Virology Group, Veterinary Infectious Disease Organization, 120 Veterinary Road, University of Saskatchewan, Saskatoon, Saskatchewan , Canada S7N 5E31
Gene Therapy Department, Transgene SA, 67000 Strasbourg, France 2
Author for correspondence: Suresh Tikoo.Fax +1 306 966 7478. e-mail tikoo{at}sask.usask.ca
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Abstract |
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Introduction |
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Five porcine adenovirus (PAV) serotypes have been identified to date (Derbyshire et al., 1975 ; Hirahara et al., 1990
). Of the five serotypes, type 3 (PAV-3) replicates to high titres in cell culture (Hirahara et al., 1990
). The prototype of this virus was first isolated from a rectal swab collected from a healthy piglet (Clarke et al., 1967
), and experimental infections of piglets with PAV-3 have been subclinical or associated with transient diarrhoea (Derbyshire et al., 1975
). The proposed use of this virus as a vector has stimulated interest in the molecular genetics of the virus. The complete nucleotide sequence of the PAV-3 genome was determined and a transcription map for the whole genome was established (Reddy et al. , 1998a
, b
).Genomic and cDNA sequence analysis identified promoters, cap sites, intronexon boundaries, poly(A) signals and poly(A) sites in the virus genome. Though the overall genome organization is similar to that of human adenovirus-2 (HAV-2), the prototype of HAVs, there were some distinct features of the PAV-3 genome. A relatively high G+C content, organization of the late region genes into six families, the absence of additional leader sequences in transcripts of the fibre, and the presence of a single small virus-associated RNA gene are some of the distinctive features of the PAV-3 genome. Availability of the complete sequence information and a transcription map for the whole genome facilitated the development of PAV-3 as a helper-independent expression vector (Reddy et al., 1999
). Using the Escherichia coli BJ 5183 recombination system, an infectious full- length clone of the PAV-3 genome was recently constructed and non- essential sites suitable for the insertion of transgene expression cassettes were identified (Reddy et al., 1999
). In the present study, we developed E1-complementing cell lines and isolated a helper-dependent PAV-3 vector. In addition, we constructed a helper-dependent PAV-3 expressing a green fluorescent protein (GFP), which was used to determine the host range of PAV-3.
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Methods |
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Construction of recombinant plasmids.
The recombinant plasmid vectors were constructed by standard procedures using restriction enzymes and other DNA-modifying enzymes as directed by the manufacturers.
(a) Construction of plasmid pFPAV201.
The left (nt 12130) and the right (nt 3265934094) terminal restriction enzyme fragments generated by NcoI were subcloned from pFPAV200 (Reddy et al., 1999 ) to generate the plasmid pPAV-101. Nucleotide numbers of the PAV-3 genome referred to in this paper are according to GenBank (accession no. AF083132; Reddy et al., 1998b
). The E1A sequences between nts 407 and 1210 were deleted using NotI and AseI digestions and a unique SrfI site was engineered into the region to create plasmid pPAV-102. The plasmid pFPAV201, which has the full-length PAV-3 genome minus E1A and part of the E3 sequences, was created by co- transformation of E. coli BJ 5183 cells with NcoI- linearized pPAV-102 and the genomic DNA of PAV-3 with a deletion of the E3 region (Reddy et al., 1999
).
(b) Construction of plasmid pFPAV202.
A 2·3 kb fragment containing the cytomegalovirus (CMV) immediate early promoter, the GFP gene and the bovine growth hormone (BGH) poly(A) signal was isolated by digesting pQBI 25 (Quantum Biotechnology) with BglII and DraIII followed by filling the ends with T4 DNA polymerase. This fragment was inserted into the SrfI site of pPAV-102 in both orientations to generate pPAV-102GFP. This plasmid was digested with PacI and SmaI enzymes, and the fragment containing part of the E1 sequence and the GFP gene was gel-purified. This fragment and Srf I-digested pFPAV201 were used to transform E. coli BJ 5183 cells to generate the full-length clone containing GFP in the E1 region (pFPAV202) by homologous recombination.
Isolation of recombinant PAV-3.
The desired PAV-3 recombinants were made as described (Zakhartchouk et al., 1998 ; Reddy et al., 1999
). Briefly, VIDO R1 cell monolayers in 60 mm dishes were transfected with 510 µg PacI-digested pFPAV201 or pFPAV202 recombinant plasmid DNA using lipofectin (Gibco- BRL). The cells showing 50% cytopathic effects were collected and freezethawed twice, before plaque-purifying the recombinant virus(es).
Western blot analysis.
Protein extracts of cells were electrophoresed (5 µg per lane) on a 10% SDSpolyacrylamide gel and transferred to a nitrocellulose membrane (Sambrook et al., 1989 ). Non-specific binding sites on the membranes were blocked with 1% BSA fraction V. The HAV-5 E1 proteins were detected by exposing the membranes to monoclonal antibodies specific to E1A (M73) or 19 kDa E1B (3D11) (Cedarlane Laboratories) followed by anti-mouse IgG conjugated to alkaline phosphatase. The GFP was detected by exposing membranes to anti-GFP serum followed by anti-rabbit IgG conjugated to alkaline phosphatase. Finally the blots were developed using an HRP or AP colour development kit (Bio-Rad).
