Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses

VIROLOGY

180,442-447(1991)

Processing,

Secretion, and lmmunoreactivity of Carboxy Terminally Truncated Dengue-2 Proteins Expressed in Insect Cells by Recombinant Baculoviruses VINCENT

DEUBEL,*~’

MARINE

BORDIER,*~’

FRANCOISE

JACOB I. SCHLESINGER,+

MEGRET,*

MARY

Virus Envelope

K. GENTRY,t

AND MARC GIRARD~

*Laboratoire des Arbovirus, lnstitut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France; tWalter Reed Army Institute of Research, Washington, D.C. 20036; *Rochester Genera/ Hospital and University of Rochester School of Medicine and Dentistry, Rochester, New York 14621; and SLaboratoire de Virologie MoEculaire, lnstitut Pasteur, 75724 Paris Cede% 15, France Received May 4, 1990; accepted August 20, 7990 Two recombinant baculoviruses were constructed by inserting via the transfer vector pAcYM1 the genes coding for the structural proteins of dengue (DEN)-2 virus downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of DEN-specific envelope (E) glycoprotein. Recombinant DEN-2 E glycoproteins were processed and transported to the surface of Spodoptera frugiperda Sf9 cells infected with both viruses. We show that about one-third of the E glycoprotein minus its whole C-terminal hydrophobic anchor domain was secreted into an endoglycosidase H-resistant form. The type-specific neutralizing epitopes were conserved in the recombinant proteins o iSSi Academic PWS, IIK. as shown with a oanel of monoclonal antibodies.

Dengue (DEN) viruses belong to the flavivirus genus of the family Flaviviridae. The viruses are transmitted by Aedes mosquitoes and cause a human disease with symptoms ranging from nondescript fever to hemorrhagic and shock syndromes (I). DEN viruses contain a nucleoprotein, C, associated with a single-stranded genome of positive sense. The nucleocapsid is surrounded by a lipid bilayer associated with the membrane protein, M, and the major envelope glycoprotein, E. This surface glycoprotein is involved in important biological functions such as cellular tropism, membrane fusion, induction of neutralizing antibodies, and cellular immunity. DEN protein E contains two mannose-rich N-linked glycans (2). Genomic RNA encodes a single polyprotein of about 3400 amino acid residues commencing with the sequence for C, prM (glycosylated precursor of M), and E (3). Primary amino acid sequences of mature structural proteins prM and E are generated by signalase cleavage of the nascent polyprotein chain in rough endoplasmic reticulum, leaving them anchored in the membrane via their carboxy-terminal hydrophobic sequence (4, 5). The molecular mechanism involved in virion assembly has not been elucidated. Unlike other enveloped viruses, a budding process does not account for released viruses and the majority of envelope protein remains associated with internal membranous structures (6, 7). The baculovirus expression system has proved its

efficiency in the expression of foreign proteins in insect cells, including proteolytic processing, glycosylation, and secretion, which seems to occur normally (8, 9). Unmodified viral glycoproteins produced in insect cells infected with recombinant baculoviruses are usually targeted to cell surface but not secreted (8, 10, 1 I). On the other hand, deletions in the transmembrane domain of viral glycoproteins resulted in secretion of the truncated polypeptide when expressed by a recombinant baculovirus (12, 13). We have investigated the possibility that DEN-2 envelope glycoprotein deleted at its carboxyl terminus could be transported to the surface of insect cells and then secreted. In addition to its use in the study of its biological properties, if such a DEN protein was obtained in large quantity it might be useful as a vaccine against dengue infection. We inserted DEN-2 cDNA coding for structural proteins into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). This DEN gene fragment, previously used for the construction of a recombinant vaccinia virus (14), was digested at its 5’ end with fcoR/ and trimmed at its 3’ end using natural fcoR/ or BamHl restriction sites (Fig.1). The DEN sequences were inserted into the unique BamHl site of the baculovirus shuttle plasmid pAcYMl (kindly provided by D. H. L. Bishop), downstream from the promoter for the baculovirus polyhedrin gene (Fig. 1). The altered polyhedrin start codon (15) was followed by 22 nucleotides before the first ATG initiation codon for the DEN proteins. Stop codons either introduced

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CopyrIght 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

442

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443

put 8

YlYrYAcNPV DNA

(9.6 Kb)

mRNA sic-q

Barn HI

lllTGTAATAA4AAAA

CCTATAA

-.--

p~Cy~1

em

GGATC

AAi-rCCGGATCTCTG@[

ATACGGATCCGGTTAR +J . - - - . <752

l .

