VIROLOGY 206, 736-741 (1995)

Expression and Characterization of Secreted Forms of Rubella Virus E2 Glycoprotein in Insect Cells

NINA O. L. SETO, 1 DAWEI Ou, AND SHIRLEY GILLAiVi 2

Department of Pathology, University of British Columbia, Research Centre, 950 West 28th Avenue, Vancouver, British Columbia, V5Z 4H4 Canada

Received July 29, 1994; accepted October 25, 1994

Two different forms of rubella virus E2 glycoproteins were expressed in insect cells: intact wild-type E2 and a soluble form of E2 (E2~Tm) glycoprotein, in which the C-terminal membrane-anchor domain was deleted. E2ATm behaved as a secretory protein and was secreted abundantly (5 mg/liter) from insect cells. In contrast to wild-type E2 (36 kDa), E2ATm was secreted into the media and was detected as two species (33 and 30 kDa). Lectin binding assays in conjunction with glycoeidase analyses revealed that both intracellular wild-type E2 and E2ATm contained only N-linked glycans, while the two secreted forms of E2ATm were found to differ in their glycosylation, with the 30-kDa form having only N-linked glycans while the 33-kDa species had both N-linked and O4inked glycans. The secreted E2ATm species were purified by precipitation between 20 and 40% saturation with (NH4)2SO4 and retained full antigenicity. The levels of antibodies elicited in mice immunized with purified E2ATm showed that the immunogenicity of secreted E2LkT m compared favorably to that of natural virion E2. © 1995 Academic Press, Inc.

Rubella virus (RV), the causative agent of German mea- sles, is a small enveloped positive-stranded RNA virus belonging to the Togaviridae family (1). infection during early pregnancy results in various birth defects, collec- tively known as congenital rubella syndrome (CRS) (1). The RV virion contains three structural proteins, E1 (58 kDa), E2 (42 to 47 kDa), and C (33 kDa) (2). E1 and E2 are membrane glycoproteins that exist as a heterodimer and form the viral spike complexes on the surface of the virion (3). The capsid (C) protein is associated with the genomic RNA forming the nucleocapsid (2). The biologi- cal role of E2 is not well defined, but strain-specific epi- topes (4) and at least one neutralizing domain have been reported (5). Antibodies to E1 are predominant following most RV infections, but antibodies to E2 are more abun- dant in ORS than in other forms of RV infection (6). Very few E2-specific monoclonal antibodies are isolated com- pared to the large number directed against E1 when mice are immunized with whole RV (5, 7), presumably due to poor immunogenicity of E2 in native virions. This may be due to the fact that E2 epitopes are buried under E1 in the heterodimeric spike complexes on the virion surface (8). E2 from the RV strain M33 contains three large, com- plex-type N-linked (9, 10) and several smaller O-linked carbohydrates (10).

Due to the nature of E2 and E1 glycoproteins that exist as a heterodimer and form the viral spike complexes on the surface of the virion, no preparations of E2 proteins

Present address= National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, K1A OR6, Canada.

~To whom correspondence and reprint requests should be ad- dressed. Fax, (604) 875-2496.

0042-6822/95 $6.00 Copyright © 1995 by Academic Press, Inc. AII rights of reproduction in any form reserved.

736

from the virion or recombinant sources have been satis- factory in terms of ease of large scale preparations or good retention of antigenicity (11- 15). The production of an antigenic, secreted form of E2 protein from insect cells will provide large quantities of material for biological and immunological studies. In order to express E2 protein abundantly in a form that can be simply purified from cell culture, we have converted the membrane-bound E2 protein into a secretory protein and expressed it using the baculovirus- insect cell system (16).