PAV-3 infection of human and animal cells.
ST, VIDO R1, 293, A549 (human), MDBK, VIDO R2 (bovine), C3HA (mouse), Cos, Vero (monkey), sheep skin fibroblasts or cotton rat lung cells were incubated with 1 p.f.u. per cell of wild-type PAV-3 or helper-dependent PAV-3 expressing GFP. The cells infected with wild- type PAV were harvested at 2 h and 3 days post-infection, subjected to two cycles of freezethaw and the virus titres were determined on VIDO R1 cells. Cells that were infected with the recombinant PAV-3 virus expressing GFP were observed with the aid of a fluorescence microscope for green fluorescence.
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Results |
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Generation of E1A deletion mutants of PAV-3
To test whether these cell lines can be used for the generation and propagation of PAV-3 mutants with deletions in E1A and E3, the full- length PAV-3 genomic DNA containing deletions in E1A (nt 4071210) and E3 (nt 2811228709) regions was cloned in a plasmid named pFPAV201 (Fig. 3a). This plasmid was digested with AseI and analysed by agarose gel electrophoresis. pFPAV200 (which contains full-length genomic DNA of PAV-3; Reddy et al., 1999
) had an AseI fragment of 2·1 kb (Fig. 4
, lane 1), which is missing in pFPAV201 (Fig. 4
, lane 2), confirming that the expected deletion has been created. A viable virus (named PAV201) was obtained following the transfection of PacI-restricted pFPAV201 into VIDO R1 cells. Virus was harvested, amplified and viral DNA was prepared by Hirt extraction (Hirt, 1967
). The structure of the viral DNA was established by cleavage with AseI (Fig. 4
). The wild-type PAV-3 had an AseI fragment of 1·280 kb (Fig. 4
, lane 4), which was missing in the recombinant PAV201 genome (Fig. 4
, lane 5). This is consistent with the expected AseI restriction pattern. The deletion mutant could only be grown in VIDO R1 and STR cells but not in ST and FPR cells.
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In order to determine the expression of GFP protein, recombinant PAV202 virus-infected cell lysates were analysed by Western blotting using GFP-specific polyclonal antibodies (Clontech). As seen in Fig. 5 , anti-GFP serum identified a band of 28 kDa protein in recombinant PAV202-infected cells (Fig. 5
, lane 5), which was similar in size to the authentic GFP protein (Fig. 5
, lane 2). No such band could be observed in mock- (Fig. 5
, lane 1) or PAV-3-infected cells (Fig. 5
, lane 3).
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Discussion |
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ST cells are commonly used for the propagation of PAV-3, since the virus grows well in this cell line. These cells were chosen first for the stable transfection with the E1 expression plasmid pTG 4671. Two weeks following transfection, several puromycin-resistant clones were obtained. The puromycin-resistant clones supported the growth of an E1A- deleted HAV-5 (Zheng et al., 1994 ) although not as efficiently as did 293 cells. One of the clones, STR, was subjected to single cell cloning and the expression of E1 proteins was confirmed by Western blotting.
Although stable cell lines expressing E1 proteins could be established, stable and sustained expression of E1 proteins has been difficult, as these cell lines tend to gradually loose the transfected DNA (Imler et al., 1996 ). This was mainly attributed to E1-mediated cytotoxicity (Graham et al., 1977
; Rao et al., 1992
). This transient expression of E1 proteins through the established cell lines is dependent on the selection markers, such as hygromycin, G418 and puromycin, which are very expensive. Moreover, even under selection pressure, the expression of E1 proteins is often lost when these cells are cultured for a long time (Imler et al., 1996
). To overcome these problems, we transformed FPRC with the E1 region of HAV-5. Transformation of cultured rodent cells by adenoviruses or adenoviral DNA has been well documented (Fisher et al., 1982
). Transformation of porcine cells either with a PAV or a HAV has not been reported due to the fact that exposure of permissive or semi-permissive cells to adenovirus normally leads to lysis of infected cells (Graham et al., 1977
). However, it was possible to transform human cells with the DNA encoding E1 proteins of HAVs. Using this approach several human cell lines, such as 293 (Graham et al., 1977
), 911 (Fallaux et al., 1996
) and PER (Fallaux et al., 1998
), have been developed and used to generate helper-dependent HAV vectors. The approach used in the present study to create an E1-complementing cell line employing the E1 region of HAV-5 for PAVs is novel as E1A proteins of HAV-5 were shown for the first time to complement for those of PAV- 3. There are several reasons for using the E1 region of HAV-5 for transformation of FPRC. The E1 region of HAV-5 was shown to transform human retina cells very efficiently (Fallaux et al., 1998
). In contrast to the E1 region of PAV-3, the E1 region of HAV-5 has been thoroughly characterized and monoclonal antibodies against E1 proteins are readily available from commercial sources. In addition, the E1A region of HAV-5 was shown to complement the E1A functions of several non-human adenoviruses (Ball et al., 1988
; Zheng et al., 1994
).
