Identification of cis-acting sequences required for selective packaging of bovine adenovirus type 3 DNA

Li Xing, Linong Zhang{dagger}, Jill Van Kessel and Suresh Kumar Tikoo

Vaccine and Infectious Disease Organization, University of Saskatchewan, 120-Veterinary Road, Saskatoon, SK, Canada S7N 5E3

Correspondence
Suresh Kumar Tikoo
Tikoo{at}Sask.Usask.CA


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The assembly of adenovirus particles is a multistep process, in which viral genomic DNA is selected and subsequently inserted into preformed empty capsids. The selective encapsidation of the adenovirus genome is directed by cis-acting packaging motifs, termed A repeats due to their AT-rich character in DNA sequence. A repeats are usually located at the left end of the viral genome. In this report, the construction and analysis of bovine adenovirus type 3 (BAdV-3) mutants containing deletion mutations introduced into the AT-rich regions are described. The main cis-acting packaging domains of BAdV-3 were localized between nt 224 and 540 relative to the left end of the viral genome. They displayed a functional redundancy and followed a hierarchy of importance. In addition, the results demonstrated that not all of the AT-rich units functioned as cis-acting packaging motifs.

{dagger}Present address: Aventis Pasteur Limited, Toronto, Canada.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adenoviruses have been studied extensively for several decades (Rowe et al., 1953; Hillemann & Werner, 1954; Russell, 2000) and have been used as a model system to explore the mechanisms of eukaryotic DNA replication, RNA transcription, gene expression and virus–host interactions. In recent years, adenovirus has received considerable attention for its potential use as a gene delivery vehicle (Berkner, 1988; Graham & Prevec, 1992; Trapnell, 1993; Trapnell & Gorziglia, 1994; Bramson et al., 1995; Hitt et al., 1997).

The assembly of adenovirus virions is a multistep process, in which viral DNA is packaged selectively into empty capsids (Edvardsson et al., 1976, 1978; D'Halluin et al., 1978a, b, 1980; D'Halluin, 1995). It has been established that packaging of the adenovirus genome involves the specific recognition of cis-acting viral DNA sequences, named packaging domains, by viral and/or cellular proteins (Daniell, 1976; Tibbetts, 1977; Hammarskjold & Winberg, 1980; Robinson & Tibbetts, 1984; Hearing et al., 1987; Grable & Hearing, 1990). The cis-acting packaging domain of human adenovirus (HAdV) was characterized at the molecular level during the last several years (Hearing et al., 1987; Grable & Hearing, 1990, 1992; Schmid & Hearing, 1997). It is usually located at the left end of the viral genome between approximately nt 194 and 380 (Hearing et al., 1987; Grable & Hearing, 1990, 1992; Schmid & Hearing, 1997) and contains at least seven functionally redundant elements, termed A repeats I to VII (AI, AII, AIII, AIV, AV, AVI and AVII) due to their AT-rich character, with AI, AII, AV and AVI being the most dominant. Repeats AI, AII, AV and AVI exhibit a bipartite consensus motif, 5'-TTTGN8CG-3', which is conserved across a number of HAdV serotypes and which can function independently (Schmid & Hearing, 1997).

To date, it was demonstrated that chicken ovalbumin upstream promoter transcription factor (a member of the steroid/thyroid hormone receptor superfamily), P-complex and octamer-1 protein could bind to minimal packaging domains of HAdV-5 (Schmid & Hearing, 1998; Erturk et al., 2003). However, only P-complex binding activity is correlated directly with packaging function (Erturk et al., 2003). Among the viral proteins, the L1 52/55 kDa protein and the IVa2 protein were demonstrated to be required for viral DNA packaging (Hasson et al., 1989; Gustin & Imperiale, 1998; Zhang & Imperiale, 2000; Zhang et al., 2001).