It has been shown that viral transmembrane glycopro- teins are secreted from cells if the transmembrane an- chor sequences are deleted (17, 18). This strategy was used to produce a soluble form of E2 protein by trunca- tion of the C-terminus of E2 before the anchor region (12). To create the soluble form of E2 protein (E2~Tm), the plasmid pE2 containing the RV E2 cDNA (12) was digested to completion with Xbal, followed by partial di- gestion with Ncol to release the DNA fragment encoding 60 C-terminal amino acids of E2 protein (19). The Xbal and Ncol termini of the truncated plasmid were made blunt with T4 DNA polymerase and recircularized using T4 DNA ligase. The ends of the EcoRI-Hindl l l fragment containing E2ATm cDNA were made blunt and inserted into the unique Nhel site (made blunt) of the transfer vector pBlueBac (/gLI) (from Invitrcgen) to form pAc- B lueBac-E2ATm (Fig. 1). The constructed recombinant plasmid was confirmed by restriction analysis. The cDNA construct contains the capsid (C) protein translation start site as well as nucleotides specifying the first eight amino acids and 55 carboxyl-terminal residues of the C protein, including the putative E2 signal sequence (19) (Fig. 1). Cotransfection of Sf9 insect cells with this recom-

SHORT COMMUNICATIONS 737

C E2 E1 E H

~4s 'r- 0 1 Y ~ I~1 v v v It ~

E C E2 H E2 ';~' u~nl YY Y ~_~

C E2 z~Tm E H

E2 Al'm '1~ Eli YY v ,'

NheI

FiG. 1. Schematic diagram of RV E2 and E2ATm cDNA. The three RV structural proteins are encoded by a 24S subgenomic mRNA and the order of translation is NH2-C-E2-E1 -COOH. Respective portions of RV capsid (C), E2, and E1 genes are indicated on the cDNA fragments 24S, F2, and E2ATm. Intergene borders are marked by vertical lines, signal peptides are indicated by open boxes, and transmembrane re- gions by solid boxes. The E2 gene was isolated from the 24S cDNA with a portion of the capsid gene and inserted into the unique BamHI cloning site in the transfer vector pAcYMI. The removal of the putative tramsmembrane domain from E2 results in the cDNA E2ATm, which was inserted into the unique Nhel cloning site of the baculovirus trans- fer vector pBlueBac (fiLl). The initiation codon of capsid protein was utilized for the expression of both E2 proteins. The RV E2 and E2z&Tm cDNA were expressed under the control of the polyhedrin promoter. The bar represents approximately 500 nucleotides; N-linked glycosyla- tion sites are indicated (Y). E and H denote the EeoRI and Hindlll restriction sites flanking the 5'- and 3'-ends of the constructs, respec- tively. The blunt-ended Pstl site, where the deoxyoligonucleotide was inserted, is marked with arrowhead.

binant plasmid and wild-type baculovirus, Autographica califomica polyhedrosis virus (AcNPV), DNA resulted in homologous recombination in vivo and replacement of the polyhedrin coding region with that of E2Z~Tm cDNA. AcNPV sequences present in the transfer vector and viral DNA give rise to recombinant baculoviruses that were identified by the formation of blue plaques, which were also viral occlusion negative (20). Clonal recombinant baculoviruses expressing intracellular and secreted BV E2 proteins were identified by immunoblot analysis (21) of infected cell extracts and culture supernatants. The recombinant baculovirus (Ac-E2Z&Tm) was purified and used for the production of E2z&Tm protein.

Pulse-chase analysis was used to evaluate the ex- pression and secretion of E2z&Tm protein. Sf9 cells in- fected with Ac-E2ATm were pulse-labeled with [35S]- methionine for 15 min and chased with 1 mM unlabeled methionine for 24 hr (22). RV proteins were immunopre- cipitated with human anti-RV sera and analyzed by SDS- PAGE as described previously (23). The amount of expressed intracellular E2zSTm protein (designated

iE2z&Tm) (32 kDa) increased from 0 to 24 hr of chase (Fig. 2A, cells), and two secreted E2Z~Tm (designated sE2/kTm) species (33 and 30 kDa) were detected in the culture medium only after 24 hr of chase (Fig. 2A, media). To determine the processing of N-linked glycosylation in expressed E2 proteins, the immunoprecipitated E2 pro- teins were digested with endoglycosidase H (endo Hi, an enzyme which selectively cleaves immature high man- nose N-linked oligosaccharides within the compartments of the endoplasmic reticulum (ER) and the mediaI-Golgi apparatus (24). Digestion of iE2z&Tm with endo H de- creased its molecular weight from approximately 32 to 25 kDa (Fig. 2A, cells, 0-hr chase). Two iE2Z~Tm species (29 and 27 kDa) were partially resistant to endo H diges- tion (Fig. 2A, cells, 2-hr chase). In contrast, the electro- phoretic mobil/ties of both sE2ATm species were not affected by digestion with endo H (Fig. 2A, media). Insect cells have the capability to convert high mannose N- linked glycans to endo H-resistant forms (25-27). The endo H-resistant oligosaccharides consist of the Man3GI- cNA% core of N-glycans that may also have a fucose attached (28).