_.--

C 0

vrM

t:fi

c H

PM

kiti

3 polyhedrin

-.

*.

Em

-EmNT2207

NT@s

pAcYM1 -D7%

GGATC AAl-rCCGGATCTCTG[ATG1( -Gai-

1GGATCGATCCTB BalllHf

GATCC

Xbal

FIG. 1. Schematic presentation of procedures used to generate plasmid pAcYM1 D2SE genes. DEN structural protein genes were excised from pKB3-D2S recombinant plasmid (4) by EC&‘/ (nucleotides 89 to 2344) or EcoRI-BarnHI (nucleotides 89 to 2207) digestions, An Xhol linker was added to the BamHl extremity to generate an early stop codon at the 3’ end of the gene, occurring two codons after the nucleotide 2207. The genes were blunt-ended using Klenow polymerase and ligated into the BamWdigested, blunt-made, and dephosphorylated site of pAcYM1 ( In to produce pAcYM 1-D2SEm and pAcYM 1D2SEs vectors, respectively. The correct transcriptional orientation of the genes relative to the polyhedrin gene promoter was checked by restriction analysis. Junctions between the polyhedrin gene (in italics) and the DEN virus genes were confirmed by sequencing. Only 16 nucleotides remained in the 5’Vtontranslated part of the DEN-2 virus genome. First ATG and stop codons for dengue gene translation are boxed.

after the BamHlsite or naturally acquired from the construction after the EcoRl site in gene E led to the construction of proteins deleted at their C-terminus of 71 (Es) or 26 (Em) amino acids, respectively (Fig. 1). The recombinant vectors pAcYM 1-D2SEm and pAcYM lD2SEs were used to transfer the DEN genes to the AcNPV genome as described elsewhere (15, 16). Briefly, substitution of recombinant DEN genes in the polyhedrin gene of the baculovirus was assayed by cotransfecting IO6 Sf9 cells with a mixture of 20 pg of purified plasmid DNA and 1 pg of purified AcNPV DNA, as previously described (15). Virus progeny in the medium harvested 5 days after transfection were plaqued, and occlusion-negative-producing viruses were plaque purified three more times in accordance with the procedure described by Summers and Smith (16). Restriction enzyme analysis of the purified viral DNA indicated that the DEN genes were fully and correctly integrated in the baculovirus genome. The synthesis of DEN virus-specific structural proteins in Sf9 cells infected with the recombinant AcD2SEm and AC-D2SEs baculoviruses was studied by immunoblotting using a pool of human convalescent sera or DEN-2 specific anti-E monoclonal antibody (MAb) 3H5 (17). Figure 2 indicates that human sera

detected a major protein in Sf9 cells infected with AcD2SEmV and AC-D2SEsV recombinants, which had a molecular size of about 59 and 54 kDa, respectively. Recombinant Em protein showed a molecular mass similar to that of the native Ev protein despite having been truncated of 26 amino acids. The possible role of the modified carbohydrate core between C6/36 and Sf9 cells may be involved in this dichotomy (see below). A faster migrating band was sometimes associated to E protein which could correspond to its breakdown during sample preparation. On the other hand, human sera recognized several DEN-2 virus-specific proteins in CG-36-infected cells, in particular glycoproteins E, NSl, and prM. Neither C and prM proteins nor any polyprotein precursor (C-prM precursor was expected at a size of about 32 kDa) was found in recombinant baculovirus-infected cells (Fig. 2), even after using radioimmunoprecipitation procedure and anti-prM MAb (data not shown). Indirect immunofluorescence with MAb 3H5 was undertaken to probe the intracellular location of the DENspecific E proteins expressed by recombinant AcD2SEm and AcD2SEs baculoviruses (Fig. 3). A strong fluorescence was observed in Sf9 cells infected with both recombinant viruses and fixed with acetone 24 hr

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444 Sf9

‘X-36

IkDa)

