I. & SHEER, E. (1988). A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Research 16, 369. IWASAKI,Y., WIKTOR,T. J. & KOPROWSKI,H. (1973). Early events of rabies virus replication in tissue cultures: an electron microscopic study. Laboratory Investigation 28, 142-148. KIENY, M. P., LATHE, R. F., DRILLIEN, R., SPEHNER,D., SKORY,S., SCHMITr, D., WIKTOR, T., KOPROWSKI,H. & LECOCQ,J. P. (1984). Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature, London 312, 163-166. KOTWAL,G. J., BULLER,R. M. C., WUNNER,W. H., PRINGLE,C. R. & GI-IOSH,H. P. (1986). Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. Journal of Biological Chemistry 261, 8936-8943.

Rabies virus glycoprotein expression

LAFON, M., WIKTOR,T. J. & MACFARLAN,R. I. (1983). Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies. Journal of General Virology 64, 843-851. LAFON, M., IDELER,J. & WUNNER, W. H. (1984). Investigation of the antigenic structure of rabies virus glycoprotein by monoclonal antibodies. Developments in Biological Standardization 57, 219-225. LEAVIT'r, R., SCHLESlNGER, S. & KORNFEL6, S. (1977). Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis and Sindbis virus. Journal of Biological Chemistry 252, 9018-9023. LECOCQ,J. P., KIENY, M. P., LI~MOINE,Y., DRILLIEN,R., WlKTOR,T., KOPROWSKi, H. & LATHE, R. (1985). New rabies vaccines: recombinant DNA approaches. In WorM's Debt to Pasteur, pp. 259271. Edited by H. Koprowski & S. A. Plotkin. New York: Alan R. Liss. LODMELL, D. L. & EWALT, L. C. (1987). Immune sera and antiglycoprotein monoclonal antibodies inhibit in vitro cell-to-cell spread of pathogenic rabies viruses. Journal of Virology 61, 33143318. MACHAMER, C. E. & ROSE, J. K. (1988a). Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. Journal of Biological Chemistry 263, 5948-5954. MACHAMER,C. E. & ROSE,J. K. (1988b). Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. Journal of Biological Chemistry 263, 5955-5960. MACHAMER,C. E., FLORKIEWIt2Z,R. Z. & ROSE, J. K (1985). A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Molecular and Cellular Biology 5, 3074-3083. MIFUNE, K., OHUCHI, M. & MANNEN,K. (1982). Hemolysis and cell fusion of rhabdoviruses. FEBS Letters 137, 293-297. MORGEAUX,S., JOFFRET, M.-L.,'LECLERC, C., SUREAU,P. & PERRIN, P. (1989). Evaluation of the induction of specific cytotoxic T lymphocytes following immunization of F1 hybrid mice with rabies antigens. Research in Virology 140, 193-206. NORRILD, B. & PEDERSEN, B. (1982). Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface. Journal of Virology 43, 395-402. PERRIN, P., PORTNOI,D. & SUREAU,P. (1982). Etude de l'adsorption et de la penetration du virus rabique: interactions avec les cellules BHK 21 et des membranes artificielles. Annales de l'lnstitut Pasteur, Virologie 133E, 403-422. PIZER, L. I., COHEN, G. H. & EISENBERG, R. J. (1980). Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production. Journal of Virology 34, 142-153. PREHAUD, C., COULON, P., LAFAY, F., THIERS, C. & FLAMAND,A. (1988). Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. Journal of Virology 62, 1-7. PREHAUD,C., TAKEHARA,K., FLAMAND,A. & BISHOP,D. H. L. (1989). Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors. Virology 173, 390-399. REAGAN, K. J. & WUNNER, W. H. (1984). Early interaction of rabies virus with cell surface receptors. In Nonsegmented Negative Strand Viruses, pp. 387-392. Edited by D. H. L. Bishop & R. W. Compans. New York: Academic Press. ROBERTSON,M. A., ETCmSON,J. R., ROBERTSON,J. S., SUMMERS,D. F. & STANLEY, P. (1978). Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistant CHO cells. Cell 13, 515-526. ' ROSE, J. K. & BERGMANN,J. E. (1982). Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell 30, 753-762.

367

SAMBROOK, J., FRITSeH, E. F. & MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SCHLUMBERGER,H. n., SCHNEIDER,L. G., KULAS,H. D. & DIRINGER, H. (1973). Gross chemical composition of strain Flury HEP rabies virus. Zeitschrift f~r Naturforschung 28C, 103-104. SEIF, I., COULON, P., ROLLIN, P. E. & FLAMAND,A. (1985). Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. Journal of Virology 53, 924-936. SMITH, J. S., SUMNER, J. W. & ROUMILLAT,L. F. (1984). Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus. Journal of Clinical Microbiology 19, 267-272. SONORA,D. L., COHEN,G. H. & EISENBERG,R. J. (1989). Influence of asparagine linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D. Journal of Virology 63, 5184-5193. SOKOL, F., STANCEK,D. & KOPROWSKI,H. (1971). Structural proteins of rabies virus. Journal of Virology 7, 241-249. SOUTHERN, P. J. & BERG, P. (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under the control of the SV40 early region promoter. Journal of Molecular and Applied Genetics 1, 327-341. STANLEY, P., CAILLIBOT,V. & SIMINOVICH,L. (1975). Selection and characterization of eight phenotypically distinct lines of lectinresistant Chinese hamster ovary cells. Cell 6, 121-128. VIDAL, S., MOTTET,G., KOLAKOFSKY,D. & Roux, L. (1989). Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendal virus glycoproteins. Journal of Virology 63, 892-900. WIKTOR, T. J. & KOPROWSKI, H. (1978). Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Proceedingsof the National Academy of Sciences, U.S.A. 75, 3938-3942. WIKTOR, T. J., COHEN, G. H., SCHLUMBERGER,H. D., SOKOL, F. & KOPROWSKI, H. (1973). Antigenic properties of rabies virus components. Journal of linmunology 110, 269-276. 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., MACKETI',M., MOSS,B. & KOPROWSKI,H. (1984). Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proceedings of the National Academy of Sciences, U.S.A. 81, 7194-7198. WUNNER, W. H. (1987). Rabies viruses -pathogenesis and immunity. In The Rhabdoviruses, pp. 361-426. Edited by R. R. Wagner. New York: Plenum Press. WUNNER,W. H., CURTIS,P. J. & WIKTOR,T. J. (1980). Rabies mRNA translation in Xenopus laevis oocytes. Journal of Virology 36, 133-142. WUNNER, W. H., DIETZSCHOLD,B., CURTIS, P. J. & WIKTOR, T. J. (1983). Rabies subunit vaccines. Journal of General Virology 64, 1649-1656. WUNNER, W. H., REAGAN, K. J. & KOPROWSKI, H. (1984). Characterization of saturable binding sites for rabies virus. Journalof Virology 50, 691-697. WUNNER,W. H., DIETZSCHOLD,B., SMITH,C. L., LAFON,M. & GOLUB, E. (1985). Antigenic variants of CVS rabies virus with altered glycosylation sites. Virology 140, 1 12. YELVERTON,E., NORTON,S., OBIJESKI,J. F. & GOEDDEL,D. V. (1983). Rabies virus glycoprotein analogs: biosynthesis in Escherichia coll. Science 219, 614-619.

(Received 16 August 1990; Accepted 6 November 1990)