Journal of General Virology

DNA sequence of frog adenovirus

Andrew J. Davison,¹ Kathleen M. Wright¹† and Balázs Harrach²

¹ MRC Virology Unit, Church Street, Glasgow G11 5JR, UK
² Veterinary Medical Research Institute, Hungarian Academy of Sciences, H-1143 Budapest, Hungary

The genome of frog adenovirus (FrAdV-1) was sequenced and found to be the smallest of all known adenovirus genomes. The sequence obtained was 26163 bp in size and contains a substantial direct repeat near the right terminus, implying that it was derived by recombination from a parental genome of only 25517 bp. The closest relative of FrAdV-1 proved to be turkey adenovirus 3, an avian adenovirus with no previously known near relative. Sequence comparisons showed that the two viruses have equivalent gene complements, including one gene the product of which is related to sialidases. Phylogenetic analyses supported the establishment of a fourth adenovirus genus containing these two viruses, in addition to the established genera Mastadenovirus and Aviadenovirus and the proposed genus Atadenovirus. Sixteen genes were identified as being conserved between these four lineages and were presumably inherited from an ancestral adenovirus.

Members of the family Adenoviridae are harboured by the full range of vertebrates (reviewed in Russell & Benkö, 1999), including fish, frogs and reptiles (Clark et al., 1973; Hedrick et al., 1985; Jacobson et al., 1984, 1996; Bloch et al., 1986; Juhasz & Ahne, 1993; Cunningham et al., 1996). To date, molecular studies have focused almost entirely on adenoviruses infecting mammals, particularly humans, and, to a lesser extent, those infecting birds. Complete genome sequences are available for 15 members of the family, as listed in Table 1. In addition, partial genome sequences are available for other adenoviruses.

The International Committee on Taxonomy of Viruses currently recognizes two genera of the family Adenoviridae, Mastadenovirus and Aviadenovirus (Benkö et al., 2000). The former contains mammalian viruses, including those with human, bovine, canine, ovine, equine, porcine and murine hosts, and the latter contains avian viruses such as fowl adenovirus type 1 (FAdV-1; also known as CELO virus). Where DNA sequence data are available, members of the two genera are clearly distinct. A third group is now apparent, containing certain bovine and ovine adenoviruses (including OAdV-287) and the duck virus DAdV-1 (also known as egg drop syndrome virus) (Vrati et al., 1996; Harrach et al., 1997; Hess et al., 1997; Benkö & Harrach, 1998; Harrach & Benkö, 1998; Dán et al., 1998; Benkö et al., 2000; Barbezange et al., 2000; M. Benkö, D. M. Thomson, P. Élö, Á. Dán, R. Szathmáry, C. Barbezange, K. Ursu, J. Meers and B. Harrach, unpublished results). The corresponding genus has been proposed as Atadenovirus, in recognition of the high A+T content of the genomes of member viruses. This nomenclature is used in the following discussion.

Table 1. Features of sequenced adenovirus genomes

* Percentage of total nucleotides that are G or C.

† Observed total proportion of the CG dinucleotide expressed as a fraction of that expected from mononucleotide composition.

‡ Contains several ambiguities and apparent errors.

§ Contains a direct repeat. Minimal genome size would be 25517 bp.

Even within this enlarged scheme, the taxonomic position of one avian virus, turkey adenovirus 3 (TAdV-3; also known as haemorrhagic enteritis virus in turkeys or marble spleen disease virus in pheasants), is problematic. Analysis of the complete genome sequence indicates that this virus is not closely related in genome organization or predicted protein sequences to any of the three genera described above (Pitcovski et al., 1998). However, the marginally closer relationship of certain proteins to those of OAdV-287, plus a small, A+T-rich genome, led to the tentative designation of TAdV-3 as an atadenovirus (Benkö & Harrach, 1998).

