Altering the surface properties of baculovirus Autographa californicaNPV by insertional mutagenesis of the envelope protein gp64 Alexandra Spenger, Reingard Grabherr, Lars To¨llner, Herm
Trang 1Altering the surface properties of baculovirus Autographa californica
NPV by insertional mutagenesis of the envelope protein gp64
Alexandra Spenger, Reingard Grabherr, Lars To¨llner, Hermann Katinger and Wolfgang Ernst
Institute of Applied Microbiology, University of Agricultural Sciences, Vienna, Austria
The envelope protein gp64 of the baculovirus Autographa
californicanuclear polyhedrosis virus is essential for viral
entry into insect cells, as the glycoprotein both mediates
pH-dependent membrane fusion and binds to host cell receptors
Surface modification of baculovirus particles by genetic
engineering of gp64 has been demonstrated by various
strategies and thus has become an important and powerful
tool in molecular biology To improve further the
presen-tation of peptides on the surface of baculovirus particles,
several insertion sites within the gp64 envelope protein were
selected by their theoretical maximum surface probability
and investigated for efficient peptide presentation The
ELDKWA peptide of the gp41 of HIV-1, specific for the
human mAb 2F5, was inserted into 17 different positions of
the glycoprotein gp64 Propagation of viruses was successful
in 13 cases, mutagenesis at four positions did not result in production of intact virus particles Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display
Keywords: Autographa californica nuclear polyhedrosis virus; baculovirus; ELDKWA epitope; gp64 envelope pro-tein; surface display
The baculovirus Autographa californica nuclear
polyhedro-sis virus has been widely used as an expression system for
eukaryotic proteins in insect cell culture [1,2], for surface
display of various peptides and proteins [3,4], and more
recently, for the transduction of mammalian cells [5–8]
Baculoviruses are large, enveloped, double-stranded DNA
viruses that replicate in the nuclei of insect cells; their
infection cycle has been studied in detail [9–12] The major
envelope protein gp64 consists of 512 amino acids [13] with
a signal peptide at the N terminus, which is responsible for
targeting the glycoprotein to the cell plasma membrane, and
a hydrophobic transmembrane domain near the C terminus
[14] Transcription is regulated by a biphasic promoter,
resulting in synthesis peaks 12 and 24 h post-infection
[15,16] After viral DNA replication and late gene
expres-sion, nucleocapsids assemble in the nucleus and migrate
through the cytoplasm to the plasma membrane, where
gp64 is concentrated The glycoprotein is then acquired by
baculoviruses as they bud through the plasma membrane [17]
Gp64 forms typical peplomeric structures consisting of three homomeric polypeptide chains linked via disulfide bonds Oligomerization occurs post-translationally inside the endoplasmic reticulum, and misfolded gp64 fails to accumulate at the cell surface Glycosylation appears to occur cotranslationally and may be the rate-limiting step in the process of gp64 maturation and transport [16] Gp64 is essential for viral entry into the host cell, as it mediates cell receptor-binding activity as well as pH-dependent mem-brane fusion [18–20] It has further been shown that gp64 is required for cell-to-cell transmission of infection in cell culture and for efficient virion budding [19,21] Although an oligomerization and a fusion domain [22] as well as a transmembrane domain [14] have been identified, no data about the X-ray crystal structure of gp64 exist However, insights about the surface structure and function of the baculovirus major envelope protein are of high relevance as baculoviruses have been demonstrated to be a valuable tool for surface display techniques, providing novel strategies for ligand screening [23], antigen display [24] and altering the viral host range [25,26] Besides the possibility to display proteins as fusions to a second copy of gp64 or its membrane anchor sequence, Ernst et al [23] have described
a novel strategy for efficient peptide display on the surface
of baculoviruses by engineering peptides directly into the native envelope protein gp64 Thereby, no duplication of the gp64 is necessary to target the foreign peptide to the surface of baculoviruses because each copy of the gp64 contains the target sequence, providing high avidity of inserted peptide By efficiently displaying specific epitopes
on the viral surface, it becomes possible to modify baculoviral tropism, e.g for specific mammalian cell transduction, and also to consider baculoviruses as an
Correspondence to W Ernst, Institute of Applied Microbiology,
University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna,
Austria Fax: +43 13697615, Tel.: +43 136006 6242,
E-mail: W.Ernst@iam.boku.ac.at, URL: http://www.boku.ac.at/
iam/baculo/
Abbreviations: AcMNPV, Autographa californica multicapsid
nuclear polyhedrosis virus; FCS, fetal calf serum; X-Gal,
5-bromo-4-chloro-3-indolyl-b- D -galactoside; m.o.i., multiplicity of infection;
d.p.i., days post-infection; h.p.i., hours post-infection; AP, alkaline
phosphatase; PO, peroxidase; BCIP, 5-bromo-4-chloro-3-indolyl
phosphate; NBT, nitro blue tetrazolium; FITC, fluorescein
isothiocyanate.
