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Journal of Virology, November 1999, p. 9137-9144, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Replication-Defective Bovine Adenovirus Type 3 as
an Expression Vector
P. Seshidhar
Reddy,1,
Neeraja
Idamakanti,1
Yan
Chen,1
Tyler
Whale,1
Lorne A.
Babiuk,1
Majid
Mehtali,2 and
Suresh
Kumar
Tikoo1,*
Virology Group, Veterinary Infectious Disease
Organization, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada S7N 5E3,1 and Gene Therapy
Department, Transgene, S.A., 67000 Strasbourg,
France2
Received 30 March 1999/Accepted 26 July 1999
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ABSTRACT |
Although recombinant human adenovirus (HAV)-based vectors offer
several advantages for somatic gene therapy and vaccination over other
viral vectors, it would be desirable to develop alternative vectors
with prolonged expression and decreased toxicity. Toward this
objective, a replication-defective bovine adenovirus type 3 (BAV-3) was
developed as an expression vector. Bovine cell lines designated VIDO R2
(HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and
6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1
proteins) were developed and found to complement the E1A deletion in
BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most
of the E1A and E3 regions was constructed. This virus could be grown in
VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results
demonstrated that the E1 region of HAV-5 has the capacity to transform
bovine retina cells and that the E1A region of HAV-5 can complement
that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A
similar replication-defective vector expressing the
hemagglutinin-esterase gene of bovine coronavirus from the E3 region
was isolated. Although these viruses grew less efficiently than the
replication-competent recombinant BAV-3 (E3 deleted), they are suitable
for detailed studies with animals to evaluate the safety, duration of
foreign gene expression, and ability to induce immune responses. In
addition, a replication-competent recombinant BAV-3 expressing green
fluorescent protein was constructed and used to evaluate the host range
of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of
replication-defective BAV-3 vectors is a major technical advancement
for defining the use of BAV-3 as vector for vaccination against
diseases of cattle and somatic gene therapy in humans.
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INTRODUCTION |
Replication-defective, recombinant
adenoviral vectors are widely used for in vivo gene transfer because of
the ability of these vectors to enter many different target cells
efficiently and to express the transgene (6). Although these
vectors are effective with respect to the entry and expression
processes, expression of the transgene is transient (8). The
transient expression is explained at least in part by the host immune
responses, both innate and acquired, to cells transduced with
adenoviral vectors (37). To overcome the problem associated
with the preexisting immune responses, nonhuman adenoviruses have been
proposed as vectors for gene therapy (21, 29, 30, 38, 39,
42). Animal adenoviruses are species specific, can enter human
cells but do not replicate, and thus have great potential as gene
transfer vectors. In addition, these vectors could be used as live
viral vaccines in animals. Currently used recombinant adenovirus
vectors are made replication defective by deletion of E1A and E1B
sequences (4). The E1A region encodes the immediate-early
proteins that transactivate all other viral early region genes. By
deleting these sequences, the expression of all other early and the
late region genes is reduced to a large extent, resulting in lower immune responses to adenoviral proteins and thus in long-term expression of a transgene. In addition, deletion of the E1 region removes the oncogenic potential of the viruses.
Bovine adenoviruses (BAVs) belong to the Mastadenovirus
genus of Adenoviridae family. Currently, the accepted 10 serotypes of BAVs are divided into two subgroups on the basis of the
differences in their biological and serological properties
(3). Serotypes 1, 2, 3, and 9 belong to subgroup I and grow
relatively well in established bovine cell lines. We have been studying
BAV type 3 (BAV-3) with a goal of developing it as an expression
vector. BAV-3 was chosen for this purpose based on the lack of
virulence and the ability of the virus to grow to high titers in cell
culture. In addition, experimental infection of calves with BAV-3
failed to produce either clinical signs or gross lesions, but all
animals seroconverted (22). Molecular studies of the genome
are essential for the development of BAV-3 as an expression vector.
Determination of the complete nucleotide sequence and transcription map
of BAV-3 was recently reported (30). Nucleotide sequence
analysis of the E1 region of BAV-3 identified open reading frames for
proteins that are homologous to the E1A and E1B proteins of human
adenovirus type 5 (HAV-5) (11, 43). Transcription analysis
of the E1 region in BAV-3 has indicated that the transcripts of the
E1A, E1B, and pIX regions are 3' coterminal (28). The
proteins produced from the E1 and neighboring pIX region of BAV-3 were
also identified and characterized by using specific antibodies raised
in rabbits (28).
Among current methods for generating adenovirus-based vectors, the
Escherichia coli BJ5183 recombination system is the most simple and efficient (5, 29, 42). We applied this method to
clone the full-length BAV-3 genome as a stable infectious bacterial plasmid and to generate the replication-competent viral vector (42). In this paper, we describe generation of
E1A-complementing cell lines, E1A deletion mutants, and
replication-defective BAV-3 vectors. This is the first report, to our
knowledge, of the development of E1A-complementing cell lines and
construction of replication-defective BAV-3 expression vectors.