In this paper, we report the complete genome sequence of frog adenovirus (FrAdV-1). We show that FrAdV-1 is a relative of TAdV-3 and use genome comparisons to update our understanding of the genetic content of TAdV-3 and to derive new insights into the genetic content and evolution of adenoviruses. Moreover, reconsideration of adenovirus phylogeny leads us to propose a fourth genus containing these two viruses.

Cells and virus. The TH-1 cell line (ATCC CCL-50), which originated from the heart tissue of the box turtle, was recovered at 20–25 °C in an atmosphere of air in basal essential medium with Hanks' basic salts solution, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 mM L-glutamine and 10 % (v/v) foetal calf serum. The cells were grown subsequently at 30–31 °C in at atmosphere of air supplemented with 10 % (v/v) CO₂ in Dulbecco's minimum essential medium with Earle's salts, 2.5 mM L-glutamine, non-essential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 % (v/v) foetal calf serum.

A lyophilized stock of FrAdV-1 (ATCC VR-896) was resuspended in 1 ml basal medium. Virus was passaged in TH-1 cells at 30–31 °C, starting with an initial inoculum of 0.1–0.2 ml added to a 25 cm² flask. Infected cells grew more slowly than uninfected cells and were subcultured approximately once a week, usually with the addition of uninfected cells at a ratio of 5:1 followed by replating at a density sufficient to permit further growth of the cells. Virus was isolated from infected cells and DNA was extracted from purified virions by proteolytic treatment and phenol extraction (Davison et al., 1993).

DNA sequencing. Random fragments of FrAdV-1 DNA were generated by sonication, cloned into bacteriophage M13mp19 and sequenced by conventional autoradiography procedures or by using an ABI Prism 377 DNA sequencer. Data were compiled by using Staden's sequence analysis program (Staden, 1987).

Authentic FrAdV-1 genome terminal fragments were not represented in the M13 library, presumably because the presence of residual terminal protein residues attached to the 5´ ends of virion DNA prevented ligation to the vector. Terminal fragments were instead isolated by treating an SphI digest of viral DNA with terminal transferase in the presence of dCTP (to 'tail' the 3´ ends of fragments with dC residues), annealing with pUC18 'tailed' at the PstI site with dG residues and obtaining appropriate clones by transformation of E. coli.

To obtain the finished sequence, each nucleotide was determined an average of 12.3 times and the entire sequence was obtained on both strands. The sequence has been deposited with the GenBank library under accession number AF224336.

DNA sequence and phylogenetic analyses. DNA sequence analyses were carried out with the GCG package (version 9.1). Phylogenetic calculations were performed with the PHYLIP program package version 3.572c (Felsenstein, 1989) on manually edited forms of amino acid sequence alignments produced by CLUSTAL W. PROTPARS was used for parsimony analysis, and PROTDIST (Dayhoff's PAM matrix) followed by FITCH (global rearrangements) was used for distance matrix analysis. For bootstrap analysis, SEQBOOT (with 100 datasets output) preceded the above calculations and, after performing them, CONSENSE was used to calculate the consensus tree. Phylogenetic relationships were visualized using TreeView (Page, 1996) as implemented previously (Harrach & Benkö, 1998). Applicable alignments, their edited format and the calculated trees are available in Newick and graphical formats (http://www.vmri.hu/~harrach).

Genome size, heterogeneity and nucleotide composition

FrAdV-1 DNA was prepared after six passages of the virus in cell culture, starting with the stock obtained from the ATCC. Restriction endonuclease digestion (data not shown) demonstrated the presence of BamHI fragments of 10.6, 10.0 and 5.6 kbp and SphI fragments of 14.2, 8.3 and 3.7 kbp, indicating that the genome size is approximately 26.2 kbp.