Bovine adenovirus type 3 (BAdV-3) was first isolated in Britain from the conjunctiva of an apparently healthy cow (Darbyshire et al., 1965). Due to its lack of virulence, BAdV-3 is being developed as a virus vector for producing recombinant animal vaccines and potential use in gene therapy (Mittal et al., 1995; Zakhartchouk et al., 1998, 1999; Reddy et al., 1999a; Baxi et al., 2000). Recently, the complete DNA sequence of the BAdV-3 genome has been determined (Baxi et al., 1998; Lee et al., 1998; Reddy et al., 1998) and transcription maps of early regions 1, 3 and 4 (E1, E3 and E4) and late regions have been established (Reddy et al., 1998, 1999b; Baxi et al., 1999; Idamakanti et al., 1999). These studies led to the development of replication-defective (Reddy et al., 1999a) and replication-competent (Zakhartchouk et al., 1998) virus vectors. A better understanding of the packaging mechanism of BAdV-3 would facilitate the construction and improvement of BAdV-3-derived virus vectors. For example, the packaging signal is required for construction of ‘gutless' BAdV-3 vectors. However, nothing is known about the cis-acting packaging sequences and trans-acting protein components involved in the encapsidation of the BAdV-3 genome into capsids. In this report, we describe the construction and characterization of mutant BAdV-3 viruses containing deletion mutations located between the left-end inverted terminal repeat (ITR) and the ATG start codon of the E1A gene.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
VIDO R2 cells (Reddy et al., 1999a) are transformed foetal bovine retina cells expressing the E1A and E1B proteins of HAdV-5. They were grown and maintained in Eagle's MEM supplemented with 10 % FBS. Mutant and wild-type BAdV-3 viruses (strain WBR-1) (Darbyshire et al., 1965) were propagated and titrated in VIDO R2 cells.

Construction of recombinant plasmids.
Plasmid pLtRtHind.Mod, which contains the 1·6 kb left-end fragment (nt 1–1653) and the 1·2 kb right-end fragment (nt 33235–34446) of the BAdV-3 genome, was used as template in PCR to create deletion mutations between the left-end ITR and the ATG start codon of the E1A gene (Reddy et al., 1998). Nucleotide numbers of the BAdV-3 genome referred to in this report are given according to the sequence under GenBank accession number AF030154. The primers used for PCR are shown in Table 1. The following conditions were used for PCR in a total volume of 50 µl: 0·5 µg template DNA, 1x PCR buffer [10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris/HCl (pH 8·75), 2 mM MgSO4, 0·1 % Triton X-100 and 0·1 mg BSA ml-1] (Stratagene), 0·4 mM dNTPs, 10 pmol each primer and 2·0 U cloned Pfu DNA polymerase (Stratagene). Cycling conditions were: 94 °C for 2 min to denature the DNA, followed by 30 cycles of 94 °C for 40 s, 37–50 °C for 40 s and 72 °C for 40 s and, finally, an extension step at 72 °C for 2 min. PCR products were separated on a 1·5 or 2 % agarose gel and visualized by ethidium bromide staining.


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Table 1. Primers used for PCR

 
To construct recombinant transfer plasmids containing the deletions between the left-end ITR and the ATG start codon of the E1A gene of BAdV-3 (Reddy et al., 1998), the DNA fragments amplified first using the primer pairs LZP53/LZP42, LZP53/BAdV-P1, LZP53/BAdV-P3, LZP53/BAdV-P5, LZP53/BAdV-P7 and LZP53/BAdV-P9 were digested with BamHI/XhoI. Secondly, the DNA fragments amplified using primer pairs LZP41/LZP43, LZP41/BAdV-P2, LZP41/BAdV-P4, LZP41/BAdV-P6, LZP41/BAdV-P8 and LZP41/BAdV-P10 were digested with XhoI/PstI. The appropriate BamHI–XhoI and XhoI–PstI DNA fragments were ligated into BamHI/PstI-digested pLtRtHind.Mod in a three-way ligation, thus creating the recombinant transfer plasmids containing the desired deletions between the left-end ITR and the ATG start codon of the E1A gene of BAdV-3. To construct the transfer plasmids containing double deletions, the second deletion mutation was introduced in the plasmids containing single deletion mutations in the same way as described above.