(Received 17 May 2002, revised 11 July 2002,
accepted 25 July 2002)
Trang 2effective antigen presenting vehicle To optimize further the
presentation of foreign peptides on baculovirus particles we
screened additional positions throughout the gp64 coding
region for insertion of a specific peptide epitope, without
removing virally encoded amino acid residues We
gener-ated mutations carrying the peptide ELDKWA [27], specific
for the human mAb 2F5 [28,29], at positions with high
theoretical maximum surface probability, as determined by
computer analysis [30] (Figs 1 and 2) Propagation of
viruses was successful in 13 out of 17 cases Titres of
recombinants ranged from 107to 108plaque forming units (p.f.u.)ÆmL)1 In some cases we observed smaller plaque phenotypes indicating a decreased viability Surface local-ization of the inserted peptide was demonstrated by flow cytometry, Western blot analysis and ELISA By increasing the binding capacity of a foreign peptide to its ligand, we were successful in providing an improved tool for applica-tions, wherever modifications of the baculovirus surface properties are of importance
M A T E R I A L S A N D M E T H O D S
Construction of transfer plasmids Cloning procedures were performed according to Sam-brook et al [31], restriction enzymes and other modifying enzymes were purchased either from Roche Diagnostics (Mannheim, Germany) or MBI Fermentas (St Leon-Rot, Germany) and used according to the manufacturer’s recommendations For PCR we used the DyNAzymeTM
EXT DNA polymerase from Finnzymes (Espoo, Finland) The plasmid used for homologous recombination at the gp64 locus was p64flank [23], which contains the 7.576-bp HindIII fragment A2 of Autographa californica multi-capsid nuclear polyhedrosis virus (AcMNPV) including the whole gp64 ORF (Entrez-Protein number NP_054158) [32] For generation of mutant vectors with insertion of the ELDKWA motif at different positions in the gp64, different cloning strategies were developed The peptides were inserted into the baculoviral envelope protein without removing viral residues
The plasmids pELD31, 43, 59, 148, 180, 234 were constructed by inserting an altered NotI fragment into the p64flank treated with NotI The altered NotI fragments were produced by two PCR reactions, amplifying two fragments using 64-(-639)-NotI-back and a 64-ELD-SacI-for primer for one reaction and a 64-ELD-SacI-back and 64-993-for primer for the second, the template was p64flank in both cases After digestion with SacI the two fragments were ligated and another PCR was carried out with the primers 64-(-639)-NotI-back and 64-939-for The PCR product was digested with NotI and after agarose gel electrophoresis the insert was ligated into NotI-treated p64flank Primers used for construction of pELD31, 43, 59, 148, 180 and 234 are listed in Table 1 (section A)
Plasmid constructs for ELDKWA-epitope insertions at codon 277, 276, 275, 274, 271 of gp64, were made as described by Ernst et al [23] for pELD278 PCRs were performed with 64-(-639)-NotI-back and primers 64-277-ELD-NotI-for, 64-276-64-277-ELD-NotI-for, 64-275-ELD-NotI-for, 64-274-ELD-NotI-for and 64-271-ELD-NotI-for, respectively (Table 1, section B) Purified NotI-digested fragments were inserted into the NotI-treated vector p64flank thereby substituting the excised fragment for the particular gp64 mutant construct (pELD277–pELD271)
The inserts for pELD279, pELD280, pELD281, pELD282, pELD283, pELD290 were generated by ligation
of two fragments made by PCR with primers 394-SacII-back and ELD-SacI-for and ELD-SacI-394-SacII-back and 64-1536-BamHI-for (Table 1, section C) The two fragments were treated with SacI, ligated, and afterwards another PCR was carried out with the outer primers 64-SacII-394-back and 64-1536-BamHI-for This PCR fragment was
Fig 1 Schematic map of the gp64 envelope protein The AcMNPV
gp64 ORF encodes a protein of 512 amino acids, with an N-terminal
signal peptide (L), a hydrophobic transmembrane domain (TM) and a
cytoplasmic tail domain at the C terminus (CTD) In addition two
functional regions have been characterized, a fusion domain (F) and an
oligomerization domain (O) within a helical region (Helix) [22] Gp64
contains five predicted N-glycosylation sites (Y) The epitopes of two
mAbs are indicated: B12D5 epitope from amino acid 277 to amino
acid 287 [22,35] and AcV5 epitope from amino acid 431 to amino acid
439 [22,36] Insertion of the ELDKWA peptide at different positions
within the gp64 is indicated by numbers corresponding to the position
of amino acids in the gp64 envelope protein Modification at positions
marked with asterisks did not succeed in production of progeny virus.
Fig 2 Amino acids of gp64 flanking the inserted peptide ELDKWA.
Numbers correspond to the amino acid position in the gp64 envelope
protein (Entrez-Protein number NP_054158) [32] The peptide was
inserted into the native envelope protein without the removal of viral
amino acids.
