Analysis of the first complete genome sequence of an Old World monkey adenovirus reveals a lineage distinct from the six human adenovirus species

Abstract

Simian adenovirus 3 (SAdV-3) is one of several adenoviruses that were isolated decades ago from Old World monkeys. Determination of the complete DNA sequence of SAdV-3 permitted the first full genomic comparison of a monkey adenovirus with adenoviruses of humans (HAdVs) and chimpanzees, which are recognized formally as constituting six of the species (HAdV-A to HAdV-F) within the genus Mastadenovirus. The SAdV-3 genome is 34 246 bp in size and has a G+C content of 55·3 mol%. It contains all the genes that are characteristic of the genus Mastadenovirus and has a single VA-RNA gene and six genes in each of the E3 and E4 regions. The genetic organization is the same as that of HAdV-12, a member of the HAdV-A species. Phylogenetic analyses showed that although SAdV-3 is related marginally more closely to HAdV-A and HAdV-F than to other species, it represents a unique lineage that branched at an early stage of primate adenovirus divergence. The results imply that the genetic layout in SAdV-3 and HAdV-12 may also have characterized the common ancestor of all sequenced primate adenoviruses.

The GenBank/EMBL/DDBJ accession number for the SAdV-3 DNA sequence reported in this paper is AY598782. Accession numbers of Third Party Annotations of the HAdV-1, HAdV-7, HAdV-35 and BAdV-1 DNA sequences are BK005234, BK005235, BK005236 and BK005277, respectively.

Members of the family Adenoviridae are non-enveloped, icosahedral viruses with linear, double-stranded DNA genomes that range in size from 26 to 45 kbp (Davison et al., 2003a). They infect a wide range of hosts from all main vertebrate groups (Benk & Harrach, 2003; Benk et al., 2000; Davison et al., 2000; Farkas et al., 2002; Kovács et al., 2003). Common features of genome organization are apparent at various taxonomic levels in the family (Davison et al., 2003a). Adenoviruses have been used as model organisms in molecular virology and important biological advances have emerged from studies of their interactions with cells. They have also formed the basis of vector systems for virus-based gene therapy and vaccination strategies (Russell, 2000).

There are five major phylogenetic lineages within the adenoviruses (Kovács et al., 2003), four of which are accepted as official genera. The genus Mastadenovirus contains all known human and chimpanzee adenoviruses as a monophyletic group, divided into the six species HAdV-A to HAdV-F (Benk et al., 2000; Davison et al., 2003a; Shenk, 2001). To date, the complete genome sequences of 14 members of this group have been released, with at least one from each species (Table 1). Many adenoviruses of apes and Old World monkeys [collectively termed simian adenoviruses (SAdVs)] have been detected or isolated (Kalter et al., 1997; Kilbourn et al., 2003; Mautner, 1989; and references therein). SAdVs have been divided into groups on the basis of haemagglutination properties (see references in the paper by Mautner, 1989).

Table 1. Available genome sequences of human and chimpanzee adenoviruses

Although early findings on the oncogenicity of SAdVs (Hull et al., 1965) induced some work on genome characterization (e.g. Dimitrov et al., 1979; Naroditsky et al., 1978), attention diminished until a recent revival of interest in chimpanzee adenoviruses (Roy et al., 2004). The E1A, E1B 19K and E1B 55K genes and the inverted terminal repeat sequences have been used for limited phylogenetic comparisons involving monkey adenoviruses (Avvakumov et al., 2002; Bailey & Mautner, 1994) and the most comprehensive analysis was based on the virus-associated RNA (VA-RNA) genes (Kidd et al., 1995). The monkey adenoviruses seem to have a closer relationship with the HAdV-A and HAdV-F species (Bailey & Mautner, 1994; Kidd et al., 1995), whereas the chimpanzee adenoviruses belong to the HAdV-B and HAdV-E species (Roy et al., 2004). The results of a preliminary analysis of a small region of the hexon gene in 25 SAdV strains supported these conclusions (Harrach et al., 2003).