The DNA sequence obtained is 26163 bp in size and has a G+C content of 37.9 %. The inverted repeat at the genome termini (ITR) is only 36 bp long. The sequence information and the restriction endonuclease cleavage data indicated the presence of a region near the right genome end that comprises a direct repeat of an internal sequence: nucleotides 25519–26127 are an exact copy of 23631–24239, a region located immediately downstream of the fibre gene. This is equivalent to direct reiteration at the genome terminus of approximately the first third of the 1888 bp region between 23631 and 25518. Moreover, a partial copy of 27 bp of the ITR is located at 25485–25511, immediately upstream of this reiteration. This structure indicates that the sequenced genome was originally derived by recombination from one which lacked the repeated sequence and had a size of 25517 bp. All analyses were carried out using the sequence obtained, rather than that of the hypothetical 25517 bp genome, which may or may not have been present in the initial virus stock.

Size heterogeneity of the FrAdV-1 genome was indicated by restriction endonuclease analysis of viral DNA isolated during an independent series of seven passages, starting with the stock obtained from the ATCC. This DNA contained an extra 1.9 kbp near the right end of the genome, a size increment that is consistent with an additional reiteration of 23631–25518 (data not shown). This finding also indicated the likelihood that the FrAdV-1 genome as sequenced was present in the ATCC stock.

The 5´-CG dinucleotide is present in the FrAdV-1 genome at a little over half the frequency expected from the mononucleotide composition (Table 1). Four other adenoviruses listed in Table 1 exhibit a level of CG depletion similar to that of FrAdV-1: TAdV-3, canine adenoviruses types 1 and 2 (CAdV-1, CAdV-2) and OAdV-287. CG depletion in each of these genomes is essentially uniform (data not shown). The phenomenon of CG depletion in cellular genomes and in certain other DNA viruses is thought to be due to methylation of cytosine in the CG dinucleotide, followed by deamination to TG and fixation of the mutation by DNA replication. Thus, it is likely that these adenovirus genomes are exposed in their entirety to cytosine methylation at some point during their life-cycles.

Genetic complement

The deduced gene layout of FrAdV-1 is shown in Fig. 1 and details of the genes are listed in Table 2. Amino acid sequence comparisons indicated that, of the adenoviruses for which sequence data are available, TAdV-3 is the closest relative of FrAdV-1. Data for the TAdV-3 genome are also presented in Fig. 1 and Table 2. Overall, the two genomes are similar in size, that of FrAdV-1 constituting the smallest yet observed among the family Adenoviridae. They share the same genetic layout and are similar in nucleotide composition (Table 1).

Fig. 1. Comparison of gene organization in FrAdV-1 with that in other adenoviruses. Each genome is marked at 5 kbp intervals and the ITRs are shown as small rectangles at the genome ends. The FrAdV-1 genome as sequenced also has a partial copy of the ITR near the right end. Predicted protein-coding regions are depicted as open arrows in the appropriate orientation and open rectangles denote protein-coding exons. Shading indicates regions that are not conserved in all adenoviruses sequenced. Nomenclature is provided for all genes in conserved regions and for selected genes in non-conserved regions (dut, deoxyuridine triphosphatase; sial, putative sialidase; hyd, hydrophobic protein).

Extensive phylogenetic analyses showed that FrAdV-1 and TAdV-3 group together and are distinct from members of the genera Mastadenovirus, Aviadenovirus and Atadenovirus. As an example, a distance matrix analysis for the hexon protein is presented in Fig. 2. Analysis of all proteins previously considered appropriate for phylogenetic calculations (Harrach & Benkö, 1998) yielded the same four clusters of viruses. The phylogenetic data, in conjunction with the presence of lineage-specific genes (see below), support the establishment of FrAdV-1 and TAdV-3 as members of a fourth genus. Having drawn this conclusion, it is also clear from Fig. 2 that the two species exhibit significant evolutionary divergence within this genus, comparable to that between the more divergent members of other genera.

Table 2. Features of FrAdV-1 genes and their counterparts in TAdV-3

* From the first base of the initiation codon to the last base of the termination codon, irrespective of orientation. Exons are given for spliced genes as appropriate.

