Department of Biological Sciences, University of South Carolina, Columbia
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Abstract |
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Key Words: Baculoviridae chitinase gene capture horizontal gene transfer UDP-glucosyl transferase viral evolution
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Introduction |
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There is evidence that a similar phenomenon has occurred in the evolutionary history of the Baculoviridae, a family of double-stranded DNA, no RNA-stage viruses parasitic on members of the phylum Arthropoda (Miller 1997; Blissard et al. 2000). Certain Baculoviridae are known to express genes belonging to the inhibitor of apoptosis (IAP) family found also in both vertebrates and insects (Crook, Clem, and Miller 1993; Clem and Miller 1994). These proteins suppress the apoptotic responses that are part of the insect's defense response against viruses (Clem, Fechhemer, and Miller 1991). Phylogenetic analysis supported the hypothesis that baculovirus IAP genes originated by capture of host genes, which occurred on at least two separate occasions in the evolution of this viral family (Hughes 2002b).
In the present paper, we used genome-wide homology searches in a set of 13 complete genomes from the viral family Baculoviridae to discover all genes that may have been horizontally transferred from cellular organisms. We were particularly interested in transfers of genes potentially important in the infection process and in genes captured from the insect host. For all gene families containing potential horizontal transfers, we used phylogenetic analysis to reconstruct the evolutionary relationships among the genes. By comparing these phylogenies with phylogenies of the viruses reconstructed on the basis of a set of conserved gene families shared by all of the baculovirus genomes analyzed and with known phylogenies of cellular organisms, we inferred the source and time of acquisition of horizontally transferred baculovirus genes relative to major cladogenetic events in the phylogeny of these viruses and in that of their hosts.
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Methods |
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The following is a list of the 22 conserved families shared by the 13 species of Granulovirus and Nucleopolyhedrovirus, with accession numbers for the protein translation from AcMNPV: polyhedrin (NP_054037); unknown protein (NP_054122); late expression factor 4 (NP_054120); unknown protein (NP_054111); very late expression factor 1 (NP_054107); p49 (NP_054173); DNA polymerase (NP_054095); late expression factor 8 (NP_054079); late expression factor 9 (NP_054092); occlusion-derived virus envelope protein ODV-E56 (NP_054179); alkaline exonuclease (NP_054163); virion protein vp1054 (NP_054084); serine-threonine kinase (NP_054039); transcriptional regulator (NP_054069); p48 (NP_054133); late expression factor 5 (NP-054129); 38k protein (NP_054128); helicase (NP_054125); capsid-associated protein vp91 (NP_054113); unknown protein (NP_054051); occlusion-derived virus envelope protein p74 (NP_054168); unknown protein (NP_054149).
Families Shared with Cellular Organisms
We used BlastP with an E-value of 10-20 to search all predicted proteins from the set of baculovirus genomes against the following sequences of cellular organisms: the set of complete protein translations (proteome) for Saccharomyces cerevisiae from http://genome-www.stanford.edu/Saccharomyces; for Arabidopsis thaliana from http://www.tigr.org; for Caenorhabditis elegans from http://www.sanger.ac.uk/C_elegans; and for Drosophila melanogaster from ftp://ftp.ebi.ac.uk/pub/databases/edgp/sequence_sets. Proteome data sets from 38 complete genomes of bacteria and all available sequences from a set of other representative organisms (the slime mold Dictyostelium discoideum, the zebrafish Danio rerio, the pufferfish Takifugu rubripes, the frog Xenopus laevis, the human Homo sapiens, and the mouse Mus musculus) were obtained from the National Center for Biotechnology Information (NCBI). A relatively strict homology search criterion was used because preliminary analyses showed that phylogenetic analyses using families assembled by less strict criteria were unlikely to show good resolution of deep branching patterns.
In each case where homology search showed evidence of homology between a baculovirus protein and one or more proteins from cellular organisms, we conducted additional homology searches against the complete NCBI nonredundant database to identify additional potential homologues. For these families, phylogenetic analyses were conducted initially including all homologs identified by these searches. For ease of presentation in figures, only selected sequences were used in the phylogenetic analyses presented here. However, the overall phylogenetic patterns presented were similar when larger data sets were used (data not shown). Organisms used in phylogenetic analyses presented here are listed in table 1.
Phylogenetic Methods
Amino acid sequences were aligned using the ClustalW program (Thompson, Higgins, and Gibson 1994). All alignments are available from the authors by request. In phylogenetic analyses, any site at which the alignment postulated a gap in any sequence was excluded from all pairwise comparisons so that a comparable set of sites was used for each comparison.