Trang 3digested with SacII and ApaI and inserted into p64flank
treated with SacII and ApaI replacing the corresponding
wild-type fragment in p64flank
The insertions into the gp64 sequence were confirmed by
screening of bacterial colonies by PCR and subsequent
analysis of the fragments by 1.5% agarose gel
electrophor-esis Primers used for amplification were 64-727-back and
64-939-for (for pELD271, pELD274, pELD275, pELD276,
pELD277, pELD279, pELD280, pELD283, pELD290), 64-53-back and 64-206-for (for pELD31, pELD43, pELD59), 64-422-back and 64-568-for (pELD148, pELD180), 64-639-back and 64-799-for (for pELD234) (Table 1, sectionD) All PCR fragments were compared to the corresponding wild-type fragment derived by amplifications using the above primer combinations and p64flank as template In addition
we sequenced 600-bp fragments of mutant p64flank, from
Table 1 Primers used for construction of gp64 mutants (A) Primers for mutagenesis at amino acids position 31, 43, 59, 148, 180 and 234 (B) Primers used for plasmids pELD271, pELD274, pELD275, pELD276 and pELD277 (C) Primers for plasmids pELD279, pELD280, pELD281, pELD282, pELD 283 and pELD290 (D) Primers used for screening and sequencing.
A
64-(-639)-NotI-back 5¢-CGGGTTGGCGGCCGCATCGTTGCTATGAACG
64-31-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGCCGTACAAGATTAAAAACTTGGAC
64-31-ELD-SacI-for 5¢-GATGACGAGCTCACCCGTCTTCATTTGCGCGTTGC
64-43-ELD-SacI-back 5-GATGACGAGCTCGACAAATGGGCGAAGGAAACGCTGCAAAAGGAC
64-43-ELD-SacI-for 5-GATGACGAGCTCGGGCGGGGTAATGTCCAAG
64-59-ELD-SacI-back 5-GATGACGAGCTCGACAAATGGGCGTACAACGAAAACGTGATTATCGG
64-59-ELD-SacI-for 5-GATGACGAGCTCGTCCGTCTCCACGATGGTG
64-148-ELD-SacI-back 5-GATGACGAGCTCGACAAATGGGCGAATAACAATCACTTTGCGCACC
64-148-ELD-SacI-for 5-GATGACGAGCTCCTGCCGCTTCACCAACTCTTTG
64-180-ELD-SacI-back 5-GATGACGAGCTCGACAAATGGGCGACGGACGAGTGCCAGGTATAC
64-180-ELD-SacI-for 5-GATGACGAGCTCGTCGTCCTGGCACTCGAGC
64-234-ELD-SacI-back 5-GATGACGAGCTCGACAAATGGGCGAAAAATAACCCCGAGTCGGTG
64-234-ELD-SacI-for 5-GATGACGAGCTCGTCATCTTTAATGAGCAGACACG
B
64-277-ELD-NotI-for 5¢-GATGACGATTGCGGCCGCTTCGCCCATTTGTCGAGCTCCTTGACTCGGTGCTCGACTTTG 64-276-ELD-NotI-for 5¢-GATGACGATTGCGGCCGCTTCTTCGCCCATTGTCGAGCTCGACTCGGTGCTCGACTTTGCG 64-275-ELD-NotI-for 5¢-GATGACGATTGCGGCCGCTTCTTGACCGCCCATTTGTCGAGCTCTCGGTGCTC
GACTTTGCGTTTAATG 64-274-ELD-NotI-for 5¢-GATGACGATTGCGGCCGCTTCTTGACTCGCGCCCATTTGTCGAGCTCGTGCTC
GACTTTGCGTTTAATGC 64-271-ELD-NotI-for 5¢-GATGACGATTGCGGCCGCTTCTTGACTCGGTGCTCGACCGCCCATTTGTC
GAGCTCTTTGCGTTTAATGCATCTGTTAAAC C
64-394-SacII-back 5¢-AACGAGGGCCGCGGCCAGTG
64-1536-BamHI-for 5¢-GCGGGATCCTTATTAATATTGTCTATTACGGTTTCTAATC
64-279-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGCCGCCCACTTGGCGCCAC
64-279-ELD-SacI-for 5¢-GATGACGAGCTCCCGCTTCTTGACTCGGTGC
64-280-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGCCCACTTGGCGCCACAACG
64-280-ELD-SacI-for 5¢-GATGACGAGCTCCGGCCGCTTCTTGACTCGG
64-281-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGACTTGGCGCCACAACGTTAG
64-281-ELD-SacI-for 5¢-GATGACGAGCTCGGGCGGCCGCTTCTTGAC
64-282-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGTGGCGCCACAACGTTAGAGC
64-282-ELD-SacI-for 5¢ GATGACGAGCTCAGTGGGCGGCCGCTTCTTG
64-283-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGCGCCACAACGTTAGAGCCAAG
64-283-ELD-SacI-for 5¢-GATGACGAGCTCCCAAGTGGGCGGCCGCTTC
64-290-ELD-SacI-back 5¢-GATGACGAGCTCGACAAATGGGCGTACACAGAGGGAGACACTGC
64–290-ELD-SacI-for 5¢-GATGACGAGCTCCTTGGCTCTAACGTTGTGGC
D
Trang 4base pair 10 to 610 (for pELD31, pELD43, pELD59,
pELD148, p180ELD) or from base pair 390 to 990 (for
pELD234, pELD271, pELD274, pELD275, pELD276,
pELD277, pELD279, pELD280, pELD283, pELD290)
Cells and viruses
Cell line Sf9 (Spodoptera frugiperda, CRL 1711; ATCC) and
AcMNPV were propagated at 27C in IPL-41 medium
(Sigma-Aldrich) supplemented with yeastolate and a lipid/
sterol cocktail containing optional 3% or 10% fetal calf
serum (FCS) Sf9Op1D stable transfected cells [33] were
cultivated in TNM-FH complete medium (Sigma-Aldrich)
containing 10% FCS and were used for propagation of
gp64 null virus (vAc64–) [19] vAc64–and the cell line SfOp1D
were both established in the laboratory of G Blissard
(Molecular Biology of Insect Viruses at the Boyce
Thomp-son Institute, Cornell University, Ithaca, NY, USA) and
kindly given to us