In COS cells transfected with RV E2 cDNA, a small amount of E2 was found in the medium (unpublished

A

chase (h)

endoH

68-

43-

29-

18-

cells media

0 24 24 - + - + - +

B H

chase(h) O 1 6 9 24 ÷

43-

2 9 -

1 8 -

FIG, 2. Pulse-chase analysis of RV E2 and RV E2z~Tm expression. Sf9 cells infected with Ac-E22~Tm(A) or Ac-E2(B) were pulse-labeled with [36S]methionine and chased with 1 mM unlabeled methionine as described (30). RV proteins were immunoprecipitated with human anti- RV sera from culture media and cellular lysates of infected cells. Sam- ples were taken after the chase periods shown above each lane. Sam- ples were treated with buffer only ( - ) or endo H (+). Two E2ATm species were secreted into the culture media and not altered by endo H digestion (single and double arrowheads). Intracellular E2 used for endo H digestion (Hi was from immunoprecipitates after a 24-hr chase period. Positions of molecular weight markers are shown on the left (kDa).

738 SHORT OOMMUNICATIONS

data). It is unclear whether proteolytic cleavage occurred to release E2 into the medium. To address the question as to whether sF2z~Tm protein present in the culture medium of Sf9 cells was due to secretion and not to proteolysis, we have isolated recombinant baculovirus (Ac-E2) expressing membrane-bound E2 protein. The plasmid pE2 containing the RV E2 cDNA (12) was di- gested with Pstl, the protruding 5'-ends were removed by treatment with T4 DNA polymerase and the plasmid was recircularized in the presence of .a deoxyoligo- nucleotide (CTAGTCTAGACTAG) with termination co- dons in all three reading frames. The blunted EcoRI- Hindlll fragment (Fig. 1) containing full-length E2 cDNA from the resulting plasmid was inserted into the blunt- ended unique BamHI site of pAcYM1 vector (29) to form the transfer vector pAcYM 1-E2. Recombinant baculovirus Ac-E2 was isolated and purified as described above. Sf9 cells infected with Ac-E2 were pulse-labeled with [sSS]methionine and chased with unlabeled methionine for 24 hr. The electrophoretic mobility of intracellular E2 (36 kDa) increased slightly from 0 to 24 hr of chase (Fig. 2B), suggesting that post-translational modification oc- curred. No full-length E2 was immunoprecipitated from the medium even after a 48-hr chase period (data not shown). The intracellular membrane-bound E2 after a 24- hr chase period was sensitive to endo H treatment (Fig. 2B), but was partially resistant to endo H digestion after a 36-hr chase period (data not shown).

To isolate sE2ATm for antigenicity and immunogenic- ity studies, Sf9 cells grown in Sf900 (Gibco-BRL) serum- free culture medium were infected with recombinant Ac- E2Z~Tm (m.o.i. = 5) and incubated at 27 ° for 78 hr. Ceils and culture supernatants were separated by low speed centrifugation and the pH of the supernatant was ad- justed to neutrality with 1 MTris-HCI (pH 8.0). An initial precipitation with (NH4)2SO4 to 20% of saturation was carried outto remove any lipid-like material in the culture medium (23). sE2ATm protein in the supernatant was fractioned by stepwise increases in the (NH4)2SO4 con- centration from 20 to 40% and 40 to 65% of saturation. The precipitates from each fraction were suspended in 1/20 the original culture volume of TNE (10 mM Tris- HCI, pH 7.6, 0.15 M NaOl, 1 mM EDTA) and dialyzed against TNE at 4 ° overnight with two buffer changes. This resulted in the purification of sE2ATm protein to 80% homogenecity in the fraction between 20 and 40% satura- tion with (NH4)2SO4, as visualized by silver staining an SDS-PAGE gel (data not shown). The yield of purified sE2ATm was approximately 70% of the secreted protein in the culture medium.