FIG. 2. lmmunoblot of DEN proteins produced in insect cells. Monolayers of 5. 1 O6 Sf9 cells were infected with baculoviruses at a multiplicity of infection (m.o.i.) of about 10 plaque forming units (PFU) per cell. C6-36 cells were infected with DEN-2V (strain 1409, jamaica 1983) under the same conditions. After 1 hr of incubation at 28”, the inoculum was removed and cells were overlayed with 5 ml of fetal calf serum (FCS)-free Grace medium. Extracts were made 2 days after infection in sample buffer (23). Proteins from lo5 cells were electrophoresed on a 10% SDS-polyacrylamide gel (23) and blotted onto a nitrocellulose membrane. The membrane was treated with incubation buffer (20 mMTris - Cl pH 7.4, 500 mM NaCI, O.OSoio Triton X-l 00) containing 3% nonfat milk and 3% FCS, then incubated with pooled DEN human convalescent sera (H) or DEN-Z E-specific mouse MAb 3H5 (M). Authentic viral Ev protein and Em and Es proteins made respectively by recombinants AcD2SEmV and AcD2SEsV are indicated. Controls prepared from mock-infected (MI) or infected with wild-type (AcNPV) Sf9 cells, and prepared from MI or DEN-2V (D2V)infected C6-36 cells were also included.

postinfection (Fig. 3A), but the staining pattern was different depending on whether E protein was expressed with or without part of its C-terminal hydrophobic region: recombinant AcD2SEmV expressing Em exhibited a diffuse cytoplasmic staining similar to that obtained with C6-36 cells infected with DEN-2V. In contrast, recombinant AcD2SEsV expressing Es exhibited irregular masses on the cell surface. When the experiments were carried out on unfixed recombinantinfected cells (Fig. 3B), fluorescence on cell membrane was visualized as a thin pericellular rim with Em protein and showed a strong positive reaction with Es protein. In contrast, a background of much reduced staining was observed in the cytoplasm and on the plasma membrane of AcNPV-infected cells. Cell-surface fluo-

rescence of DEN-2V-infected C6/36 cells showed no obvious fluorescence. These results suggest that recombinant E proteins were processed and transported at the cell surface. On the other hand, anti-prM MAb 2H2 (18) did not stain any specific structure in recombinant-infected cells (data not shown). The antigenic properties of the recombinant E proteins were investigated by indirect immunofluorescence assay using a collection of anti-E MAbs. Among 21 MAbs that recognized authentic DEN-2 E protein in C6-36 cells, 4 MAbs lost their major reactivity with recombinant E proteins in Sf9 cells (Table 1). The other MAbs showed a reactivity with the recombinant proteins that was similar to but often weaker than that observed with DEN-2 E protein. On the other hand, all MAbs that were tested recognized the recombinant proteins in the Western-blot procedure (data not shown). The analyses of structure and antigenicity of the recombinant E proteins by immunofluorescence using MAbs are puzzling. The fact that Es and Em proteins were recognized by 4 of the 21 MAbs in Western blotting but not in immunofluorescence assays suggests that a modification of antigenic domains occurred on the proteins. This may be due either to the lack of the C-terminus or to the modified glycan core structure processed in Sf9 cells. As DEN-2V did not grow in these cells, we were not able to verify this last hypothesis. However, MAb 4G2 reacted with a conformational domain ranging from amino acids 298 to 397 (F. Megret, unpublished results), a region which does not contain a N-glycosylation site. The 3 other MAbs were not mapped and we do not know their recognition

FIG. 3. Visualizaton of DEN-2 E protein by indirect immunofluorescence. Virus-infected Sf9 or C6-36 cells were harvested 24 hr postinfection and fixed with acetone (A) or unfixed (B). then incubated sequentially with anti-E MAb 3H5 and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody. 1, wild AcNPV-infected Sf9 cells; 2, recombinant Ac-D2SEmV-infected Sf9 cells; 3, recombinant AcD2SEsV-infected Sf9 cells; 4, DEN-2Winfected C6-36 cells.

445

SHORT COMMUNICATIONS TABLE 1 IMMUNOREACTIVIT~TESTEDBYIMMUNOFLUORESCENCEOFTHE GLYCOPROTEINSEXPRESSEDINSFS 0~05-36

DENGUE CELLS

MAb

Specificity

AcNPV

Ac-D,SEmV

AC-D,SEsV

D,V

4E11 4D2/5 7Cl 7Dl 7El 6C12 2G4 4G2 1664 13G9 167 9D12 7c3 13B7 lH6 6B6 3H5 D21Fl 4E5 2H3 3F6