Viruses were isolated by plaque purification and amplified
using standard procedures [1] Budded viruses were prepared
by ultracentrifugation of supernatants [harvested 5 days
post-infection (d.p.i.)] over a 30% sucrose cushion Pellets
were resuspended in phosphate buffered saline (NaCl/Pi)
(8 gÆL)1 NaCl, 0.2 gÆL)1 KCl, 1.44 gÆL)1 Na2HPO4,
0.24 gÆL)1KH2PO4, pH 7.4)
Generation of recombinant viruses
Cloning procedures yielded a set of transfer plasmids that
encoded mutant forms of the AcMNPV gp64 coding
regions containing the sequence for the ELDKWA peptide
at different positions To generate recombinant viruses, Sf9
cells were plated in 25 mm2T-flasks (2· 106cells per flask)
and cotransfected with 100 ng Ac64– DNA and 500 ng
transfer plasmid by liposome-mediated transfection [34]
using the CellFECTINTMtransfection reagent (Life
Tech-nologies) Ac64–DNA, the parental viral DNA, lacking the
entire gp64 reading frame (it is substituted by a lacZ
expression cassette) was extracted and prepared from
SfOp1D-infected cells
Recombinant viruses were purified from the
transfec-tion supernatant by plaque assay After 5 days petridishes
were overlaid with agarose containing
5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal), for identification of
viruses lacking the lacZ expression cassette White
plaques were amplified, and to confirm the insertions
into the gp64 envelope protein, viral DNA was
prepared from infected Sf9 cells DNA fragments from
base 53 to base 939 were amplified by PCR and sequence
analyses were performed using primers 64-53-back and
64-939-for
Western blotting of infected cells and budded virions
Samples were prepared for the Western blotting analysis in
the following manner Sf9 cells were infected with a
multiplicity of infection (m.o.i.) of 10 and harvested 24 h
post-infection (h.p.i.), washed with NaCl/Pi and lysed in
1· sample buffer (100 mM Tris/HCl pH 6.8, 4% SDS,
0.2% Bromophenol blue, 20% glycerol, 200 mM
b-merca-ptoethanol) containing 2· 105 cells per 10 lL Budded
virus preparations were also diluted and mixed with sample
buffer resulting in 2 lg protein per 10 lL(determined with
a Bio-Rad Protein assay) Samples were heated to 95C for 10 min prior to SDS/PAGE (10% polyacrylamide) Proteins were transferred to a PVDF-Membrane (Bio-Rad) using a semidry transfer cell (Bio-(Bio-Rad) Membranes were blocked with 3% BSA in NaCl/Pi including 0.1% Tween-20 (TPBS) prior to gp64 and ELDKWA detection The native and the mutant gp64 proteins were probed with B12D5 mAb [35] (1 : 1000) or with AcV5 mAb [36] (1 : 1000) After several washing steps with TPBS, mem-branes were incubated with goat anti-(mouse IgG) alkaline phosphatase (AP) conjugate (Sigma) diluted 1 : 1000 Reactive bands were detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro blue tetrazolium (NBT) as substrate The inserted epitope ELDKWA was detected by human mAb 2F5 (1 lgÆmL)1) and goat anti-(human IgG) AP conjugate (Sigma; 1 : 1000) followed by detection with BCIP and NBT Molecular masses were estimated by comparing the reactive bands with the bands from a prestained high range molecular mass marker (Bio-Rad)
FACS analysis of infected cells For FACS analysis 106cells were infected in 6-well plates at
an m.o.i 10 and harvested 24 and 48 h.p.i After washing with NaCl/Pi cells were probed with specific antibodies B12D5 (1 : 100) and 2F5 (10 lgÆmL)1) diluted in NaCl/Pi
containing 10% FCS for 1 h at room temperature Cells were washed with 1 mL NaCl/Piand after centrifugation cells were resuspended in goat anti-(mouse IgG) fluorescein isothiocyanate (FITC) conjugate (Sigma) (for B12D5 pre-treated samples) or goat anti-(human IgG) FITC conjugate (Sigma) (for 2F5-treated samples) diluted 1 : 50 in NaCl/Pi containing 2% FCS After 1 h of incubation with FITC conjugates cells were washed again and resuspended in
300 lLNaCl/Pi containing propidium iodide (1 lgÆmL)1) for staining of dead cells Labelled cells were analysed on a FACS Calibur (Becton Dickinson) Data were analysed withCELLQUESTsoftware
ELISA of budded virions 96-well-plates MaxiSorpTM (Nunc) were pre-coated with
100 lL human mAb 2F5 (5 lgÆmL)1) in coating buffer (8.4 gÆL)1NaHCO3, 4 gÆL)1Na2CO3, pH 9.6) overnight at
4C The plates were washed with TPBS and preparations