The antigenic properties of baculovirus-expressed RV E2z~Tm proteins were investigated. Both iE2ATm and sE2ATm forms were recognized by an E2-specific mono- clonal antibody and human anti-RV sera (data not shown). The purified sE2ATm protein was used as an antigen in immunoblot analysis to detect E2 antibodies

in human sera from individuals of known immune status (30). The intensity of the reaction varied slightly between individuals with weak to no recognition in sera 4, 10, and 14 and strong recognition by an individual with CRS (serum 9) (Fig. 3A). ORS patients are known to have ex- ceptionally high levels of anti-E2 antibodies (6). The anti- genic properties of purified sE2ATm were compared with those of E2 from purified RV virions. Approximately equal amounts of RV proteins from RV virions versus sE2ATm protein were used in the immunoblotting. The correlation of the reactivities between E2 from RV virions and the recombinant sE2ATm was good (Fig. 3A), indi- cating that sE2ATm protein has similar antigenic activity to the wild-type E2 from RV virion. We have used sE2ATm as a target antigen in enzyme-linked immunosorbent assays (ELISA) for serological detection of E2-specific antibodies in the same sera. The ELISA mean ab- sorbance values obtained for sera 1 to 15 were 0.46, 0.67, 0.32, 0.31, 0.63, 0.32, 0.52, 0.7, 0.8, 0.21, 0.56, 0.27, 0.33, 0.44, and 2.27, respectively, with pooled human neg- ative sera having the value of 0.057. Although the com- parison could not be made quantitatively due to the na- ture of experimental conditions used, it demonstrated the potential of sE2ATm to be used as a target antigen in serological assays for RV infection.

To study the immunogenicity of sE2ATm, BALB/c mice were subcutaneously inoculated with 5 #g of purified sE2ATm emulsified in 100 #1 of complete Freund's adju- vant (CFA), or phosphate-buffered saline in 100 #1 CFA in control animals. Mice received three additional injec- tions of antigen or saline in Freund's incomplete adjuvant at 3-week intervals. Mice were bled and sera collected for analysis. The presence of antFRV E2 antibodies was determined by immunoblot analysis. Mice immunized wi:th sE2Z~Tm produced antibodies against sE2ATm as well as E2 from RV virions (Fig. 3B), indicating that the antigenic structure of sE2ATm is similar to that of E2 in the virion. In order to further confirm the antigenic properties of sE2ATm, we have analyzed the reactivity of anti-E2 sera raised by immunizing mice with RV E2 isolated from RV virion by preparative SDS-PAGE (11). Anti-sera raised from denatured E2 from RV virions failed to recognize E2 from RV virion or sE2ATm (data not shown), confirming that the antigenicity of sE2ATm is preserved. The presence of virus neutralizing antibodies in mouse sera was determined by immunocytochemical focus assay. No viral neutralization activity was observed in the sera from mice immunized with sE2z~Tm (data not shown). This finding is consistent with our data obtained from mice immunized with a vaccinia recombinant virus expressing full-length RV E2 protein (unpublished data).

The glycosylation of RV E2 in the virion is heteroge- neous (31). The two species of sE2Z~Tm observed may be due to the difference in oligosaccharide processing. Lectin-binding assays in conjunction with treatment with glycosidases were used to define the gtycans present

SHORT COMMUNICATIONS 739

A RV

1 2 3 4 5 6 7 8 9101112131415 M - - + B A

E2ATM

1 2 3 4 5 6 7 8 9101112131415M --

:E=

1 2 3 4 1 2 3 4

68

43

29 E2&Tm

FiG. 3. Immunogenicity of sE2ATm. (A) Immunoblot analysis of purified sE2ATm with human sera. Purified RV particles or sE2ATm were separated on 11% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and tested for their immunoreactivities to human sera from individuals with wild-type RV infection (lanes 1-5), with congenital rubella syndrome (lanes 6-10), or healthy normal (lanes 11-15). M is the strip probed with E2 monoclonal antibodies. Strips probed with pooled human positive (+) and negative (-) sera, are indicated. Human sera were used at 1,60 dilution. (B) Immunoblot analysis of sE2ATm antisera. Strips containing separated RV proteins (lanes 1 and 3) or sE2ATm (lanes 2 and 4) were probed with human negative sera (A, lanes 1 and 2); human anti-RV sera (1,100 dilution) (A, lanes 3 and 4), mouse preimmune sera (B, lanes 1 and 2) or sE2ATm mouse antisera (1,50 dilution) (19, lanes 3 and 4). Positions of proteins corresponding to viral El, E2, and C as well as secreted sE2ATm are indicated. Positions of molecular weight markers are shown (kDa).