Plasmids containing the full-length genome of BAdV-3 with deletions in the putative packaging domain were generated by homologous recombination in Escherichia coli strain BJ5183 (Chartier et al., 1996) between an HindIII-linearized individual recombinant transfer plasmid and the genomic DNA from wild-type BAdV-3. These plasmids were characterized by restriction enzyme analysis. The endpoints of deletion mutations introduced into plasmids were determined by nucleotide sequence analysis.

Isolation of BAdV-3 mutants.
VIDO R2 cell monolayers seeded in 35 mm dishes were transfected with 5 µg PacI-digested, individual, full-length plasmid DNA using Lipofectin, according to the manufacturer's instructions (Invitrogen). After 10–15 days of incubation at 37 °C, transfected cells were collected and freeze-thawed three times. Lysates were used to infect freshly prepared VIDO R2 cells until CPE appeared. Mutant viruses were plaque-purified (Hitt et al., 1997) three times and then characterized by PCR, restriction enzyme analysis and sequence analysis of virion DNA.

Determination of virus packaging efficiency.
The packaging efficiency of the mutant viruses was determined by coinfection of VIDO R2 cells with both mutant and wild-type BAdV-3, according to the method described earlier (Grable & Hearing, 1990, 1992) with little modification. VIDO R2 cells were infected with 5 p.f.u. of each of the viruses per cell. At 48 h post-infection (p.i.), one half of the cells was used to isolate total DNA and the other half of the cells was used to prepare viral DNA. For isolation of total DNA, infected cells were lysed by addition of Nonidet P-40 to 0·4 % and then digested with proteinase K at 50 °C for at least 2 h. High molecular mass DNA was isolated as described previously (Sambrook et al., 1989). For isolation of viral DNA from virions, infected cells were precipitated and suspended in lysis buffer [20 mM Tris/HCl (pH 8·0), 0·2 % deoxycholate and 10 % ethanol]. After incubation for 60 min at room temperature, the lysate was cleared at 10 000 g for 30 min. The supernatant was adjusted to 2 mM CaCl2 and 2 mM MgCl2 and was digested with 40 µg RNase A ml-1 and 10 µg DNase I ml-1 at 37 °C for 30 min. The reaction was stopped by addition of EDTA and EGTA to a final concentration of 50 mM each. Virus particles were lysed by addition of sarkosyl to 0·5 % and samples were digested with 1 mg proteinase K ml-1 at 50 °C for 1–2 h. After phenol/chloroform extraction, viral DNA was precipitated with ethanol. DNA isolated from virus-infected cells or virions was digested with XhoI/PstI and analysed by Southern hybridization.

Southern hybridization.
XhoI/PstI-digested DNAs were separated on 1·5 % agarose gels and then transferred to Gene Screen Plus hybridization transfer membranes (Perkin-Elmer) using the high-salt, capillary-transfer method, according to the instructions of the manufacturer. The 280 bp DNA fragment corresponding to nt 560–839 was amplified by PCR with the primer pair LZP41/BAdV-P10, labelled with [32P]dCTP using the Random Primers DNA labelling system (Invitrogen) and used as a probe for Southern hybridization. Blots were prehybridized in ULTRAhyb ultrasensitive hybridization buffer (Ambion RNA) at 42 °C for 30 min and 32P-labelled probes were added. Hybridization was performed at 42 °C overnight. After washing extensively with 0·1x SSC and 0·1 % SDS, blots were exposed to X-ray film (Kodak) without an intensifying screen. The bands in the autoradiograms were scanned and their relative intensities were determined and analysed by computer densitometry using the Bio-Rad phosphorimager program. The data presented for packaging efficiency based on coinfection experiments represent the averages of three independent experiments. The values in three experiments varied by an SD of 1·5.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the left-end genomic sequence of BAdV
The cis-acting packaging domains of HAdV-5 (Grable & Hearing, 1990, 1992) and canine adenovirus type 2 (CAdV-2) (Soudais et al., 2001) are located at the left end of the viral genome. Due to the similarity of genome organization (Reddy et al., 1998), we also focussed on the left-end region of the BAdV-3 genome (Fig. 1). However, analysis of the region especially between the left-end ITR and the ATG start codon of the E1A gene did not show any identity to the consensus bipartite structure of the dominant cis-acting packaging elements (5'-TTTGN8CG-3') of HAdV-5 (Schmid & Hearing, 1997). To determine if this region contained the cis-acting packaging domain of BAdV-3, mutant viruses containing deletions in this region of the viral genome were isolated and analysed for packaging efficiency.