of budded viruses harvested at both 3 and 5 d.p.i were added and serially diluted 1 : 2–1 : 128 starting with samples containing 2 lgÆmL)1 protein in dilution buffer (TPBS including 1% BSA) After incubation for 1 h at room temperature, the plates were washed with TPBS and
100 lLB12D5 mAb (1 : 1000 in dilution buffer) per well was added After a further 1 h of incubation at room temperature, the plates were washed and goat anti-(mouse IgG) peroxidase (PO) conjugate (Sigma) was applied as second antibody Plates were incubated for a further 1 h and after washing 100 lL of substrate (1 gÆL)1 1.2-o-phenylendiamindihydrochlorid in citrate buffer pH 5.0 containing 0.03% H2O2) was added to each well The reaction was stopped by the addition of 1.25MH2SO4and the product was measured at 492 nm in a multichannel photometer (EAR 400 AT, SLT)
Trang 5R E S U L T S
Construction of ELDKWA mutant viruses
The peptide epitope ELDKWA [27] derived from the HIV-1
glycoprotein gp41 specifically binds to the human mAb 2F5
[28,29] This ligand binding interaction served as a model to
identify and compare several positions within the
baculo-virus major envelope protein for surface accessibility (Figs 1
and 2) Recombinant viruses were generated by
cotransfec-tion of Sf9 cells with transfer plasmids containing the
modified gp64 coding sequence and Ac64– DNA, a
baculovirus mutant where gp64 had been deleted by
insertion of a b-galactosidase expression cassette [19]
Thereby, only peptide insertions that maintained the
functionality of gp64 and thus, had restored the deletion,
produced virus progeny Amplification of ELDKWA
presenting viruses was successful in most cases The four
out of 17 positions that did not tolerate insertion of the
short peptide were amino acids 31, 43, 59 and 148 The fact
that no infectious virus could be generated in these four
cases was tested by repeated cotransfection of viral Ac64–
DNA together with the corresponding transfer plasmid into
Sf9 cells In addition, subsequent amplification of
transfec-tion supernatant did not lead to the productransfec-tion of virus
progeny either Visual examinations of Sf9 cells following
transfection and amplification over a period of more than
14 days did not show any signs of virus growth Successful
virus propagation could be achieved for insertions at
position 180, 234, 271, 274, 275, 276, 277, 279, 280, 281,
282, 283 and 290 These mutant viruses were amplified and
characterized further
Virus growth
After transfections, plaque assays were performed to isolate
single virus clones Mutants which had substituted the
b-galactosidase expression cassette were identified by
over-laying plates from plaque assays using X-Gal-containing
agarose Recombinants with a white phenotype were used
for further amplification Sequence analysis of PCR
prod-ucts from these mutant viruses confirmed the correct
insertion of the ELDKWA peptide into the gp64 gene To
observe the growth of viral mutants on Sf9 cells, viruses
were amplified in two successive steps and subjected to
plaque assay for determining plaque forming units per mL
(p.f.u.ÆmL)1) Each viral stock was titrated twice and
medians of these two analyses are shown in Table 2 Titres
of the recombinants ranged from 5· 106 to 1.7· 108
p.f.u.ÆmL)1 The lowest number of p.f.u in viral stocks
contained virus AcELD180; its titre was 30 times lower
than wild-type virus titre Most viruses reached titres of
between 2· 107 and 8· 107 p.f.u.ÆmL)1 Insertions at
positions 234 and 275 had no effect on viral growth,
their stocks showed titres like that of wild-type AcMNPV
(1–2· 108p.f.u.ÆmL)1) In some cases we observed smaller
plaque phenotypes, indicating a decreased viability [26]
Stability of AcELD283
Because the ELDKWA peptide is inserted into the native
envelope protein gp64, all viruses produced contain the
insertion in their envelope protein In order to investigate
the generation of progeny virions over multiple passages, we sequentially passed one representative mutant, AcELD283, through Sf9 cells by infection of 107cells with 500 lL of the previous virus stock and thereby produced five successive virus stocks These stocks were examined for virus titre and surface expression of the ELDKWA peptide on infected cells using FACS (Fig 3) For FACS, triplicate infections of
