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
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ABSTRACT |
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Present address: Aventis Pasteur Limited, Toronto, Canada.
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INTRODUCTION |
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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.
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METHODS |
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Construction of recombinant plasmids.
Plasmid pLtRtHind.Mod, which contains the 1·6 kb left-end fragment (nt 11653) and the 1·2 kb right-end fragment (nt 3323534446) 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, 3750 °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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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 1015 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 12 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 560839 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.
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RESULTS |
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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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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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DISCUSSION |
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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
).
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ACKNOWLEDGEMENTS |
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Received 11 June 2003;
accepted 3 August 2003.
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