1 Veterinary Medical Research Institute, Hungarian Academy of Sciences, PO Box 18, H-1581 Budapest, Hungary
2 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
3 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3
Correspondence
Gábor M. Kovács
gkovacs{at}vmri.hu
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ABSTRACT |
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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.
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INTRODUCTION |
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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
).
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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.
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METHODS |
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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).
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RESULTS AND DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 26 April 2004;
accepted 28 June 2004.