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Fig. 1. Nucleotide sequence of the left-end region of the BAdV-3 genome. The left-end ITR is shown in italics. The transcriptional initiation site and ATG start codon for the E1A gene are underlined. AT-rich motifs are shown in bold.

 
Isolation and analysis of mutant viruses
As the E. coli strain BJ5183 recombination system (Chartier et al., 1996) is the most simple and efficient method of generating adenovirus-based mutants, we also used this system to construct and isolate mutant BAdV-3. Initially, deletions in the BAdV-3 genome were constructed in a recombinant transfer plasmid by deleting DNA sequences using PCR by insertion of two PCR products into the BamHI/PstI sites of plasmid pLtRtHind.Mod (Fig. 2A). The deletions were then rebuilt into an intact viral genome by homologous recombination between wild-type BAdV-3 genomic DNA and HindIII-linearized recombinant transfer plasmids in E. coli strain BJ5183 cells (Fig. 2B). The resulting full-length plasmids containing the whole genome of BAdV-3, with deletions in the desired region, were characterized by restriction enzyme and DNA sequence analysis. Mutant BAdV-3 viruses containing the desired deletions were isolated by transfection of VIDO R2 cells with PacI-digested full-length plasmid DNA (Fig. 2B). Mutant viruses were plaque-purified and propagated in VIDO R2 cells. To confirm the presence of specific deletion(s), we first analysed virion DNA by PCR using the primer pair LZP41/PLB5 (Table 1). Mutant viral DNA fragments were observed for a shift in the size of their DNA in agarose gel due to the deletions. Compared to the wild-type BAdV-3 control, all deletion mutants yielded the expected smaller-size DNA fragments (Fig. 3A–D). Secondly, deletions were confirmed by DNA sequence analysis of virion DNA.



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Fig. 2. (A) Schematic diagram of plasmid pLtRtHind.Mod. BAdV-3 sequences are from the extreme left and right ends of the viral genome. E1A, E1B and E4 mRNAs, and their directions of transcription, are shown as open arrows. The plasmid backbone is shown as a thin line. ITRs are shown as filled boxes. (B) Schematic representation of the strategy used for the construction of full-length plasmids and mutant viruses. Plasmid DNA is shown as a thin line. BAdV-3 genomic DNA is indicated with boxes. The ITR is shown as a filled box. Hatched boxes represent regions in which deletions were introduced.

 


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Fig. 3. PCR analysis of mutant viruses. Comparison of PCR products generated from mutant viruses and wild-type BAdV-3 using the primer pair LZP41/PLB5. The expected sizes of the PCR products are shown at the bottom. Molecular size markers are indicated on the left. Schematic views of mutant viruses analysed in (A)–(D) are shown in Figs 4–7, respectively.

 
The proteins encoded by BAdV-3 E1 are required for activation of transcription of other early genes and virus replication (Zhou et al., 2001). Since these deletions also contain regulatory element(s) that affect the expression of the E1 genes (Xing & Tikoo, 2003; unpublished data), we used VIDO R2 cells (foetal bovine retina cells transformed with HAdV-5 E1) (Reddy et al., 1998) for the present study.

To compare the packaging efficiency of the mutant viruses, coinfection experiments were used, as described earlier (Grable & Hearing, 1990, 1992). VIDO R2 cells were coinfected with a mutant and wild-type virus. The coinfecting wild-type virus provides normal levels of the viral early and late gene products to complement the mutant with impaired gene expression. At 2 days after infection, one half of the infected cells was used to prepare total DNA and the other half of the cells was used to prepare viral DNA. The coinfecting mutant and wild-type viral DNAs were distinguished by restriction enzyme digestion followed by Southern hybridization. By comparing the relative amounts of mutant and wild-type viral DNA present in the infected cells with the relative amounts of each viral DNA present in intact virions, the packaging efficiency of the mutant viruses could be measured accurately, independent of other effects resultant from early and late gene expression.