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MATERIALS AND METHODS |
Virus and viral DNA.
The WBR-1 strain of BAV-3 was
cultivated in Madin-Darby bovine kidney (MDBK) and VIDO R2 cells. VIDO
R2 is a transformed fetal bovine retina cell (FBRC) line expressing the
E1 proteins of HAV-5. The cells were grown in Eagle's minimum
essential medium supplemented with 5% fetal bovine serum. The viral
DNA was extracted from virus-infected cell monolayers by the method of
Hirt (17). Replication-defective HAV-5 expressing
-galactosidase (Ad5d1E1AlacZ) was propagated as described
previously (43).
Construction of recombinant plasmids.
The recombinant
plasmid vectors were constructed by standard procedures (31)
using restriction enzymes and other DNA-modifying enzymes as directed
by the manufacturers.
(i) Construction of plasmid pFBAV500.
Plasmid pTG5435,
containing the full-length BAV-3 genome in pPOLYIIsn14, was digested
with HindIII, and the restriction enzyme fragment
harboring the terminal fragments (nucleotides [nt] 1 to 1656 and
33232 to 34446) of BAV-3 was gel purified and self-ligated to produce
plasmid pBAV-101. Nucleotide numbers of the BAV-3 genome referred in
this report are according to GenBank accession no. AF030154. To delete
a part of the E1A sequence between the nt 536 and 1077, pBAV-101 was
digested with AccI and SpeI, and ends were blunt
repaired and dephosphorylated. To facilitate the cloning of foreign
genes, an XbaI linker was inserted into the deleted region,
and the resulting plasmid was designated pBAV-102. The deletion of the
E1A region was then rescued into the full-length infectious clone by
homologous recombination in E. coli BJ5183 between the
HindIII-linearized pBAV-102 and the genomic DNA from BAV3.E3d, a virus lacking the E3 region (42), creating
plasmid pFBAV500.
(ii) Construction of recombinant plasmid pFBAV501.
The gene
coding for glycoprotein D (gD) (33) of bovine herpesvirus
type 1 (BHV-1) under the control of the simian virus 40 (SV40)
immediate-early promoter, a 137-bp length chimeric intron, and SV40
late poly(A) signal was first cloned into the blunt-ended XbaI site of pBAV-102 to generate plasmid pBAV-102.gD. A
full-length plasmid (E1A-E3 deletion) with the gD gene in the E1A
region (pFBAV501) was generated by homologous recombination in E. coli BJ5183 between the HindIII-digested
pBAV-102.gD and the genomic DNA of BAV3.E3d (42).
(iii) Construction of recombinant plasmid pFBAV502.
The
genome of recombinant BAV333 (27) contains the
hemagglutinin-esterase (HE) gene under the control of SV40 early
promoter, a 137-bp chimeric intron, and SV40 poly(A) signal inserted in the E3 region of the BAV3.E3d genome (42). To construct a
replication-defective recombinant BAV-3 expressing the HE gene of
bovine coronavirus (BCV) from the E3 region of BAV-3, the genomic DNA
of recombinant BAV333 was recombined with
HindIII-digested plasmid pBAV-102 in E. coli
BJ5183 to create plasmid pFBAV502.
(iv) Construction of plasmid pFBAV304.
The green fluorescent
protein (GFP) gene under the control of the cytomegalovirus (CMV)
immediate-early promoter and bovine growth hormone poly(A) signal was
obtained from pQBI 25 (Quantum Biotechnologies) by BglII and
DraIII digestions followed by blunt ending with T4 DNA
polymerase. This fragment was first cloned into the SrfI
site of the E3 transfer vector pBAV-301 (plasmid constructed by
ligating the 7,635-bp KpnI-SspI fragment from
pFBAV302 to KpnI-NotI [T4 treated]-digested
plasmid PpolyIIsn14) in the same orientation as the E3 transcription
unit to generate pBAV301.gfp. A KpnI-SwaI
fragment encompassing the modified E3 region was isolated from
pBAV301.gfp and recombined with SrfI-digested pFBAV.302 DNA (42) in E. coli BJ5183, creating plasmid pFBAV304.
Isolation of recombinant BAV-3.
VIDO R2 cell monolayers in
60-mm-diameter dishes were transfected with 5 to 10 µg of
PacI-digested pFBAV500, pFBAV501, pFBAV502, and pFBAV304
recombinant plasmid DNAs by using Lipofectin (Gibco/BRL). After
incubation at 37°C, the transfected cells showing cytopathic effects
were collected and freeze-thawed twice, and the recombinant viruses
were plaque purified on VIDO R2 cells.
Western blot analysis.
Extracts of mock- or virus-infected
cells were resolved (5 µg per lane) by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a
nitrocellulose membrane (Bio-Rad). Nonspecific binding sites on the
membrane were blocked with 1% bovine serum albumin before incubation
of the blots with protein-specific monoclonal or polyclonal antibodies.