† Percentage amino acid sequence identity calculated using GAP at default settings. –, Insufficient similarity to allow sequence alignment.

‡ A version of ORF 7 that is modified near its 3´ end and lacks a stop codon is present in the repeated region at the right end of the genome.

Pitcovski et al. (1998) listed nine open reading frames (ORFs) in the TAdV-3 genome that are greater than 100 codons in size and lack counterparts in other adenoviruses (ORFs 1–8 and E3). On the basis of their conservation in FrAdV-1, we consider that four (ORFs 1, E3, 7 and 8) encode proteins. In each genome, ORF 1 encodes a protein that is significantly related to bacterial sialidases, the most similar being that of Clostridium sordellii. The relationship of the TAdV-3 ORF 1 protein to sialidases was not recognized previously. Conserved regions include four partially repeated elements noted in the C. sordellii protein by Rothe et al. (1989) and highlighted in Fig. 3. It is not known whether the adenovirus proteins function as sialidases, but it is intriguing that pathogen-specified sialidases have been implicated in many diseases, including those mediated by bacterial agents and influenza viruses.

Fig. 2. Distance matrix analysis of adenovirus hexon proteins. The final edited alignment had a length of 820 residues. The two established and two proposed genera are circled. Bootstrap values were calculated for 100 data sets and those relating to non-human adenoviruses are shown. The bar represents a divergence of 0.1 (i.e. 10 %) between pairs of sequences. Virus types are represented by shortened forms of the ICTV abbreviations (Benkö et al., 2000) containing the host designation and type number (B, bovine; C, canine; D, duck; E, equine; F, fowl; Fr, frog; H, human; M, murine; O, ovine; P, porcine; T, turkey). The recently characterized Odocoileus (black-tailed deer) adenovirus and a human adenovirus B strain are denoted Od1 and HCardosa, respectively. Data were accessed from the complete genome sequences listed in Table 1, with the addition of hexon sequences for: B4, AF036092; E1, L79955; E2, L80007; F10, U26221; H1, AF161559, X67709, Y17244; H3, X76549; H4, AF065062; H6, X67710, Y17245, AF161560; H7, X76551; H8, AF161561, AB023546, Y17246; H9, AF161562, X74657, Y17247; H16, X74662; H19, AF161565, X98359, Y17250; H37, AF161567, AB023555, Y17252; H41, X51783; H48, U20821; HCardosa, AJ012091; and Od1, AF198354.

The E3 genes of FrAdV-1 and TAdV-3 are so named because they are located in the region corresponding to that containing E3 genes in mastadenoviruses. However, the encoded proteins appear unrelated to mastadenovirus E3 proteins. Similarity between the FrAdV-1 and TAdV-3 E3 proteins is confined largely to 19 conserved cysteine residues. The ORF 7 and ORF 8 genes are followed in FrAdV-1, but not in TAdV-3, by a small ORF of 79 codons (25307–25068) that probably does not encode a protein. The five smallest ORFs (ORFs 2, 3, 4, 5 and 6) identified in the TAdV-3 sequence by Pitcovski et al. (1998) probably do not encode proteins. ORFs 2, 3, 5 and 6 overlap recognized protein-coding regions and are not conserved in FrAdV-1. TAdV-3 ORF 4 is also not conserved in FrAdV-1, but a shorter ORF that almost entirely overlaps it in another reading frame would encode a hydrophobic protein, as would a similarly located ORF in FrAdV-1. These proteins are not related significantly in amino acid sequence (Table 2), but their similarity in location, size and hydrophobicity profile indicates that they are likely to be genuine viral proteins. The comparative analysis also revealed coding regions expressed by splicing that were not identified previously in the TAdV-3 genome, the 33K gene and short upstream coding exons in the pTP and DBP genes. In addition, each genome contains a U exon and a 22K gene.