Analysis of mutant viruses with internal deletions
In the first set of mutant viruses, small deletions were introduced individually between nt 224 and 560 of the BAdV-3 genomic DNA (Fig. 4A). In addition, Southern blot analysis of total DNA (Fig. 4B) suggests that these mutant viruses could replicate as efficiently as wild-type BAdV-3 in coinfection experiments. However, Southern blot analysis of virion DNA (Fig. 4A, B) suggested that the deletions of sequences between nt 224 and 311 (Bav3-224/311), nt 311 and 382 (Bav3-311/382), nt 383 and 468 (Bav3-383/468) or nt 467 and 541 (Bav3-467/541) resulted in a 2- to 4-fold decrease in packaging efficiency in coinfection experiments. In contrast, deletions of sequences between nt 537 and 552 (Bav3-537/552) or nt 553 and 560 (Bav3-553/560) did not result in a detectable decrease in the packaging efficiency of mutant viruses in coinfection experiments. These results indicate that the sequences between nt 224 and 541 may contain sequence motif(s) necessary for packaging of viral DNA into virions. However, the data could not rule out the possibility that the sequences between nt 537 and 560 may also contain the functional packaging motif. Since the cis-acting packaging motifs are functionally redundant and show an important hierarchy (Schmid & Hearing, 1997), it is possible that the region between nt 537 and 560 in BAdV-3 contains a very weak packaging motif, the effect of which could not be detected in coinfection experiments.



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Fig. 4. Analysis of mutant viruses carrying individual deletions. (A) Schematic view of mutant viruses. The top of the figure shows the positions of AT-rich motifs (filled boxes), the E1A ATG start codon (arrowhead) and the proximal portion of the left-end ITR (open rectangle) on the structure of the BAdV-3 genome. Individual deletion mutant names are given on the left. Nucleotide numbers correspond to the first nucleotides present on either side of the deletion. The deleted sequences are indicated by a dotted line. Mutant virus packaging efficiency (COINF.) is expressed as the fold reduction in packaged mutant DNA relative to the packaged coinfecting wild-type DNA. Data were normalized to the amount of each viral DNA (mutant and wild-type) present in total DNA. (B) Southern hybridization analysis of total DNA and virion DNA isolated from VIDO R2 cells coinfected with wild-type and mutant viruses. Total and virion DNA were digested with XhoI/PstI and subsequently subjected to Southern hybridization using the BAdV-3 left-end fragment between nt 560 and 839 as a 32P-labelled probe. The corresponding wild-type and mutant left-end DNA fragments are indicated. The mutant viruses tested were Bav3-224/311 (lane 1), Bav3-311/382 (lane 2), Bav3-383/468 (lane 3), Bav3-467/541 (lane 4), Bav3-537/552 (lane 5) and Bav3-553/560 (lane 6). WT, Wild-type; MU, mutant.

 
Analysis of mutant viruses with progressive deletions
To characterize further the packaging motifs of BAdV-3, we constructed and analysed two additional sets of BAdV-3 mutants. One set contained mutant viruses with unidirectional deletions that progress from a common site at nt 224 towards the downstream border of the packaging domain. The data obtained with these mutants in virus infections are shown in Fig. 5(A, B). Bav3-224/382, which contains a deletion from nt 224 to 382, showed a 10-fold decrease in packaging ability in coinfection experiments when compared to wild-type virus. The deletion of an additional 87 nt (Bav3-224/468) resulted in a reproducible increase in packaging efficiency in coinfection experiments when compared with Bav3-224/382.