106cells were performed, using 500 lL of each virus stock, which corresponds to an m.o.i ranging from 10 (AcELD283 stock 5) to 20 (AcELD283 stock 2) Titres remained similar over five passages (Table 3); in addition,
as concluded from FACS histograms the amount of ELDKWA on the surface of infected cells only varied slightly approving the stable insertion of the ELDKWA peptide into the native envelope protein gp64 (Fig 3A) Staining with B12D5 mouse mAb showed no binding demonstrating the disruption of the epitope of this mouse mAb and the absence of any wild-type gp64 (Fig 3B) Expression of recombinant gp64 in infected insect cells The expression levels of recombinant gp64 were first compared using Western blot analysis Sf9 cells were infected
at an m.o.i of 10, harvested 24 h post-infection, and subjected to SDS/PAGE After blotting, the membranes were probed either with the mouse mAb B12D5 specific for gp64 (Fig 4A), or with the human mAb 2F5, which recognizes the ELDKWA epitope (Fig 4B) It was shown that gp64 expression levels of recombinants were comparable
to gp64 levels expressed by wild-type AcMNPV (Fig 4A) Constructs with insertions at positions 279–283 showed no or only weak reactive bands indicating that modification at these positions leads to disruption of the specific epitope Monsma and coworkers [22] have mapped the mouse mAb B12D5 epitope to 11 amino acids spanning positions 277–287 of the AcMNPV gp64 sequence Our experiment revealed that amino acids 277 and 278 are not required for antibody binding By using a Western blot stained with 2F5
we could demonstrate that all constructs that yielded infectious progeny showed a reactive band at 64 kDa confirming the successful insertion of the ELDKWA peptide into the native envelope protein (Fig 4B)
Table 2 Virus stock titres of recombinant viral mutants and wild type AcMNPV determined by plaque assay on Sf9 cells.
Titer (p.f.u.ÆmL)1)
Trang 6Presentation of the ELDKWA on surface of infected cells
The presentation of the inserted epitope ELDKWA on the
surface of infected cells was determined by FACS analysis
24 and 48 h.p.i For this purpose Sf9 cells were infected
three times, independently with an m.o.i of 10 using 6-well
plates and 106 cells per well Fig 5 shows ELDKWA
detection using the human mAb 2F5 and goat anti-human IgG FITC conjugate Nearly all constructs gave a higher fluorescence signal than the construct containing the insertion at position 278, previously described by Ernst
et al [23], indicating a better presentation or exposition of the inserted peptides The relative fluorescence intensity determined by FACS was three to six times higher in the constructs AcELD180, AcELD271, AcELD274, AcELD283 and AcELD290 We also analysed cells
48 h.p.i for ELDKWA expression (data not shown) Results were similar to analyses 24 h.p.i., confirming that viruses AcELD180, AcELD271, AcELD274, AcELD283 and AcELD290 were the best candidates for surface presentation of the ELDKWA peptide
gp64 levels were measured by FACS using the mouse mAb B12D5 Ac64–, the gp64 knockout mutant served as negative control These results showed that most viruses expressed comparable amounts of gp64 Lower binding capacity was detected for constructs AcELD279 and AcELD280 Viruses AcELD281, AcELD282 and
Fig 3 Stability of AcELD283 Virus
AcELD283 was sequentially passed through
Sf9 cells and thereby five successive virus
stocks were produced (AcELD283/1–
AcELD283/5) These stocks were investigated
for ELDKWA expression and virus titre.
ELDKWA expression of infected cells was
investigated with FACS (A) Staining with
human mAb 2F5, specific for the ELDKWA
and anti-human FITC conjugate (histogram
1–4) (B) staining with mouse mAb B12D5,
which is specific for the viral glycoprotein gp64
and anti-mouse FITC conjugate (histogram
5–8) AcELD283 virus stocks are depicted in
blue, the AcMNPV control virus is shown in
black Selected histograms are representative
of three independent experiments.
Table 3 Virus stock titres of recombinant viral mutants and wild type
AcMNPV determined by plaque assay Results shown are the means of
two analyses Depending on the virus titre m.o.i ranged from 10
(AcELD283 stock 5) to 20 (AcELD283 stock 2).