The U exon was first identified from an analysis of the human adenovirus type 40 (HAdV-40) sequence as a conserved feature of several mastadenoviruses (Davison et al., 1993). It is apparent as a coding region extending from an initiation codon to a splice donor site in the region immediately upstream of, and on the other strand from, the fibre gene. It is now apparent for the first time that a U exon is present in FrAdV-1 and TAdV-3 and also in all aviadenoviruses and atadenoviruses for which data are available. Its genome location in representatives of each genus is shown in Fig. 1. The alignments shown in Fig. 4 indicate that predicted amino acid sequences are conserved within, but not between, genera. In each case, the U exon is proposed to encode the N terminus of a protein, but downstream exons remain undefined. The current state of analysis indicates that the U exon is present in all sequenced adenoviruses except two mastadenoviruses, murine adenovirus type 1 (MAdV-1) (Meissner et al., 1997) and bovine adenovirus type 10 (BAdV-10) (K. Ursu, B. Harrach, B. Adair and M. Benkö, unpublished results). These observations are most satisfactorily interpreted as due to inheritance of an ancient exon, with loss in certain lineages. Expression of the U exon is yet to be investigated in detail in any adenovirus genome.

Fig. 3. Amino acid sequence alignment of the putative sialidases of FrAdV-1 and TAdV-3 with the sialidase of Clostridium sordellii. The most conserved region of each protein is shown: for C. sordellii, residues 41–284 of a 404 residue protein (P15698); for TAdV-3, residues 44–281 of a 517 residue protein; for FrAdV-1, residues 74–313 of a 568 residue protein. Fully conserved residues are shown in the consensus (con) line. Repetitive elements noted previously in the C. sordellii sequence are underlined.

Human adenoviruses potentially encode a protein (termed 22K) production of which would require the lack of internal splicing in the 33K protein-coding region and expression of a substantial coding sequence in the intron (Davison et al., 1993), yielding a protein that is identical to the 33K protein in its N-terminal sequence (see HAdV-40 in Fig. 1). Patterns of expression in this region of the genome are not fully understood for any adenovirus. The region appears to encode at least one protein in all adenoviruses (Fig. 1), but it is not clear in every case whether expression involves splicing in the protein-coding region (to yield 33K) or not (to yield 22K), or whether both forms are expressed. Against this complex background, it is evident that experimental data are required to resolve expression patterns in this region and to identify the most ancient genetic elements.

In addition to proposed splicing in the protein-coding regions of the pTP, DBP, U exon and 33K genes, the initiation codons of most FrAdV-1 and TAdV-3 genes with mastadenovirus counterparts that encode late proteins are preceded close upstream by potential splice acceptor sites, as expected. It is anticipated that these viruses express other coding and non-coding exons that cannot easily be located merely by examination of sequence data. Promoter elements, including the major late promoter, also could not be identified with confidence in the A+T-rich genomes.

Adenovirus evolution

Of the 22 genes proposed for FrAdV-1 and TAdV-3, 16 have counterparts in all sequenced adenoviruses. In general, conserved genes are those involved in DNA replication and virion formation. This constitutes strong evidence that extant adenoviruses evolved from an ancestral virus that contained these genes and was recognizably an adenovirus. Although not a complete gene, the U exon is probably also an ancient feature.

Fig. 4. Amino acid sequence alignments of polypeptides predicted to be encoded by the U exon. Conserved residues are shown in the 'con' line and are indicated in lower case for aviadenoviruses in the region for which the relevant FAdV-10 sequence is unavailable. Data were accessed from the complete genome sequences listed in Table 1, with the addition of partial sequences for: HAdV-3, X01998; HAdV-7, Z48954; BAdV-1, AF038868; BAdV-2, S75673; porcine adenovirus type 1 (PAdV-1), L43364; PAdV-2, L43365; PAdV-4, L23218; FAdV-8a, U40587; FAdV-10, AF006739; and BAdV-4 (Á. Dán, M. Benkö, A. Bánrévi, Gy. Berencsi and B. Harrach, unpublished results). Single frameshift errors are proposed in the BAdV-1 and PAdV-1 sequences. The polypeptide sequence in this region for HAdV-41 (M85254) is identical to that of HAdV-40 and those for HAdV-11 (L08232) and HAdV-35 (U32664) are identical to that of HAdV-7.