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Fig. 5. Analysis of mutant viruses carrying progressive deletions with a common start site at nt 224. (A) Mutant names, endpoints of deletions and in vivo packaging analysis are as described in the legend to Fig. 4(A). (B) Southern hybridization analysis of total and virion DNA isolated from VIDO R2 cells coinfected with wild-type and individual mutant viruses. Southern hybridization was performed as described in the legend to Fig. 4(B). The mutant viruses tested were Bav3-224/382 (lane 1), Bav3-224/468 (lane 2), Bav3-224/541 (lane 3), Bav3-224/552 (lane 4) and Bav3-224/560 (lane 5).

 
The deletion of an additional 74 nt (Bav3-224/541) resulted in a 25-fold decrease in packaging efficiency when compared to wild-type BAdV-3. Bav3-224/552, which carries an additional deletion between nt 541 and 552 on the background of Bav3-224/541, showed an 11-fold decrease in packaging efficiency. Bav3-224/560, containing the deletion between nt 224 and 560, displayed a phenotype similar to that of Bav3-224/552 in coinfection experiments.

Another set of mutant viruses contained viruses with unidirectional deletions that progress from a common site, nt 560 towards the upstream border of the packaging domain (Fig. 6A). The results are shown in Fig. 6(B). The deletion of sequences between nt 537 and 560 in Bav3-537/560 did not affect packaging efficiency. An additional deletion of 70 nt (Bav3-467/560) resulted in a 3-fold reduction in packaging efficiency. However, an additional deletion of 84 nt (Bav3-383/560) or 156 nt (Bav-311/560) did not lead to an increase in the reduction in packaging ability in coinfection experiments. In addition, the sequences between nt 541 and 560 appear to have no packaging function.



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Fig. 6. Analysis of mutant viruses carrying progressive deletions with a common start site at nt 560. (A) Mutant names, endpoints of deletions and in vivo packaging analysis are as described in the legend to Fig. 4(A). (B) Southern hybridization analysis of total and virion DNA isolated from VIDO R2 cells coinfected with wild-type and individual mutant viruses. Southern hybridization was performed as described in the legend to Fig. 4(B). The mutant viruses tested were Bav3-537/560 (lane 1), Bav3-467/560 (lane 2), Bav3-383/560 (lane 3) and Bav3-311/560 (lane 4).

 
Analysis of mutant viruses carrying double deletions
To evaluate further the sequences between nt 224 and 541, we constructed and analysed three mutant viruses with double deletions (Fig. 7A, B). Bav3-224/311 : 383/541 (containing deletions between nt 224 and 311 and between nt 384 and 541) showed an 8-fold reduction in packaging efficiency in coinfection experiments. Bav3-224/311 : 467/541 (containing deletions between nt 224 and 311 and between nt 467 and 541) showed a 5-fold reduction in packaging efficiency. Bav3-224/382 : 467/541 (containing deletions between nt 224 and 382 and between nt 467 and 541) showed a 7-fold reduction in packaging efficiency. The results suggested that the sequences between nt 311 and 383 are important, similar to the sequences between nt 382 and 467, for virus packaging.



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Fig. 7. Analysis of mutant viruses carrying double deletions. (A) Mutant names, endpoints of deletions and in vivo packaging analysis are as described in the legend to Fig. 4(A). (B) Southern hybridization analysis of total and virion DNA isolated from VIDO R2 cells coinfected with wild-type and individual mutant viruses. Southern hybridization was performed as described in the legend to Fig. 4(B). The mutant viruses tested were Bav3-224/311 : 383/541 (lane 1), Bav3-224/311 : 467/541 (lane 2) and Bav3-224/382 : 467/541 (lane 3).

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The packaging of adenovirus DNA requires recognition of specific viral DNA sequences by viral and cellular proteins (Schmid & Hearing, 1995). The cis-acting packaging motif(s) among a subgroup of HAdVs appear to be conserved (Schmid & Hearing, 1997). However, characterization of cis-acting packaging motif(s) of CAdV-2 (Soudais et al., 2001), a nonhuman adenovirus, suggests that packaging motifs may be different in nonhuman adenoviruses. The viral DNA elements involved in DNA packaging might have adapted to interact with species-specific viral and/or cellular factors. In this report, we describe the construction, characterization and analysis of BAdV-3 mutants for the identification of packaging sequences.