G/NT” G/NT G/NT G/NT G/NT G/NT G/NT G/NT GlGlSG/NT C/NT Clc/TINT T/NT T/NT T/NT T/NT T/NT Tl-

-b

++ + + + + +/+/-

t++ + + + ++ +/+I-

++ ++ -

++ +++ -

+ + ++ -t+ + +I+/+

++ ++ ++ ++ ++ +I+I+

++ ++ +++ +++ +++ ++ +++ +++ +++ +++ ++ +++ ++ ++ + ++ +++ + ++ ++ +

-

the amount of intracellular to extracellular Es protein was about 3 (data not shown). We also investigated the influence of oligosaccharide processing on the secretion of DEN Es protein from the Sf9 cells. Ac-D2SEsV-infected cells were radiolabeled with [35S]methionine in the absence or the presence of tunicamycin, and the intracellular and extracellular fractions were analyzed by immunoprecipitation using MAb 3H5 (Fig. 4). When compared to the untreated product, the 54-kDa intracellular Es protein showed a reduced mobility to 50 kDa in the presence of the tunicamycin inhibitor and was not secreted. On the other hand, authentic intracellular DEN-2 virus E glycoprotein was reduced in size by 4000 Da (data not shown). To investigate the possibility of modifications in the glycosylation pathways during Es translocation, labeled immunoprecipitated intracellular and extracellular proteins were tested by digestion with endoH and endof. EndoH (endo-@I-acetyl-b-glucosaminidase H)

D2V

I

-M-T EEMEMEMEM

AeD2SEsV

EndoH EndoF --

I Tm

Note. Sf9 cells infected either with wild-type AcNPV or with recombinants AcD2SEmV and AcD2SEsV were treated for immunofluorescence 24 hr p.i. using 21 monoclonal antibodies (MAbs). C6-36 cells infected with DEN-2V (DZV) were used as positive control. a MAbs were of group (G), subgroup (SG), complex(C), and type(T) specificity and exhibited neutralizing (NT) or no neutralizing (-) activity. b Relative intensity of fluorescence: +++, strong reaction; ++, good reaction; f, moderate reaction; +/-, weak reaction; -, no reaction,

site. Further characterization of the MAbs is needed to confirm our tentative interpretation. The kinetics of processing and secretion in Sf9 cells infected with AcD2SEmV and AC-DZSEsV revealed that Em and Es proteins were detected by 12-l 6 hr p.i. and reached a maximum level at 16-28 hr p.i. (data not shown). The analysis of extracellular fraction of recombinant baculovirus-infected cells detected no Em protein, but, in contrast, Es protein was present in the supernatant fluid by 16-20 hr p.i. and reached a maximum level at 24-32 hr p.i. (data not shown). The amount of DEN virus-specific E protein in recombinant Sf9 cultures was estimated by comparison with serial known amounts of authentic DEN-2 envelope protein from purified DEN virus, as described elsewhere (19). We estimated that the yield of expressed Em and Es proteins in recombinant baculovirus-infected cells 48 hr p.i. was of the order of 3 to 6 pug and 5 to 10 pg of protein per 10” cells, respectively, and that the ratio of

attached to recombinant FIG. 4. Analysis of the carbohydrate DEN-2 Es protern by using endoglycosidase H (EndoH), endoglycosidase F (EndoF), and tunicamycin (Tm). Sf9 cells infected at a m.o.i. of 10 PFU per cell with recombinant AC-D2SEsV were labeled with 100 PCi of [?Jmethionine from 20 to 24 hr postinfection, and cell extract (E) or medium (M) was prepared. Cells were lysed in 1 ml of RIPAbuffer(lOmMTris,pH7.5, l%NaDOC, l%NP40.0.1%SDS, and 1 mM phenylmethylsulfonyl fluoride) per 1O6 cells. Dengue-specific proteins from 1O6 infected cells and corresponding supernatant fractions were precipitated wrth 1 ~1 of DENZ-specific MAb 3H5 and Incubated overnight at 4O. The precipitates were collected on protein A-Sepharose beads by an incubation with 50 ~1 of beads at room temperature for 1 hr. Purified recombinant DEN-2 Es glycoprotein was subjected to EndoH or EndoF treatment or mock-treated (M-T). To do this, the immunoprecipitated E proteins were recovered from protein A-Sepharose beads by boiling 5 min in 0.1% SDS and 0.1 M P-mercaptoethanol in water. The eluates were diluted threefold with 50 mM sodium phosphate, pH 5.5, and incubated overnight at 37” with 20 mu/ml endoH or 1 U/ml endoF (9). The effect of tunicamycin on synthesis and secretion of Es protean in Sf9 cells was analyzed by an additionof 5 fig/ml of the drug in the medium during 2 hr of methionine star\/ing and 4 hr of labeling periods. C6-36 cells were infected with DEN-2V (D2V) and were collected 36 hr postinfection. Samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis and fluorography.