The membranes were washed and exposed to anti-mouse or anti-rat
immunoglobulin G conjugated to horseradish peroxidase or alkaline
phosphatase and developed by using a horseradish peroxidase or alkaline
phosphatase color development kit (Bio-Rad).
Immunoprecipitation.
Confluent monolayers of VIDO R2 cells
in six-well dishes were infected with the virus at a multiplicity of
infection (MOI) of more than 5. The cells were preincubated for 2 h in minimal essential medium free of methionine and cystine prior to
labeling with 50 µCi of [35S]methionine
(Tran35S-label; phosphate-buffered saline, 1,000 Ci/mmol; ICN Radiochemicals Inc., Irvine, Calif.) for 4 h. The
cells were washed once with phosphate-buffered saline, harvested by
scraping, and then lysed with ice-cold modified
radioimmunoprecipitation assay buffer. The radiolabeled proteins were
immunoprecipitated with a pool of anti-BHV-1 gD monoclonal antibodies
(MAbs) (18) or polyclonal anti-BCV rabbit antibodies
(9, 10) and analyzed by SDS-PAGE. The gels were dried, and
protein bands were visualized by autoradiography.
Infection of human and animal cells with BAV-3.
MDBK, VIDO
R2 (bovine), STR (swine testicular cells expressing the E1 proteins of
HAV-5), VIDO R1 (fetal porcine retina cell line expressing E1 proteins
of HAV-5), 293, A549, HeLa, HepG2 (human), Cos, Vero (monkey), C3HA
(mouse), sheep skin fibroblast, dog kidney, or cotton rat lung cells
were infected with more than 5 PFU of wild-type BAV-3 or BAV304 per
cell. The cells infected with wild-type BAV-3 were harvested at 2 h and 3 days postinfection, and the virus titers were determined in
MDBK cells. Cells infected with BAV304 were examined for green
fluorescence with the aid of a fluorescence microscope.
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RESULTS |
Development of MDBK cell line 6.93.9.
Our initial goal was to
develop an MDBK cell line expressing E1 proteins of BAV-3, as these
cells are commonly used for the propagation of BAV-3. MDBK cells are
also permissive to wild-type HAV-5 infection, and the available E1
deletion mutants of HAV-5 would allow us the selection of the clones
that are expressing E1 proteins of BAV-3 by cross-complementation
(43). MDBK cells were transfected with plasmid pVD-Neo,
containing a SalI B fragment of BAV-3 covering 0 to 23.4 map
units on the viral genome and a Neor marker under the
control of the long terminal repeat from Rous sarcoma virus. The
SalI B fragment contains all of the E1 region, pIX, IVa2,
and pol genes and part of the terminal protein (pTP) gene.
The Neor system permits the selection of cells that take up
the plasmid vector containing the Neor marker. Several
G418-resistant clones were isolated, expanded, and tested for the
ability to support the growth of Ad5dlE1AlacZ (43). Five clones supported the growth of an E1A deletion
mutant, and a clone (6.93.9) which supported the growth of the virus to the highest titer was selected and subjected to single-cell cloning. However, even though these cells supported the growth of an E1A deletion mutant, we could not detect E1 protein expression in any of
the clones including 6.93.9 by Western blotting using polyclonal antibodies specific to E1 proteins of BAV-3 (28). Clone
6.93.9 cells grew slowly, rapidly acidified the medium, and never
formed a confluent monolayer.
Development of FBRC line VIDO R2.
Since MDBK cells were not
useful for the generation of replication-defective BAV-3 vectors, we
attempted to develop an FBRC-derived cell line. Early-passage secondary
FBRCs were prepared by standard techniques. Two attempts to transform
FBRCs by the E1 region of BAV-3 (pVD-Neo) with or without G418
selection were not successful. As our experience with clone 6.93.9 indicated that the E1A region of BAV-3 could complement the E1A of
HAV-5, we assumed that the FBRCs transformed with the E1 region of
HAV-5 should also complement the E1 region of BAV-3. Human fetal retina
cells are efficiently transformed with the E1 region of HAV-5
(13). To develop a bovine cell line expressing the E1
protein of HAV-5, subconfluent monolayers of FBRCs were transfected
with plasmid pTG4671(pTG6559 [20] without the
E1blarge mutation) by the calcium phosphate technique. Plasmid pTG4671 contains the entire E1A and E1B coding sequences of
HAV-5 (nt 505 to 4034). The transcription of E1A is under the control
of the constitutive mouse phosphoglycerate kinase gene (PGK)
promoter, and transcription of the E1B region is under the control of
its natural promoter and a globin poly(A) signal. This plasmid also has
a selection marker, the puromycin acetyltransferase gene, under the
control of the SV40 early promoter and SV40 poly(A) signal. Several
morphologically transformed colonies were observed 4 weeks after
transfection without selection for puromycin resistance. The cells in
transformed foci showed altered morphology, smaller and rounded,
whereas the untransformed FBRCs were long and slender (Fig.
1). The transformed cells expressed
Vimentin, not cytokeratin, indicating that they are mesenchymal in
origin (data not shown). Transformed cell line VIDO R2 was established
from a separated foci and then subjected to single-cell cloning.