Titer (p.f.u.ÆmL)1)
Trang 7AcELLD283 showed no binding to the mouse mAb B12D5
confirming the results obtained from Western blot analysis
Expression on viral particles
Recombinant viral constructs which gave the highest signals
of ELDKWA on the surface of virally infected Sf9 cells
were selected for further investigations These constructs
were AcELD180, AcELD271, AcELD274, AcELD281,
AcELD283 and AcELD290
The packaging and uptake of modified gp64 into the viral
particle was investigated by immunoblot analysis of budded
virus preparations Blots were probed with the gp64-specific
antibodies mouse mAb B12D5 and mouse mAb AcV5, and
the ELDKWA specific human mAb 2F5 (Fig 6A–C)
AcMNPV served as positive control for gp64 detection
Corresponding to the results obtained from infected cells
AcELD281 and AcELD283 showed no reactivity with
mouse mAb B12D5, but gp64 could be detected by using
mouse mAb AcV5 which binds to denatured gp64
recog-nizing amino acids 431–439 [22] (Fig 6A and B)
The binding capacity of ELDKWA was highest in viral constructs AcELD274, AcELD283 and AcELD290 (Fig 6C) For further analysis, virus preparations were subjected to ELISA plates precoated with human mAb 2F5 Detection of bound virus was done using mouse mAb B12D5 and anti-mouse IgG PO conjugate As mouse mAb B12D5 no longer recognized AcELD281 and AcELD283, these viruses could not be analysed Highest binding capacity was detected for AcELD274 (Fig 7)
D I S C U S S I O N
Modification of virus envelope proteins often results in the loss of infection as envelope proteins are required for spread
of infection In the baculovirus AcMNPV, the envelope glycoprotein gp64 is responsible for viral entry into the host cell, has receptor binding activity and is required for efficient budding of viral particles However, targeted surface modification of infectious particles is a desired goal in molecular biology, as this may result in novel presentation and delivery tools Baculovirus surface display holds a great potential for drug screening, investigations of protein– protein interactions, antigen presentation and altering cell tropism To exploit fully the possibilities of the baculovirus surface display system, it becomes necessary to understand and investigate the functional and structural domains of the baculovirus major envelope protein Insertional mutagene-sis by Monsma and coworkers [22] have previously revealed
an oligomerization domain, which is located within an alpha-helical region, and a fusion domain of gp64 Further,
a transmembrane domain has been mapped to the C terminus of the glycoprotein [14] Most attempts to display foreign proteins on the surface of baculovirus virions and infected insect cells were done by expressing the target protein as a N-terminal fusion to a second copy of gp64 [37– 39] Boublik et al [37] suggested that incorporation of the target protein was a result of co-oligomerization of the gp64 fusion proteins with wild-type gp64 Additionally, they
Fig 4 Western blot analysis of infected insect cells (A)
Immuno-staining of infected cells with mAb B12D5, which is specific for the
envelope protein gp64: 2 · 10 5
infected cells were used per lane Ac64– the gp64 deletion mutant (lane 2) showed no reactivity with B12D5.
Additionally, constructs AcELD279 to AcELD283 (lane 11, 12, 13, 15,
16) did not react with B12D5, indicating B12D5 epitope destruction by
insertion of ELDKWA Other recombinants (lane 3, 4, 5, 6, 7, 8, 9, 10,
17) and wild-type AcMNPV (lane 1, 14) showed binding to B12D5 (B)
Detection of ELDKWA epitope in the gp64 envelope protein with
human mAb 2F5: Reactive bands at 64 kDa confirm the expression of
ELDKWA presenting envelope protein AcMNPV and Ac64 – did not
react with 2F5 antibody.
Fig 5 FACS analysis of ELDKWA epitope in infected insect cells A total of 10 6 cells were infected with mutant viruses at an m.o.i 10 and harvested 24 h.p.i Three independent infections were made for each virus and stained separately with human mAb 2F5, which is specific for the ELDKWA epitope, and anti-human FITC conjugate A total of
10 000 events were measured for FACS analysis Dead cells stained with propidium iodide were gated out Uninfected Sf9 cells and cells infected with AcMNPV served as negative control for ELDKWA detection On the Y-axis the mean intensity of fluorescence for infected Sf9 cells, expressed as the median of fluorescence of three independent infections, is shown Error bars correspond to the SD of the medians of three independent infections.