The genome comparisons shown in Fig. 1 highlight the evolution of lineage-specific genes at the genome ends. For example, at the left end, these include the E1A genes in mastadenoviruses, the p32K gene in atadenoviruses and the sialidase-related gene in the fourth genus. Evidence that mastadenoviruses and atadenoviruses may have diverged more recently than the other genera is based on weak similarities between the E1B genes and between one of the E4 genes (encoding the 34K protein). In addition, lineage-specific, unrelated genes have arisen in the E3 region of mastadenoviruses and in FrAdV-1 and TAdV-3. It is quite possible that the common ancestor also had genes in these regions (perhaps evolutionarily unrelated to those in extant adenoviruses), but it is likely that an earlier progenitor lacked them and had a genome of approximately 20 kbp. Additional genes are present at the gene V and IX loci of mastadenoviruses and probably arose at an early stage within that lineage.

The means by which lineage-specific genes have developed is obscure. Gene capture is evidenced by the presence of a deoxyuridine triphosphatase (dut) gene in aviadenoviruses (Chiocca et al., 1996; Fig. 1) and possibly in certain mastadenoviruses and the sialidase-related gene in FrAdV-1 and TAdV-3. Indeed, this may be the major means whereby additional genes have been incorporated, followed by the development of novel functions through divergence. Other possible mechanisms include generation of genes de novo and duplication and divergence of genes that were either captured or generated de novo.

The phylogeny of mastadenoviruses (Fig. 2) is largely similar to that of their hosts, supporting the view that most of these viruses have evolved exclusively with their hosts or have switched hosts only between closely related species. The relationships within the atadenoviruses or the fourth genus do not fit comfortably with this model, however, and instead suggest that certain lineages in both genera may have originated with drastic interspecies transmission events. In exploring this further, it is worthwhile recollecting that Clark et al. (1973) isolated FrAdV-1 from a renal tumour obtained from a leopard frog (Rana pipiens) by growth on a reptilian cell line (TH-1) known to be particularly susceptible to adenovirus infection. The virus did not cause cytopathic effect on any other reptilian cell line or on cell lines of mammalian, avian, fish or amphibian origin, and did not result in pathology when injected into tadpoles or into chick embryos. Consequently, these workers undertook reisolation experiments to confirm that the virus originated from the tumour. Their ability to isolate the agent repeatedly and only from the tumour tissue constitutes good evidence that FrAdV-1 is indeed a frog adenovirus. Nevertheless, the current lack of additional information about this or other amphibian adenoviruses does leave a grain of doubt. This prompts caution in regard to the proposal that interspecies transmission events are of evolutionary significance within the fourth genus. It does not, however, compromise the important insights gained from the FrAdV-1 sequence into adenovirus phylogeny and gene content.

In conclusion, robust evaluation of the parts played by coevolution and interspecies transmission requires the derivation of a timescale for adenovirus evolution and further sampling of adenovirus genomes, especially in the more sparsely populated genera. It is encouraging that the amount of available adenovirus sequence data is approaching that required for such an evaluation and that studies of other amphibian, reptilian and fish adenoviruses are likely to extend our understanding of ancient events in adenovirus evolution.

We are grateful to Duncan McGeoch for critical reading of the manuscript. The Hungarian part of the work was supported by grant OTKA T022405.

† Present address: Police Forensic Science Laboratory (Dundee), Tayside Police Headquarters, West Bell Street, Dundee DD1 9JU, UK

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