Initially, the expression of E1 in VIDO R2 cell line was analyzed by
reverse transcription (RT)-PCR using primers specific to E1A and E1B
regions of HAV-5. The PCR products generated using the E1 region DNA as templates were used as size controls (Fig. 1C, lanes 3 and 6). As seen
in Fig. 1C, HAV-5 E1A (lane 2) and 19-kDa E1B (lane 5) genes of
expected lengths were transcribed in VIDO R2 cells. The RNA samples
without reverse transcriptase added did not show any bands (lanes 1 and
4), indicating that genes were amplified from E1 mRNAs and not from
residual DNA.
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FIG. 1.
VIDO R2 cell line. Morphological features of
untransformed FBRCs (A) and transformed VIDO R2 cells (B). Cells were
stained with crystal violet and photographed at a magnification of
×100. (C) Analysis of ethidium bromide-stained RT-PCR products.
Products of RT-PCR using DNase-treated RNA (lanes 1, 2, 4, 5, and 7) or
of PCR using DNA (lanes 3 and 6) as a template were synthesized by
using primer pairs for E1A (sense, 5'-ATGAGACATATTATCTGCCA-3';
antisense, 5'-CTTACTGTAGACAAACATGC) (lanes 1 to 3) and
19-kDa E1B (sense, 5'-ATGTTTAACTTGCATGGCGT-3'; antisense,
5'-ATTCCCGAGGGTCCAGGCCG-3') (lanes 4 to 6). Lanes 1 (E1A
primers) and 4 (19-kDa E1B primers) shows RT-PCR of RNA without reverse
transcriptase; lane 7 shows RT-PCR using RNA from FBRCs. Sizes of
marker (M) DNA are shown in base pairs.
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For complementation of an essential viral protein by a cell line, the
protein of interest must be synthesized in sufficient
quantities by the
host cells. To determine the level of E1A proteins
produced by VIDO R2,
Western blot analysis was carried out. The
mouse monoclonal antibody
M73, which recognizes all HAV-5 E1A
proteins, and antibody 3D11
(Calbiochem), directed against the
19-kDa HAV-5 E1B protein, were used.
In VIDO R2, the levels of
E1A proteins produced were similar to or
little higher than those
produced in 293 cells (Fig.
2A). However, the amount of 19-kDa
E1B
protein produced was slightly less in VIDO R2 cells (Fig.
2B). Western
blot analysis using the same antibodies did not recognize
any proteins
from extracts from FBRCs (Fig.
2, lanes 2). Attempts
to detect the
HAV-5 E1B 55-kDa protein in 293 and VIDO R2 cells
in
immunoprecipitation assays using a rat MAb (DP 08; Cedarlane)
specific
to the protein were not successful.
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FIG. 2.
Western blot analysis of E1 proteins. Proteins from 293 cells (lane 1), FBRCs (lane 2), and VIDO R2 cells (lane 3) were
separated by SDS-PAGE (10% gel) under reducing conditions and
transferred to nitrocellulose. The separated proteins were probed in
Western blots by MAbs against HAV-5 E1A (A) and 19-kDa E1B (B). Sizes
are indicated in kilodaltons.
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To investigate the complementing properties of the VIDO R2, the cells
were infected with the E1A deletion mutant of HAV-5
(Ad5
dlE1AlacZ [
43]). This cell line
supported the growth of
the deletion mutant to 10
7 PFU/ml.
To determine whether the VIDO R2 cell line could support
plaque
formation, cells cultured in 35-mm-diameter dishes were
infected with
BAV-3 or HAV-5 and incubated in a CO
2 incubator.
Clear
plaque formation was evident on days 5 and 7 postinfection
with HAV-5
and BAV-3, respectively. We also observed a substantially
more rapid
onset of viral cytopathic effect in E1-expressing cell
lines than in
MDBK cells and FBRCs. In addition, VIDO R2 supported
the formation of
clear plaques by recombinant BAV-3.
Transfectability of E1-expressing cell lines.
To test the
ability of the cells to take up large DNA, MDBK, 6.93.9, and VIDO R2
cells in 35-mm-diameter dishes were transfected with 1 to 3 µg of
PacI-restricted plasmid pFBAV304 DNA (described below) by
using Lipofectin (GIBCO/BRL). This plasmid contains the entire BAV-3
genome with the E3 region replaced by a GFP gene under the control of a
CMV immediate-early promoter. When observed under a fluorescence
microscope 24 h posttransfection, more than 3% of VIDO R2 cells
showed fluorescence, as opposed to less than 0.1% cells in MDBK and
6.93.9 cultures. Further incubation of the transfected VIDO R2 cells
for 10 to 14 days resulted in the production of a recombinant virus
(named BAV304) expressing GFP. These observation suggest that VIDO R2
is a better cell line for the generation of recombinant BAV-3 due to
its greater transfection efficiency and/or the presence of HAV-5 E1A/B sequences.
Construction of E1A deletion mutant of BAV-3.