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removes only high-mannose N-linked core sugars, leaving one molecule of asparagine-linked N-acetylglucosamine, while endoF (endolycosidase F) cleaves complex and hybrid glycans as well as endoH-resistant Man3GlcNAc2 core from glycoproteins (20). Figure 4 shows that intracellular Es protein was partially sensitive to endoH and fully sensitive to endof, resulting in a protein migrating similarly to the tunicamycin-treated protein. In contrast, extracellular Es protein was clearly resistant to endoH relative to untreated or endof-deglycosylated products. Partial endoH resistance to intracellular Es protein suggests the presence, perhaps at the cell surface, of secretable protein from its endoHsensitive precursor. This is consistent with the indication that the nonsecreted Em protein is fully sensitive to endoH treatment (data not shown). Our results are also consistent with previous well-documented findings concerning the abolishment of protein secretion after tunicamycin treatment, and the processing of N-linked oligosaccharide to a state of endoH resistance (9). Moreover, the extracellularform of Es was smaller than the cellular form probably due to the cleavage of the terminal mannose group prior to release. Conversely, the electrophoretic mobility of the endof-treated form of secreted Es protein was slower than cell-associated forms after endoF or tunicamycin treatment. These results lack satisfactory explanation and further studies are needed to determine the processing of the oligosaccharides. We have demonstrated in this study that in vitro alteration of the DEN E protein C-terminal hydrophobic domain leads to the surface expression and secretion of a glycoprotein by SfS-recombinant baculovirus-infected cells. Our results suggest that the structural features involved in membrane anchoring are present within a hydrophobic peptide of 71 amino acids constituting the C-terminus of the protein E. It has been shown in previous studies that a C-terminal truncation engineered in the DEN-4 specified E glycoprotein to delete 39 amino acids of the putative hydrophobic anchor did not result in its secretion from recombinant vaccinia virus-infected cells (21). This discrepancy may be due to the presence of remaining internal hydrophobic sequences in E involved in its adherence to membranes, which were removed by our construction for Es secretion. Nevertheless, the possibility that the differences observed may be linked to the properties of the expression vector and the host cells cannot be excluded. Moreover, we have shown that glycan residues of secreted Es glycoprotein were processed in Sf9 cells to an endoH-resistant form. Failure to detect C and prM proteins in infected cells is not understood. It has been shown that the capsid protein is matured by the release from the membrane

by a putative viral nonstructural peptidase (5). In the absence of such a protease, C protein may remain anchored in the membrane via its hydrophobic C-terminus, which functions as a signal peptide in prM translocation, and could rapidly be degraded. Protein prM was expressed only at a low level in the early stages p.i. in Sf9 cells infected with recombinant baculovirus expressing JEV structural proteins (IO), but was not detected in cells infected with recombinant baculovirus expressing DEN-4 structural proteins (22). It has been suggested that prM may be rapidly cleaved into M protein or degraded by cellular enzymes (10). Further investigations are needed to establish the nature of prM processing in insect cells. We have demonstrated that the MAbs which exhibit type-specificity and neutralizing activity recognized the Es protein expressed in Sf9 cells. Moreover, the DEN recombinant Es protein is synthesized at a higher level than the membrane-anchored Em protein, it seems to be more soluble than authentic E protein, and it can be easily purified (M6gret and Deubel, manuscript in preparation). In another study, this protein will be investigated as a diagnostic antigen and as a vaccine subunit.

ACKNOWLEDGMENTS We thank Thuy Nang Vu for helping with the engineering of the D,SEs gene, Michsle Bouloyfor providing baculovirus-purified DNA, and Philippe Despres for helping with the study of protein glycosylation. The generosity of David H. L. Bishop in providing the pAcYM1 vector and of Kenneth H. Eckels in providing some monoclonal antibodies is gratefully acknowledged. We thank LBon Rosen for advice on the preparation of the manuscript. This investigation was supported partly by Pasteur Vaccins, Contract IP/PV dengue 2; partly by the Direction des Recherches Etudes et Techniques, Grant 88/230; and partly by the World Health Organization, Programme for Vaccine Development, Grant V22/181/18.

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