The main aim of the present work was to determine the first complete genome sequence of an Old World monkey adenovirus and to compare it with available human and chimpanzee adenovirus sequences. SAdV-3 was chosen because in a pilot study that focused on a short part of the hexon gene, this strain clustered into a monophyletic group of monkey adenoviruses in haemagglutination group II (Harrach et al., 2003). This virus (originally called S.V.15) was isolated by Hull et al. (1956) during tissue culture of rhesus macaque kidney cells; antibodies against it are common in primates (Mautner, 1989). Our analysis showed unambiguously that SAdV-3 represents a unique lineage that is distinct from the six HAdV species.

Cells and virus.
Rhesus macaque kidney cells (LLC-MK2, ATCC CCL-7) were grown at 37 °C in an atmosphere of 5 % (v/v) carbon dioxide in Dulbecco's minimum essential medium (DMEM) supplemented with 100 IU penicillin ml¹, 100 µg streptomycin ml¹ and 10 % (v/v) fetal calf serum. A lyophilized stock of SAdV-3, purchased from the American Type Culture Collection (VR-1449), was resuspended in 1 ml DMEM and passaged in LLC-MK2 cells, starting with an initial inoculum of 0·10·2 ml added to a 25 cm² flask.

Virus DNA was isolated by using a modification of the method of Hirt (1967). Cells (approx. 10⁶) in a 25 cm² flask were infected with SAdV-3 and, 24 h after infection, were pelleted and resuspended in 100 µl medium. The resuspended cells were mixed with 200 µl extraction buffer [400 mM NaCl, 5 % (v/v) Triton X-100, 50 mM Tris/HCl (pH 7·5)], incubated at 4 °C for 30 min and centrifuged at 4000 g for 15 min at 4 °C. After transferring the supernatant to a new tube, SDS and proteinase K were added to final concentrations of 1 % (w/v) and 0·8 mg ml¹, respectively, and the mixture was incubated at 37 °C for 1 h. Nucleic acids were extracted once with phenol and twice with chloroform, ethanol-precipitated and resuspended in a small volume of 10 mM Tris/HCl (pH 7·5), 1 mM EDTA.

DNA sequencing.
A random library of SAdV-3 DNA fragments was prepared by using a Novagen M13mp18 Perfectly Blunt Cloning kit and sequenced by using an ABI PRISM 377 instrument. Regions of ambiguity were resolved by preparing three to four independent clones of PCR products in vector pGEM-T (Promega) and sequencing the inserts on both strands by using an ABI 3100 sequencer. The sequence database was compiled from electropherograms by using Pregap4 and Gap4 (Staden et al., 2000) and Phred (Ewing & Green, 1998; Ewing et al., 1998). Each nucleotide in the genome was determined an average of eight times and the entire sequence was obtained on both strands. The purity of the SAdV-3 DNA was estimated to be approximately 6065 %, from the proportion of random clones whose sequences matched the SAdV-3 genome.

Phylogenetic analysis.
The genetic content of SAdV-3 was deduced by comparison with previously sequenced primate adenoviruses [utilizing the Third Party Annotations (TPAs) in Table 1] and predicted amino acid sequences were used for phylogenetic analyses. Additional sequences were obtained from GenBank and aligned by using MultAlin (Corpet, 1988). Genealogies were inferred by using MEGA 2.1 (Kumar et al., 2001) with the Poisson correction model (Nei & Kumar, 2000) and deletion of residues at gaps. Trees were tested by bootstrapping with 1000 replications and visualized and edited by using the Tree Explorer facility of MEGA 2.1. Analysis by the unweighted pair-group method with arithmetic mean analysis (UPGMA) was also carried out to study protein distances among HAdV and SAdV strains. This approach reliably represents the distance relations among the groups and strains. Variable evolutionary rates along sequence alignments were checked by calculating entropy by using the DAMBE program (Xia & Xie, 2001) and the results were visualized with STATISTICA 6.0 (StatSoft).