To construct a
replication-defective recombinant BAV-3, full-length BAV-3 genomic DNA
containing deletions in the E1A (nt 536 to 1077) and E3 (nt 26456 to
27701) regions was cloned in a plasmid named pFBAV500 (Fig.
3a). Plasmid pFBAV500 DNA digested with
PacI (to release the recombinant viral genome from the
plasmid) was used to transfect VIDO R2 cells. A recombinant virus
(named BAV500) with the E1A and E3 regions deleted was obtained 10 days following transfection. The virus was amplified in VIDO R2 cells, and
the viral DNA was extracted from infected cells. The DNA was analyzed
after digestion with restriction enzyme ClaI. The wild-type BAV-3 had ClaI fragments of 3.1, 6, and 7.5 kb (Fig.
4, lane 1), which were missing in the
recombinant BAV500 genome, which instead had fragments of 2.5 and 12.2 kb (lane 2). This is consistent with the expected ClaI
fragments of the wild type and the deletion mutant.
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FIG. 3.
Strategy used for the generation of recombinant BAV-3.
Different plasmids were constructed from different genomic clones as
described in the text. Origins of DNA sequences: plasmid DNA, thin
line; BAV-3 genomic DNA, hollow box; inverted terminal repeats, filled
box. The plasmid maps are not drawn to scale.
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FIG. 4.
Restriction enzyme analysis of the recombinant BAV-3
genome. (A) The DNAs were extracted from VIDO R2 cells infected with
BAV-3 (lane 1), BAV500 (lane 2), BAV501 (lane 3), or BAV502 (lane 4) as
described previously (17) and digested with ClaI.
(B) The fragments shown in panel A were transferred to Nytran membranes
and probed with  -32P-labeled gD and HE probes. Lane M, 1 Kb Plus DNA Ladder (Gibco/BRL) used for sizing the viral DNA
fragments.
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Construction of E1A deletion-based recombinants.
To determine
the usefulness of E1A-deleted (replication-defective) BAV-3
recombinants as gene delivery vehicles, we constructed recombinant
BAV-3 expressing BHV-1 gD (32) or BCV HE (26). The full-length gD gene [flanked by the SV40 early promoter, chimeric intron, and SV40 poly(A) signal] was inserted into the E1A region of
the BAV500 genome in the same transcriptional orientation as E1 (using
the homologous recombination machinery of E. coli), (5), creating plasmid pFBAV501 (Fig. 3b). Earlier, we
constructed a replication-competent recombinant BAV-3 (BAV333)
expressing BCV HE (27). To construct a replication-defective
recombinant BAV-3 expressing BCV HE, the full-length HE gene [flanked
by the SV40 promoter, chimeric intron, and SV40 poly(A) signal] was
inserted into the E3 region of the BAV500 genome in the same
transcriptional orientation as E3 (using the homologous recombination
machinery of E. coli, (5), creating plasmid
pFBAV502 (Fig. 3c). The PacI-digested pFBAV501 or pFBAV502
DNA was transfected into VIDO R2 cells to isolate a recombinant virus
named BAV501 or BAV502, respectively. BAV501 and BAV502 were amplified
in VIDO R2 cells, and viral DNAs were extracted from the infected
cells. The presence of foreign genes in viral genomes (gD in BAV501 and
HE in BAV502) was confirmed by ClaI restriction enzyme
analysis. The BAV500 genome had a ClaI fragment of 2.5 kb
(Fig. 4A, lane 2) that was missing in the recombinant BAV501 genome,
which instead had a fragment of 4.4 kb (Fig. 4A, lane 3). This
suggested that recombinant BAV501 contained a gD gene, a possibility
confirmed by Southern blot analysis (Fig. 4B, lane 3). As expected, the
12.2-kb BAV500 DNA fragment (Fig. 4A, lane 2) was replaced with a
14.1-kb DNA fragment in BAV502 (Fig. 4A, lane 4). This suggested that
recombinant BAV502 contains an HE gene in the E3 region, a
possibility confirmed by Southern blot analysis (Fig. 4B, lane 4).
To demonstrate that infection with the E1 mutant viruses is abortive in
noncomplementing cell lines, MDBK, FBRC, 6.93.9, and
VIDO R2 cultures
were used for infection studies. Cells were infected
at an MOI of less
than 1, grown for a week, subjected to two freeze-thaw
cycles, and
titrated on the VIDO R2 cells. Wild-type BAV-3 and
BAV-3.E3d (E3
deletion [
42]) grew to high titers (10
9
PFU/ml) in all cells tested, whereas the replication-defective
recombinant viruses (E1A-E3 deletion) grew only in the VIDO R2
(Fig.
5) and 6.93.9 (10
6 to
10
7 PFU/ml) cells. This reduction in titer of
replication-defective
BAV-3s could be due to lower than optimum level
of E1A expression
in cell lines or differences in the transcriptional
activation
property of different E1As in different cell lines, which
may
affect the efficiency of virus production. Alternatively, it is
possible that certain unspecified replication functions of bovine
E1A
cannot be efficiently replaced by HAV-5 E1A.