Phylogenetic analyses were conducted by three methods: (1) minimum evolution (ME) (Rzhetsky and Nei 1992); (2) maximum parsimony (MP) (Swofford 2000); and (3) quartet maximum likelihood (QML) (Strimmer and von Haeseler 1996), using the TreePuzzle-5.0 program. ME trees were based on the gamma-corrected amino acid distance (Ota and Nei 1994), using the parameter for the gamma distribution estimated by TreePuzzle-5.0. QML trees were based on the JTT model of amino acid sequence evolution (Jones, Taylor, and Thornton 1992) with rates assumed to vary among sites according to a gamma distribution. Nei, Xu, and Glazko (2001) provide theoretical justification for use of gamma distances for concatenated amino acid sequences.
The significance of internal branches in ME trees was tested by the standard error test, and the standard errors of branch length were computed by the bootstrap method implemented in the MEGA2 program (Kumar et al. 2001). The reliability of each internal branch in MP trees was assessed by bootstrapping; 1,000 bootstrap samples were used. The reliability of each internal branch in QML trees was assessed by the proportion of 10,000 puzzling steps supporting the branch (Strimmer and von Haeseler 1996).
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Results |
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DNA Ligase
In the DNA ligase family, homologs were found in all three species of Granulovirus but in only one species of Nucleopolyhedrovirus, LdMNPV (fig. 2). However, the sequence from LdMNPV did not cluster with the Granulovirus sequences (fig. 2). Although the rooting of the tree was not certain, there were two major clusters separated by a significant internal branch (fig. 2). One cluster included DNA ligase of Granulovirus and DNA ligase I of eukaryotes; the other cluster included DNA ligase of LdMNPV, DNA ligase of poxviruses, and DNA ligase III of eukaryotes (fig. 2).
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IAP
Because the phylogeny of baculovirus inhibitor of apoptosis (IAP) was the subject of a recent paper (Hughes 2002b), the phylogenetic tree is not shown here. In the phylogentic tree of IAP, baculovirus IAP clustered with those of insects, and at least two separate events of transfer of genes in this family from insect hosts to baculoviruses were supported (Hughes 2002b).
Ribonucleotide Reductase 1
In the phylogeny of ribonucleotide reductase 1, there were two major clusters, separated by a highly significant internal branch (fig. 3). One cluster included sequences from one species of Granulovirus (CpGV) and two species of Nucleopolyhedrovirus (LdMNPV and OpMNPV), together with bacterial sequences from the Firmicutes (fig. 3). The other cluster included sequences from Archaea, from other groups of bacteria, from eukaryotes, and from poxviruses, along with sequences from two species of Nucleopolyhedrovirus, SeMNPV and SlMNPV (fig. 3). Within the latter cluster, SeMNPV fell outside a cluster that included SlMNPV and sequences from insects and other animals, which was supported by a highly significant internal branch (fig. 3). Ribonucleotide reductase 1 from SlMNPV clustered with those of insects, although the branch supporting this pattern was not significantly supported (fig. 3).
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Chitinase
Chitinase sequences from Granulovirus and Nucleopolyhedrovirus formed a monophyletic group that clustered with sequences from the gamma subdivision of Proteobacteria, and this pattern was supported by a highly significant branch (fig. 4). A chitinase from a member of the viral family Phycodnaviridae (PbCV1) fell outside the cluster of sequences from baculoviruses and the gamma division of Proteobacteria, as did other bacterial sequences and eukaryotic sequences (fig. 4). Thus, the phylogeny supported the hypothesis that the gene encoding chitinase was transferred from the gamma division of Proteobacteria to the common ancestor of Granulovirus and Nucleoplyhedrovirus. In the phylogenetic tree, chitinases of baculoviruses clustered with one clade of bacterial chitinases but within a larger clade that included other bacterial sequences (fig. 4). This topology supports the hypothesis that horizontal gene transfer occurred from bacteria to baculoviruses rather than in the opposite direction.
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Discussion |
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In a survey of the complete genomes of the 13 species of Granulovirus and Nucleopolyhedrovirus, six protein families showed phylogenetic tree topologies indicative of horizontal gene transfer. In the case of DNA ligase, the phylogenetic tree showed two clusters, each of which contained sequences from both baculoviruses and cellular organisms (fig. 2). Theoretically, this topology is consistent with the hypothesis that horizontal transfer occurred from baculoviruses to cellular organisms. However, given the widespread occurrence of DNA ligases in all groups of eukaryotes, the hypothesis that horizontal transfer occurred from eukaryotes to DNA viruses seems much more plausible. By contrast, in the other five families, the baculovirus sequences fell within clusters of sequences from cellular organisms (figs. 36 and Hughes 2002b). Thus, in these families, the phylogenies supported horizontal transfer from cellular organisms to viruses, rather than in the opposite direction.