Table 1 compares genomic features of sequenced members of the HAdV species, Table 2 lists characteristics of the SAdV-3 genome that can be deduced from the sequence and Fig. 1 shows its genetic layout. The genome of SAdV-3 is similar in size (34 246 bp) to the genomes of HAdV-40 and HAdV-12 (Table 1). Chimpanzee adenoviruses have larger genomes than SAdV-3: that of SAdV-21, which belongs to HAdV-B, is larger by 1278 bp and those of other chimpanzee viruses, which belong to HAdV-E, are larger by 22162358 bp. The G+C content of the SAdV-3 genome is 55·3 mol%, which is closest to that of HAdV-C. In a wider context, differences in base composition between genomes can be detected on various taxonomic levels and may result from mutational bias or selection processes (see Mooers & Holmes, 2000; and references therein). In viruses, such differences may be associated with the hosts (e.g. flaviviruses; Jenkins et al., 2001). However, the G+C content of HAdV genomes is known to correlate with the viral species, rather than with the host. This is clear in the case of HAdV-B and HAdV-E, where both human and chimpanzee adenoviruses cluster (Table 1). Indeed, the fact that one of the adenovirus genera (Atadenovirus) is named after the low G+C content of its members' genomes (Benk & Harrach, 1998) attests to the historical importance of this feature in the taxonomy of the family (Benk et al., 2000). At an intragenomic level, nucleotide composition varies along the SAdV-3 genome, with the region from the 52K to pVI genes having the highest G+C content and the E3 region the lowest (Table 2). A 35 mol% range in G+C content is apparent between complete coding regions with extreme values (pVII and E3 CR1-β1).

Table 2. Features of SAdV-3 genes

(9K):

Fig. 1. Genetic content of SAdV-3. The genome is depicted as a central horizontal line marked at 5 kbp intervals, with the E1A, E1B, E3 and E4 regions shaded. Protein-coding regions are shown as arrows (for entire coding regions or 3'-coding exons) or rectangles (for other exons).

SAdV-3 exhibits all the principal genomic features of the genus Mastadenovirus (Wadell, 2002). The characteristic, genus-specific genes in the E1A, E1B, E3 and E4 regions, plus the IX and V genes, are present. The main differences among the HAdV species concern the number of genes in the VA-RNA, E3 and E4 regions (Bailey & Mautner, 1994; Davison et al., 2003a, b; Kidd et al., 1995; Ma & Mathews, 1996; Ursu et al., 2004). VA-RNAs are short regulatory RNAs, transcribed by RNA polymerase III, that are found in the cytoplasm of adenovirus-infected cells (Kidd et al., 1995; Ma & Mathews, 1996; Mathews & Shenk, 1991). Primate adenoviruses contain one or two tandem VA-RNA genes upstream of 52K and single VA-RNA genes have also been identified at different genome locations in one member each of the genera Aviadenovirus and Atadenovirus (Hess et al., 1997; Larsson et al., 1986). SAdV-3 has one VA-RNA gene (Kidd et al., 1995) upstream of 52K, like HAdV-A, HAdV-F and a subgroup of HAdV-B viruses (Kidd et al., 1995; Ma & Mathews, 1996). At the level of gene organization, the E3 and E4 regions are the most variable parts of HAdV genomes (Bailey & Mautner, 1994; Davison et al., 2003a, b; Ursu et al., 2004). They are a focus of interest, as the genes therein are involved in interactions between the virus and its host and are often removed or manipulated during vector development (Horwitz, 2004; Leppard, 1997). SAdV-3 has counterparts of all six HAdV E4 genes (five in the case of HAdV-40, which appears to have lost E4 ORF1). The six genes in the SAdV-3 E3 region are homologous to the six HAdV-12 E3 genes and five have counterparts in HAdV-40 (which appears to have lost E3 12.5K). HAdV-C has seven E3 genes, but three lack detectable homologues in SAdV-3. HAdV-D has eight E3 genes, whereas HAdV-B and HAdV-E have nine (Davison et al., 2003a). SAdV-3 has one fibre, which is shorter that in HAdV-12 which has 22 fibre shafts, similarly to HAdV-2 (van Raaij et al., 1999), whereas SAdV-3 seems to have only 19. In summary, the gene layout in the SAdV-3 genome is the same as that in HAdV-12, which belongs to species HAdV-A, and similar to that in HAdV-40, which belongs to species HAdV-F. However, the G+C content of the SAdV-3 genome is significantly different from those of both species (Table 1).