The construction of PAV201 is the first step towards the development of replication-defective PAV-3 containing deletions in E1 and E3 regions. The construction of PAV202 further demonstrated the feasibility of using this vector system for foreign gene expression. In addition, deletion of 1·4 kb (E1A+E3) should also increase the capacity of the resulting vector, PAV201, to accommodate 3·2 kb of foreign DNA.
The presence of low levels of helper-independent vectors in the batches of helper-dependent adenoviruses that are grown in 293 or 911 cells have been reported (Fallaux et al., 1998 ). This occurs as a result of recombination events between the viral DNA and the integrated adenoviral sequences present in the complementing cell line (Hehir et al., 1996
). This type of contamination constitutes a safety risk, which could result in the replication and spread of the virus. Complete elimination of helper- dependent adenoviruses in the batches of helper-dependent vectors can be achieved using two approaches. First, by developing new helper cell lines and matched vectors that do not share any common sequences (Fallaux et al., 1998
). Second, by using cross- complementation that exists between two distantly related adenoviruses such as HAV-5 and PAV-3. VIDO R1 cells contain the E1 coding sequences of HAV-5. Although there is no significant identity between the E1 regions of HAV-5 and PAV-3 at the nucleotide sequence level, the proteins produced from the region can complement the function of each other. Thus, the problem of helper-independent vector generation by homologous recombination should be rare when VIDO R1 cells are used for the propagation of recombinant PAV-3.
HAV serotypes belonging to all subgroups can transform rodent cells in tissue culture (Tooze, 1981 ). The transforming region is comprised of E1A and E1B and both are necessary for complete transformation (Van den Elsen et al., 1983
). The E1A proteins of HAV-2 have been extensively characterized. Three conserved regions (CR1 to CR3) of the E1A proteins have been identified that are required for the interaction of the protein with cellular proteins. CR1 and CR2 are required for transformation and serve as binding sites for the product of the retinoblastoma susceptibility gene (Rb) and p300 protein respectively. This activity is responsible for freeing of E2F transcription factor from the clutches of Rb and p300, which is responsible for induction of S phase events (Bagchi et al. , 1990
). Sequences within CR3 are critical for the transcriptional activation capacity of the E1A protein. The expression of E1A stimulates apoptosis, an important cellular defence against virus infection and perturbation of cell growth control. Both proteins encoded by the E1B region are involved in preventing the E1A-mediated apoptosis (Rao et al., 1992
; Debbas & White, 1993
). Thus, the expression of E1B protein makes cells tolerant of levels of E1A that are required for cell transformation (Lowe & Ruley, 1993
). The ability of E1 proteins of HAV- 5 to transform FPRC into a stable cell line suggests that similar molecular mechanisms of transformation are used in foetal porcine cells.
The E1 region in PAV-3 is located within the leftmost 12% of the genome and encodes proteins that are analogous to those found in HAV-5 (Reddy et al., 1998a ). There is a high degree of conservation in the CR2 and CR3 between the E1A proteins of PAV-3 and HAVs. In this study, we have demonstrated that the E1A proteins of HAV-5 could complement for the E1A of PAV-3. Earlier, functional similarities between the E1A proteins of HAV-5 and simian adenovirus-7 (Kimelman et al., 1985
); between HAV-5 and mouse adenovirus-1 (Ball et al., 1988
); and also between HAV-5 and bovine adenovirus-3 (Zheng et al., 1994
) were demonstrated in transient transfection assays or using co-infections.
The use of the GFP as a marker for recombinant virus allows the visualization of infected cells by fluorescence microscopy. Since the recombinant GFP gene, isolated from the jellyfish Aequorea victoria was first used as a genetic marker in Caenorhabditis elegans , it has been used in a variety of organisms (Cubitt et al. , 1995 ). GFP is a valuable biological marker, which allows a non-evasive detection that requires only illumination by UV light to yield green fluorescence. Future investigations on the virus tropism will likely involve the study of cell populations in which only a subset of cells may be infected by PAV-3. In such cell populations, the GFP can allow the isolation of virus-infected cells for subsequent experiments regarding the study of virus persistence.
The finding that PAV-3 was effective in entering human, canine, sheep and bovine cells in which it does not replicate or replicates poorly is an important observation. This may have implications in designing a PAV-3 vector for vaccination in human and animal species. Recombinant HAVs expressing virus antigens have been shown to induce protective immune responses in animals such as mice and dogs, in which HAV replicates poorly or not at all (Prevec et al., 1989 ).
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Acknowledgments |
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This paper is published with the permission of VIDO as journal series no. 257.
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Footnotes |
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References |
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Received 27 April 1999;
accepted 2 July 1999.