Trang 8could not rule out the possibility that some fusions with
gp64 may affect virus growth These data suggest that levels
displayed on the surface of viral particles depend on the
amino acid sequence, the length of the insertion and
secondary structure If gp64 fusion proteins are not able to
build a secondary structure that allows oligomerization with
wild-type gp64, the viral particle presents only low levels or
even fails to display the target protein on the viral surface;
however, growth of the resulting viruses is not affected To
circumvent these problems of low level presentation on viral
particles, we established a method for display of target
peptides, that allows incorporation into the viral envelope
only when modification does not inhibit functional
homo-oligomerization of the gp64 envelope fusion protein [23], the
main basis for viral infectivity The strategy was to modify
the native envelope protein itself so that only modifications which do not affect the essential functions of the glycopro-tein would result in production of viral particles We could demonstrate that direct insertion into the native gp64 leads
to high avidity display of short peptides on the surface of virions and infected cells [23] To gain further insight into the structural properties of gp64 and to increase the accessibility of peptide insertions to their ligands, we inserted the peptide epitope ELDKWA, specific for the human mAb 2F5, at 17 different positions within the baculovirus major envelope protein Of these, 13 positions yielded infectious virus progeny Four insertions apparently were lethal, indicating that these positions (31, 43, 59 and 148) are located within an essential domain, that is either directly responsible for some specific interaction or is structurally important for the protein’s function
Presentation of the ELDKWA peptide was shown to be best in constructs modified at positions 274 and 283 In comparison, AcELD283 had better growth characteristics,
as virus titres and plaque phenotypes were comparable to those of wild-type AcMNPV The titre of AcELD274 was
10 times lower and plaques were considerably smaller In conclusion, efficient surface display of desired epitopes on the surface of baculovirus particles can be achieved, however, for the price of somewhat slower growth and lower virus titres N-terminal fusions into a second copy of gp64 have frequently been proven to be useful for efficient display of various proteins [37–39] on infected insect cells, however, N-terminal insertion into the singular wild-type copy of gp64 must not necessarily be expected to result in the production of infectious virus In the course of our research we generated viruses containing a streptag peptide
at the N terminus of the native envelope protein, which grew
up to 2· 107p.f.u.ÆmL)1, but ligand display on the virus surface was weaker than constructs containing the streptag
Fig 6 Western blot analysis of budded virions (A) Immunoblotting of
virus samples with gp64-specific mAb B12D5 Two lg protein
(determined with Bio-Rad Protein assay) were loaded per lane For
AcELD281 and AcELD283 (lane 6 and 7) no reactivity with B12D5
could be detected, confirming the disruption of the B12D5 epitope (B)
All recombinant viral mutants showed reactive bands at 64 kDa
probed with mAb AcV5, which recognizes amino acids 431–439 within
the gp64 envelope protein (C) Detection of ELDKWA epitope in viral
samples was carried out with human mAb 2F5 Lane 1 represents
AcMNPV as control Reactive bands of viral clones at 64 kDa are
marked by arrows.
Fig 7 ELISA of mutant viruses Purified ELDKWA-containing viri-ons (AcELD180, AcELD 271, AcELD274, AcELD278, AcELD281, AcELD283, AcELD290) and AcMNPV were applied to 2F5 pre-coated plates and bound virions were detected by the anti-gp64 specific mAb B12D5 and an anti-mouse peroxidase conjugate Two virus preparations, harvested 3 and 5 d.p.i., were analysed independently The mean optical density of these two analyses is plotted on the Y-axis Error bars correspond to the SD As the B12D5 has no reactivity with AcELD281 and AcELD283 ELDKWA surface levels of these con-structs could not be determined in this assay.
Trang 9at position 278 (data not shown) Hence, in this study
only insertions within the gp64 coding region and not a
N-terminal fusion construct were taken into account and
were investigated
The principle feasibility of linear peptide insertion could
be successfully demonstrated in this approach A general
validity of this concept can be concluded from ongoing
experiments where more complex structures were inserted
into selected sites 274 and 283 which had been identified
during the course of this project Further examples include
the insertion of a 17-amino acid epitope of mAb 3D6 [40],
containing a loop structure between two cysteine residues, at
position 274, and a 23-amino acid portion of the envelope
protein of the Foot and mouth disease virus into site 283 of
native gp64 (unpublished results) Both insertions were
compatible with the function of the gp64 envelope protein
as we concluded from viral titres and kinetics of
recombin-ant peptide expression on the surface of infected Sf9 cells
Having identified sites within the baculovirus major
envelope protein gp64, that allow insertions of target
peptides and provide efficient surface presentation without
loosing infectivity and/or normal propagation, these
candi-dates may serve for various useful applications in molecular
biology Surface presentation of relevant epitopes for in vivo
antigen presentation requires proper presentation and high
accessibility Peptides that bind certain mammalian cell
receptors or mediate cell entry through the membrane may
serve to improve baculoviral vectors for mammalian cell
transduction Evaluation of other peptides designed for
specific functions, e.g virus targeting to selected tissues,
extension of host range or enhancement of transduction
efficiency of nonpermissive cells are envisaged as future aims
and therefore would go beyond the scope of this present
study Also larger proteins could be presented on the
baculovirus surface, by fusion to protein which binds to the
epitope present in the major envelope protein Preliminary
experiments have shown that this strategy leads to a drastic
increase in avidity of proteins as compared to previously
described methods (unpublished results)
A C K N O W L E D G E M E N T S
The authors thank G Blissard for providing the cell line SfOp1Dand
gp64null virus vAc64– We thank R Voglauer and N Borth for FACS
analysis This project was funded by the FWF (Fonds zur Fo¨rderung
der wissenschaftlichen Forschung) project Nr P14538 MOB.
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