The adenovirus protease cleaves precursor proteins (denoted by a p suffix) in steps that are required for appropriate functioning of the mature proteins (Weber, 1995). Based on known and predicted cleavage sites for the protease, consensus motifs consisting of (M/L/I/V/F)XGG'X and (M/L/I/V/N/Q)X(A/G)X'G have been described (Anderson, 1990; Davison et al., 2003a; Farkas et al., 2002; Vrati et al., 1996; Webster et al., 1989). These sites can be considered as shared derived characters in studies of adenovirus phylogeny (Benk & Harrach, 2003; Farkas et al., 2002). Putative protease cleavage sites were identified in all the precursor proteins of SAdV-3. pTP and pIIIa have three sites each, and pX and pVI have two. Grydsuk et al. (1996) mapped one common epitope in pVI from HAdV-A and HAdV-F and one unique epitope from HAdV-F. The common epitope (EEKLPPLE) is present in pVI of SAdV-3, with the K substituted by an N residue. The mature product of pVII (VII) enters the host-cell nucleus with virus DNA during infection and may bind to human chromosomes (Lee et al., 2003). It is formed by cleavage of a 24 aa N-terminal segment from pVII; a second cleavage site within this segment has been identified and shown to be conserved among adenoviruses (Vrati et al., 1996).

The N-terminal fragment (VIII) that is produced by cleavage of pVIII by the protease is a structural component that associates with the internal face of hexon molecules and stabilizes the capsid (Everitt et al., 1975; Hannan et al., 1983; Liu et al., 1985; Weber, 1976). Fig. 2 shows an amino acid sequence alignment of the central region of pVIII from various members of the genus Mastadenovirus. This region contains three putative cleavage sites although, in many cases, the two types of consensus overlap. The extent of variation along pVIII from primate adenoviruses is illustrated in Fig. 2 and shows strong conservation, not only of the N-terminal product of pVIII (i.e. VIII), but also of the C-terminal sequence. This indicates that the C-terminal sequence is likely to have an important function, either prior to cleavage or as a mature product. The 2526 residue fragment that would be generated by cleavage at the N-terminal and central sites (the precise size depends on which of the overlapping motifs at the latter directs cleavage) is only one to two residues longer in HAdV-40 and HAdV-12, but five residues shorter in the other primate adenoviruses. The variability of the amino acid sequence encoded by this region may reflect the fact that the DNA sequence is bifunctional, as it also contains the TATA element of the E3 promoter.

Table 1, with additional data from: HAdV-4, AF361223; HAdV-21, AB073222; PAdV-3, AF083132; CAdV-1, Y07760; CAdV-2, U77082; BAdV-1, BD269513; BAdV-2, AF252854; PAdV-5, AF289262; PAdV-4, L23218; TSAdV-1, AF258784; and MAdV-1, NC_000942. The TPAs prepared by Davison et al. (2003a) were consulted when necessary.

Genealogies were calculated for the amino acid sequences that are encoded by all SAdV-3 genes, in comparison with their homologues in human and chimpanzee adenoviruses. They showed similar phylogenetic relationships, with differences (if apparent) in early branches, where topologies were generally not supported by high bootstrap values. The results of the analyses of four genes, which represent the most frequently observed topologies and were relatively well-supported, are shown in Fig. 3. The overall phylogenies support earlier findings (Bailey & Mautner, 1994; Kidd et al., 1995), indicating that HAdV-B, HAdV-E and HAdV-D (the former two species including chimpanzee adenoviruses) are related more closely to each other than they are to HAdV-A and HAdV-F, which are closer to the monkey adenoviruses. Phylogenetic analyses consistently demonstrated the unambiguous separation of SAdV-3 from the HAdV species. All of the genes studied showed the same distance relationships as the four that are presented in Fig. 3, with the distances between SAdV-3 and the HAdV species always being significantly greater than the smallest distance between HAdV species. SAdV-3 is thus a novel species that represents an early branch of the primate adenovirus lineages. The observation that SAdV-3 has the same genetic content as HAdV-12 (and HAdV-40, taking into account the loss of a gene in each of the E3 and E4 regions and the gain of a second fibre gene) suggests that this layout may have been present in the common ancestor of all primate adenoviruses for which genome sequences are available. Sequencing of additional monkey adenovirus genomes would allow this hypothesis to be tested.

Table 1).

Technical advice contributed by C. Cunningham and A. Dolan is acknowledged gratefully. Part of this work was conducted during a period of study by G. M. K. at the MRC Virology Unit, sponsored by the Federation of European Microbiological Societies. The work was also supported by Hungarian research grants OTKA T043422 and MEH 4767/1/2003.

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Received 26 April 2004; accepted 28 June 2004.