Although the primary sequence of cis-acting packaging motif(s) appears to be different, packaging motifs are usually located at the left end of the adenovirus genome between the left-end ITR and the ATG start codon of the E1A gene (Schmid & Hearing, 1997; Soudais et al., 2001). However, although Bav3-224/560, containing the largest deletion between nt 224 and 560, showed significantly reduced packaging efficiency, it still remained viable. These results suggest that upstream and downstream boundaries of the cis-acting packaging domain of BAdV-3 may be located in the left-end ITR and the E1A-encoding sequence, respectively. Despite the flexibility in the position and orientation of the packaging domain, it must be located within 600 bp of the genomic terminus of HAdV-5 (Hearing & Shenk, 1983; Hearing et al., 1987) for its proper function. This implied that the left-end ITR may be involved in the packaging of adenovirus (Schmid & Hearing, 1998). However, recent reports suggested that the ITRs are not required for the efficient packaging of HAdV-5 (Ostapchuk & Hearing, 2003). Taken together, these results suggest that some packaging motifs of BAdV-3 may be located downstream of nt 560.

Analysis of the DNA sequence of the left end of the BAdV-3 genome did not show any identity to the consensus sequence of the cis-acting packaging domain of HAdV-5 (Schmid & Hearing, 1997). Recently, we identified the packaging domain of porcine adenovirus type 3 (PAdV-3), which contains six AT-rich motifs flanked by GC-rich sequences (Xing & Tikoo, 2003). Mutational analysis of these motifs suggests that continuous AT nucleotides are more important for virus packaging than GC nucleotides (unpublished data). Interestingly, we found 20 AT-rich motifs between the left-end ITR and the ATG start codon of the E1A gene of BAdV-3. However, 18 of these AT-rich motifs are located between nt 224 and 541, a region that appears to contain packaging motif(s). Two of these AT-rich motifs, located between nt 541 and 560, do not appear to contain packaging motif(s). Aside from the AT-rich character, none of the potential packaging motifs of BAdV-3 shows any primary sequence identity. These results confirm earlier speculations (Grable & Hearing, 1990; Soudais et al., 2001) and suggest further that packaging of BAdV-3 may require recognition of overall DNA structure rather than the primary sequence of packaging motif(s).

In HAdV-5, the packaging domain is located upstream of the TATA box of the E1A promoter and overlaps two distinct enhancer elements (Hearing & Shenk, 1983, 1986). In PAdV-3, the packaging domain (Xing & Tikoo, 2003) overlaps the promoter region of E1A (Reddy et al., 1998, 1999b) and enhancer-like regulatory element(s) (unpublished data). Like HAdV-5 and PAdV-3, the packaging domain of BAdV-3 overlaps the transcriptional control region of E1A (Xing & Tikoo, 2003; unpublished data). This organization could correlate the regulation of E1 gene expression with the packaging process. As speculated previously (Grable & Hearing, 1990), the binding of trans-acting packaging proteins to the cis-acting packaging sequences at late times of infection may repress transcription by altering the conformation of viral DNA or preventing the interactions of transcriptional proteins with this region. If this is true, this may represent a common mechanism employed by these adenoviruses to control its replication cycle.

Although gutless or helper-dependent adenovirus vectors have demonstrated great promise in reducing the host immune response against virus-encoded proteins and extending the expression of therapeutic genes (Kochanek et al., 2001), isolation of a gutless vector without helper virus contamination has not been achieved. Based on the availability of the cis-acting packaging domain of BAdV-3 and the growth properties of BAdV-3 in human cells, a system can be developed whereby vector DNA is packaged specifically (Xing & Tikoo, 2003).


   ACKNOWLEDGEMENTS
 
We thank Wayne Connor and Caron Pyne for their technical assistance and Alexandre Zakhartchouk for valuable suggestions. This research was supported by a Discovery grant from the Natural Science and Engineering Research Council (NSERC) of Canada to S. K. T. Published as VIDO journal series no. 352.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 11 June 2003; accepted 3 August 2003.



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