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FIG. 5.
Production of recombinant BAV-3 by VIDO R2 and FBRC
cells. Near-confluent monolayers were infected with less than 1 MOI of
wild-type or recombinant BAV-3. After a week, the cells were
freeze-thawed and the virus was titrated on VIDO R2 cells as described
in the text.
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Kinetics of expression of gD and HE.
The kinetics of gD
expression by recombinant BAV501 was determined at three different time
points postinfection in VIDO R2 and MDBK cells by immunoprecipitation
assay. Electrophoretic analysis of metabolically radiolabeled
immunoprecipitates from recombinant BAV501-infected VIDO R2 cells
lysates revealed immunoreactive proteins with approximate molecular
masses of 63 kDa (unglycosylated) and 71 kDa (glycosylated) (Fig.
6A, lanes 5 and 6). No corresponding proteins could be detected in mock (lane 1) or wild-type BAV-3 (lane
2)-infected cells. The molecular weight of the unglycosylated and
glycosylated gD corresponds to that of the authentic gD
immunoprecipitated from BHV-1-infected cell extracts (lane 3). In
BAV501-infected cells, expression of gD was first detected at 24 h
postinfection (lane 5) and continued to be produced up to 36 h
postinfection (lane 6), the last time point used in the study. Similar
kinetics of gD expression was observed in MDBK cells (Fig. 6B, lanes 5 and 6).
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FIG. 6.
Kinetics of gD expression by recombinant BAV-3. Proteins
from lysates of [35S]methionine-labeled mock-infected
(lane 1), BAV-3-infected (lane 2), BHV-1-infected (lane 3), or
BAV501-infected cells harvested at 12 (lane 4), 24 (lane 5), and 36 (lane 6) h postinfection were immunoprecipitated with anti-gD MAbs
(18) and separated by SDS-PAGE 10% gel under reducing
conditions. (A) VIDO R2 cells; (B) MDBK cells. Positions of size
markers (in kilodaltons) are shown to the left of each panel.
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The kinetics of HE expression by recombinant BAV502 was determined in
VIDO R2 cells. Anti-BCV polyclonal rabbit serum immunoprecipitated
a
65-kDa polypeptide from the cells infected with BAV502 (Fig.
7, lanes 5 and 6). This polypeptide
comigrated with the authentic
HE protein produced from BCV-infected
cells (lane 3). No such
protein was immunoprecipitated from mock (lane
1)- or wild-type
BAV-3 (lane 2)-infected cells. The kinetics of HE
expression (lanes
5 and 6) was similar to that observed for gD in VIDO
R2 cells
(Fig.
6A, lanes 5 and 6). Glycosylation of the recombinant gD
and HE proteins was examined by labeling the virus-infected cells
with
[
3H]glucosamine and then performing immunoprecipitation
assays.
The results confirmed that the proteins produced by recombinant
adenoviruses are glycosylated and are indistinguishable in migration
rate from the authentic proteins produced from virus-infected
cells
(data not shown).
View larger version (101K):
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[in a new window]
|
FIG. 7.
Kinetics of HE expression by recombinant BAV-3 in VIDO
R2 cells. Proteins from lysates of
[35S]methionine-labeled mock-infected (lane 1),
BAV-3-infected (lane 2), BCV-infected (lane 3), or BAV502-infected
cells harvested at 12 (lane 4), 24 (lane 5), and 36 (lane 6) h
postinfection were immunoprecipitated with anti-BCV polyclonal
antibodies (9, 10) and separated by SDS-PAGE (10% gel)
under reducing conditions. Positions of size markers (in kilodaltons)
are shown on the left.
|
|
Construction of replication-competent BAV-3 expressing GFP.
Plasmid pFBAV304 DNA (Fig. 3d) was digested with PacI and
transfected into VIDO R2 cells to isolate recombinant virus BAV304. BAV304 was amplified in MDBK cells, and viral DNA was extracted from
the infected cells. The viral DNA was analyzed by agarose gel
electrophoresis after digestion with restriction enzyme
BamHI. We observed a 2.3-kb DNA fragment in the BAV304
genome (Fig. 8A, lane 3) which was absent
in the BAV3.E3d genome (Fig. 8B, lane 2). This suggested that the
BAV304 genome contained a GFP gene, a possibility confirmed by Southern
blot analysis (Fig. 8B, lane 3). A similar 2.3-kb DNA fragment was also
observed in the pFBAV304 genome (Fig. 8A, lane 4) but not in
pFBAV302 (lane 5). This suggests that pFBAV304 contained a GFP gene, a
possibility confirmed by Southern blot analysis (Fig. 8B, lane 5).
View larger version (75K):
[in this window]
[in a new window]
|
FIG. 8.
Analysis of recombinant BAV304 genome. (A) The DNAs were
extracted from BAV-3 (lane 1), BAV3.E3d (lane 2), BAV304 (lane 3),
pFBAV304 (lane 4), and pFBAV302 (lane 5) digested with
BamHI and analyzed by ethidium bromide staining of an
agarose gel. (B) The fragments shown in panel A were transferred to a
Nytran membrane and probed with a -32P-labeled GFP gene.