on the sE2ATm. The lectins used for glycan differentia- tion were the agglutinins of Galanthus nivalis (GNA), Sarnbucus nigra (SNA), Maaekia amurensis (MAA), Da- tura stramonium (DSA), and peanut (PNA) (32). GNA, which interacts with terminally linked mannose, bound both forms of sE2ATm even after digestion with O-gly- cosidase,-an enzyme which-releases the galactose/~(1- 3) N-acetylgalactosamine (Gal/~(1-3)GalNAc) unit from 0-glycans (Figs. 4A and 40). GNA did not bind sE2ATm after digestion with N-glycanase (Fig. 40), an enzyme that cleaves all N-linked glycans, suggesting that both

A B

<[ q:~ ,<" <~

~ I ~ ~ c . . o

C D

O N H C C H N 0

43

29

FIG. 4. Differentiation of glycan linkages on secreted E2~&Tm. Purified sE2ATm (33 and 30 kDa) was separated by SDS-PAGE, transferred to nitrocellulose, and probed with (A) lectins GNA, SNA, MAA, PNA, or DSA. sE2ATm was treated with buffer, lane C; H, endo H; N, N-glyca- nase; or O, O-glycosidase and then probed with human anti-RV sera (B), lectin GNA (C), or lectin PNA (D). Positions of molecular weight markers are shown (kDa).

forms of sE2ATm contained terminal mannose residues in high-mannose and/or hybrid-type oligosaccharide chains. SNA and MAA, which bind to ~(2-6) and ez(2- 3) sialic acid residues, did not bind sE2ATm (Fig. 4A). DSA, which binds to galactose /g(1-4) N-acetylglucos- amine (Gal~(1-4)GIcNAc) present in complex and hybrid N-glycan structures but not to high mannose type struc- tures, was found not to recognize recombinant sE2ATm (Fig. 4A), indicating that no complex N-linked structures were present on these glycoproteins. Complex oligosac- charide side chains found on glycoproteins from verte- brate hosts are usually replaced in insect cells by small truncated side chains and it has been shown that insect cells cannot sialylate oligosaccharide chains (28). PNA, which recognizes Gal/g(1-3)GalNAc, the core unit of O- glycans, bound only to the 33 kDa but not the 30 kDa form of sE2ATm (Fig. 4A). Digestion with O-glycosidase abolished the binding of PNA to the 33-kDa sE2ATm glycoprotein, whereas digestion with N-glycanase did not (Fig, 4D). These results suggest that the 33-kDa sE2ATm contained both N-linked and O-linked glycans, while the 30-kDa sE2ATm contained only N-linked glycan.

Removal of both O-linked and N-linked glycans by treatment with N-glycanase followed by O-glycosidase did not completely reduce the molecular weight of sE2ATm from 33 to 25 kDa as expected for unglycosyl- ated sE2ATm (Fig. 5A, lane N + O). In contrast, a 29- kDa form of sE2ATm was observed which did not bind to either PNA (Fig. 5B) or GNA (Fig. 40, and data not shown). It is possible that this 29-kDa form of sE2ATm may contain more complex O-glycan structures where

740 SHORT COMMUNICATIONS

A E2 - ~ E 2ATm - ~

N N 4. ÷ o o N C O 0 N C

29

N N -4- ÷ O O N C O O N C

18

-29

-18

FIG. 5. Glycan linkages on intracellular E2 proteins. Intraceltular membrane-bound E2 (E2) or secreted E2ATm (sE2ATm) was digested with glycosidases, separated on 11% SDS-PAGE under reducing con- ditions, and transferred to nitrocellulose membranes. The membranes were probed with human anti-RV serum (A), or with PNA (B). Samples treated with N-glycanase (lane N), O-glycanase (lane O), N-glycanase followed by O-glycosidase (lane N + O), or buffer only (lane C) are indicated. Mobilities of protein standards are shown (kDa).