Two of the six families showing evidence of horizontal gene transfer, DNA ligase and ribonucleotide reductase 1, play roles in the metabolism of DNA. The animal lodestar protein, of which a homolog is found in certain baculovirus species, is involved in transcription termination (Liu, Xie, and Price 1998). The other three families for which there is evidence of horizontal transfer are all known to be directly involved in the infection process. The role of baculovirus IAP in suppressing the insect host's apoptotic defense response (Clem, Fechhemer, and Miller 1991) has been mentioned previously. Chitinase breaks down chitin, the polysaccharide that is a major constituent of arthropod cuticle. During infection of insect cells by AcMNPV, chitinase is localized mainly in the endoplasmic reticulum and is released into the extracellular medium on cell lysis. There it is important for the liquefaction of tissues that characterizes infection of insect larvae by these viruses (Savile et al. 2002). Baculovirus UDP-glucosyltransferase is involved in viral suppression of host metamorphosis through inactivation of host ecdysosteroid hormones (Evans and O'Reilly 1999).
Phylogenetic analyses provided evidence that genes encoding baculovirus IAP (Hughes 2002b) and UDP-glucosyltransferase (fig. 6) were horizontally transferred from insects. These transfers thus presumably occurred after the ancestor of baculoviruses began to infect insects, and the acquisition of these genes improved the adaptation of this family of viruses to their insect hosts. The baculovirus gene encoding chitinase, by contrast, was shown to be of bacterial origin, being derived from an ancestor from the gamma division of Proteobacteria. Hatwin et al. (1995) previously proposed that the baculovirus chitinase gene was transferred from a species of Proteobacteria. However, they included only one baculovirus chitinase in their phylogenetic analysis, and that sequence clustered with a bacterial chitinase from the genus Serratia (Hatwin et al. 1995). Thus, their tree suggested a transfer directly from one bacterial species. By contrast, our analysis included a much larger number of species, and baculoviruses chitinases clustered with a group of species from the gamma division of Protobacteria. This result places the horizontal transfer event before speciation of this group of bacteria. Hatwin et al. (1995) speculated that since Proteobacteria include enteric pathogens of insects, horizontal transfer from bacteria to virus may have occurred in the insect host.
The evolutionary origin of viruses remains mysterious (Morse 1994). Since all viruses are parasitic on cellular organisms and require molecular machinery produced by their cellular hosts in order to complete their life cycles, it seems reasonable to hypothesize that viruses originated after cellular organisms and indeed that they originated from genetic elements originally present in cellular organisms. However, it is uncertain how many independent evolutionary events have given rise to new viral lineages. Strauss and Strauss (1998) proposed that all or most RNA viruses constitute a monophyletic group and thus have a common origin. On the other hand, the monophyly of the large DNA viruses (double stranded DNA, no RNA-stage viruses) is much more difficult to establish. Iyer, Aravind, and Koonin (2001) have recently proposed that four families of large DNA viruses (Poxviridae, Asfarviridae, Iridoviridae, and Phycnoviridae) share a common origin. However, these authors did not test this hypothesis with phylogenetic methods; and, even if their hypothesis is correct, the relationship of these four families to other large DNA viruses, including Baculoviridae, remains uncertain.
Phylogenies of genes of large DNA viruses in the families Poxviridae and Herpesviridae with homologs in vertebrates were consistent with the hypothesis that these viruses have captured genes from cellular organisms repeatedly over the course of their evolution (Hughes 2002a). Similarly, an extensive phylogenetic analysis of dUTPase genes from viruses and cellular organisms suggested that different viral lineages have captured dUTPase genes independently at least five times (Baldo and McClure 1999). The present analyses suggested that different families of large DNA viruses independently acquired several genes from cellular organisms. There was evidence of independent acquisition of genes for DNA ligase (fig. 2) and ribonucleotide reductase 1 (fig. 3) in Baculoviridae and Poxviridae and of independent acquisition of genes for chitinase in Baculoviridae and Phycnoviridae (fig. 5). Because homologous genes may have been acquired independently by two different viral lineages, many of the genes of large DNA viruses may not provide reliable phylogenies of the viruses themselves. Indeed, these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.
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Acknowledgements |
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Footnotes |
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William Jeffery, Associate Editor
E-mail: austin{at}biol.sc.edu.
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