Lane M, 1 Kb Plus DNA Ladder (Gibco/BRL) used for sizing the DNA
fragments.
|
|
To examine the expression of GFP, recombinant BAV304-infected cell
lysates were analyzed by Western blotting using GFP-specific
polyclonal
antibodies (Clontech). The anti-GFP serum identified
a band of 28 kDa
in recombinant BAV304-infected cells (Fig.
9,
lanes 1 to 3). No such band was
observed in mock (lane 4)- or
wild-type BAV-3 (lane 5)-infected cells.
In BAV304-infected cells,
GFP was detected from 12 to 36 h
postinfection (lanes 1 to 3).
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 9.
Western blot analysis of GFP expression. Proteins from
mock (lane 4)-infected, BAV-3-infected (lane 5), or BAV304-infected
MDBK cells harvested at 12 (lane 1), 24 (lane 2), and 36 (lane 3) h
postinfection were separated by SDS-PAGE (10% gel) under reducing
conditions and transferred to nitrocellulose. The separated proteins
were probed in Western blots by GFP-specific polyclonal antiserum.
Molecular masses (lane M) are indicated in kilodaltons.
|
|
Replication of BAV-3 and expression of GFP in different cell
types.
To determine the host species restriction of BAV-3, 13 different cell types obtained from six different mammalian species were
infected with wild-type BAV-3. BAV-3 replicated in cells of bovine
origin and cotton rat lung fibroblasts. In contrast to the earlier
observations (23), BAV-3 was able to replicate in cotton rat
lung cells as well as or better than in bovine cells. No virus
replication was apparent in cells derived from all other species, since
titers were lower than those of input virus. To further characterize
the host species restriction of BAV-3, we examined GFP expression in
cells infected with BAV304. The greatest number of GFP-expressing cells
were obtained in MDBK, VIDO R2, STR, and cotton rat lung fibroblast
cultures, where as very few sheep skin fibroblasts and VIDO R1 cells
showed fluorescence. Human cell lines 293 and HeLa infected with BAV304
showed mild fluorescence, while A549 and HepG2 cells showed no
fluorescence (data not shown). All other virus-infected cells had
low-level or no fluorescence (data not shown).
| |
DISCUSSION |
Human 293 (15), 911 (12), HAV-5
E1-expressing A549 (20), and PER (13) cells have
been used to generate replication-defective HAVs. These cell lines are
not suitable for BAV-3 infection, as they are nonpermissive for BAVs.
To develop a bovine cell line for the generation of
replication-defective BAV-3 vectors, initially MDBK cells were
transfected with a plasmid, pVD-Neo, containing E1 sequences of BAV-3.
A number of G418-resistant clones which supported the growth of an E1A
deletion mutant of HAV-5 were selected. However, attempts to detect the
E1 proteins of BAV-3 by Western blot analysis were unsuccessful. We
have also not been able to generate E1 deletion mutants of BAV-3 by
using this cell line. We hypothesized that this failure is due to the
low transfection efficiency of MDBK cells. Consequently, we developed a
second series of E1-complementing cell lines derived from FBRCs. Retina cells were chosen for the purpose because they are considered easy to
transfect and transform with the E1 region of adenoviruses (13,
14). Two attempts to transform FBRCs with plasmid pVD-Neo containing the left end of the BAV-3 genome were not successful. Reasons for our failure to transform FBRCs with the E1 region of BAV-3
are unknown. However, several transformed foci were evident following
the transfection of FBRCs with a plasmid, pTG4671, containing the E1
region of HAV-5 under the control of the mouse PGK promoter. The resulting transformed cells contained the integrated E1 sequences of HAV-5 in their genomic DNA as determined by PCR. These cells also
produced E1A and at least one of the E1B proteins in amounts roughly as
high as in 293 cells. The results of this study indicate that the
diploid cultures of FBRCs could be immortalized with the E1 region of
HAV-5. The resultant cell line, called VIDO R2, has been passaged more
than 50 times and shown to be stable with respect to retention of the
E1 region, morphology, and the ability to support the replication of
E1A deletion viruses.
Functional homologies between the E1A proteins of the mouse adenovirus
type 1 and HAV-5 were demonstrated in transient transfection assays
(2). Similarly, Zheng et al. (43) showed
transactivation activity of BAV-3 E1A proteins on the E2 and E3
promoters of HAV-5 in MDBK cells coinfected with E1A-deleted HAV-5 and
BAV-3. In the present study, we demonstrated that the E1A deletion
mutants of HAV-5 could be grown on 6.93.9 cells, suggesting that E1A
proteins of BAV-3 could complement those of HAV-5. Using the VIDO R2
cell line and E1A deletion mutants of BAV-3, we further demonstrated that the E1A proteins of HAV-5 could complement those of BAV-3 during
viral replication. This may be explained by strong homologies noticed
among the E1A proteins of adenoviruses, especially in conserved region
3 (2, 28, 43). The amino-terminal part of conserved region 3 contains a zinc finger motif required for the transactivation function,
and the carboxy terminus contains a domain required for association
with various transcription factors (7). Once it was
established that the E1A proteins of HAV-5 could complement for those
of BAV-3, two BAV-3 recombinants expressing gD of BHV-1 and HE of BCV
were generated from the same cell line. These viruses grow exclusively
in bovine cell lines expressing E1 proteins of either BAV-3 (6.93.9) or
HAV-5 (VIDO R2) but not in MDBK cells and FBRCs.