Gal/~(1-3)GalNAc is substituted by fucose, galactose, or N-acetyl-glucosamine (33), which must be removed by the respective exoglycosidases in order to make the core disaccharide accessible to PNA. However, treatment of 29-kDa sE2ATm with ~-L-fucosidase or a-galactosidase prior to O-glyc.osidase digestion did not affect its electro- phoretic mobility (data not shown), suggesting that this 29-kDa species may not contain more complex O-gty- cans and may contain another form of oligosaccharide chain which is not cleaved by N-glycanase (34). Further studies are required to address this question.

O-linked oligosaccharides are added monosaccharide by monosaccharide to the polypeptide backbone, starting typically with N-acetylgalactosamine (GalNAc) and ga- lactose. The available data suggest that the addition of the core GalNAc can take place both in the ER and in the Golgi apparatus (35, 36). The acquisition of O-linked glycosylation on both intracellular membrane-bound E2 and iE2ATm was studied by treatment of both proteins (harvested after 70 hr postinfection) with N-glycanase and O-glycosidase, followed by binding to PNA after the digested protein samples were transferred to the mem- branes, No O-linked sugars were detected in either mem- brane-bound E2 (Fig. 5) or iE2ATm (data not shown). It appears that O-linked glycosylation of RV E2 protein in insect cells may occur in late stages of glycoprotein pro- cessing beyond the medial Golgi compartments as both intracellular membrane-bound E2 and iE2ATm were par- t ial ly endo H-sensitive (24). It is interesting that a protein species partially resistant to N-glycanase was also ob- served in membrane-bound E2 (Fig. 5).

in this study, we have expressed RV E2 envelope pro-

tein as a soluble and membrane-bound protein in recom- binant baculovirus-infected insect cells. Our results dem- onstrate that the antigenicities of purified sE2/kTm corre- late well with the wild-type E2 antigen from RV virion. The requirement for fetal bovine serum in conventional cell culture media hinders the direct use and purification of recombinant proteins, as fetal bovine serum contains a spectrum of proteins. The production and secretion of sE2/kTm into the serum-free medium using an insect cell system resulted in an antigen with very low concentration of extraneous proteins that facilitated isolation of large quantities of antigenic E2 (5 mg/liter), not easily obtained by using the COS cell expression system (13) or isolating E2 protein from purified RV virions. The secreted form of E2 is valuable for biochemical studies of E2 and as a target antigen for the laboratory diagnosis of acute and congenital rubella infections.

it has been shown that under certain conditions, insect cells are capable of producing recombinant proteins that contain N-linked complex-type oligosaccharides equiva- lent to those found in mammalian ceils (37), However, no complex N-linked structures were found on sE2ATm (Fig. 4). We have shown previously that N-linked glycosyl- ation is essential for proper intramolecular disulfide bonding to promote correct folding of RV E2 as well as for the secretion of E2 from COS cells (9). It appears that the small N-linked oligosaccharide side chains in sE2ATm can function as well in maintaining proper con- formation of E2 for its antigenicity and secretion. The role of heterogeneous glycosylation and O-linked glycosyla- tion in the antigenicity and processing of RV E2 protein remains to be determined. Further characterization of the two soluble forms of E2ATm may provide information on the importance of the glycosylation pattern of E2 to the biological properties of RV.

ACKNOWLEDGMENTS

We thank Dr. Aubrey J. Tingle (Departments of Pediatrics and Pathol- ogy, University of British Columbia) for providing human' sera, Dr. John Safford (Abbott Laboratories) for E2 monoclonal antibodies, and Dr. David Bishop (NERO Institute of Virology and Environmental Microbiol- ogy, Oxford, LIK) for providing baculovirus vector pAcYMI. This work was supported jointly by grants from the Medical Research Council of Canada and the British Columbia Health Research Foundation. Shirley Gillam is an investigator of the British Columbia's Children's Hospital Foundation.

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