The presence of replication-competent adenoviruses (RCAs)
in batches of replication-defective adenoviruses is a major problem for
the use of these vectors in gene therapy and is potentially a safety
risk. RCAs are generated by recombination between sequences in the
adenovirus vector and the matching sequences in complementing cell
lines such as 293 and 911, resulting in acquisition of the E1 region by
the vector (16). The RCAs, in addition to providing helper
function to replication-defective vectors, will aggravate the host
immune response and enhance tissue damage (19). Although problems with RCAs can be overcome by carefully designing the vectors,
an advantage of the VIDO R2 cells is that they contain the E1 region of
HAV-5 and do not contain sequences that are present in
replication-defective BAV-based vectors. Thus, the absence of
homologous sequences should completely eliminate the problem of RCA
generation by homologous recombination and allow the production of safe
recombinant BAV-3 vectors.
The demonstration of a mucosal immune response following mucosal
delivery of recombinant HAV-5 expressing BHV-1 gD or BCV HE in cotton
rats (1, 24, 25) has stimulated interest in the development
of BAV-3, vector-based vaccines in cattle. Recently, we constructed
replication-competent BAV-3 recombinants expressing BHV-1 gD
(42) and demonstrated that intranasal immunization of calves
with these recombinants induced protective immune responses against
BHV-1 (41). However, these vectors have the potential to
shed recombinant virus into the environment.
Ideally, a recombinant vaccine antigen should be produced in the target
animal without virus replication, thus making the live vectors safe by
eliminating transmission of the virus to other farm animals and humans.
Cell lines complementing E1A function allowed us to construct
replication-defective BAV-3 recombinants that may serve as safe
expression systems or nonreplicating vaccine vectors for cattle. For
our initial experiments, we chose genes coding for protective antigens
from BHV-1 (a virus involved in the development of a complex
respiratory disease syndrome called shipping fever [33-35,
40]) and BCV (a virus involved in causing neonatal diarrhea
[9, 10, 26]). The availability of these recombinant
viruses expressing vaccine antigens should help in determining the
potential of the replication-defective recombinant BAV-3 for delivery
of vaccine antigens to mucosal surfaces of cattle.
Recombinant BAV-3 also has potential as a viral vector for somatic gene
therapy in humans. In this respect, a recombinant BAV-3 expressing GFP
represents an important tool for characterizing and potentially
improving its properties as a vector. The GFP gene was chosen as a
marker because it provides a simple and reliable method for the
detection of transgene expression in cells. The observation of low
levels of GFP expression in human cells even though the gene was placed
under the control of a strong CMV immediate-early promoter indicates
that the entry of BAV-3 to human cells is restricted. For successful
somatic gene therapy, we need to develop gene transfer vectors with
very high transduction efficiency. There are several ways to improve
the transduction efficiency of BAV-3 into human cells, by utilizing
high doses of the virus or by combining the virus with cationic
liposomes. An alternative strategy is to replace the knob region of the
fiber of BAV-3 with that of an HAV. This seems to be possible, as the
entry of an ovine adenovirus into human cells was enhanced when the
knob region of the fiber was replaced with that of HAV-5
(38). The RGD motif, which interacts with surface integrins
(36) and facilitates the entry of virus into cells, is
present in the penton base protein of HAV-5 but absent in BAV-3
(30). Introduction of such a motif into BAV-3 may facilitate
the entry of BAV-3 into human cells.
| |
ACKNOWLEDGMENTS |
We thank members of the laboratory, including A. N. Zakhartchouk, Mohit Baxi, and Caron Pyne for help.
This work was supported by grants from National Science and Engineering
Research Council of Canada, Medical Research Council of Canada,
Saskatchewan Agriculture Development Fund, Saskatchewan Beef
Development Fund, Western Economic Diversification, and Alberta Agriculture Research Institute. P.S.R. was the recipient of a postdoctoral research fellowship from the Health Services Utilization and Research Commission, Saskatoon, Saskatchewan, Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: VIDO, 120 Veterinary Rd., University of Saskatchewan, Saskatoon, Saskatchewan,
Canada S7N 5E3. Phone: (306) 966-7482. Fax: (306) 966-7478. E-mail:
tikoo{at}sask.usask.ca.
Published with the permission of the Director of VIDO as journal
series no. 258.
Present address: Genetic Therapy, Inc., Gaithersburg, MD 20878.
| |
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Journal of Virology, November 1999, p. 9137-9144, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
