Phylogenetic position of the Diadromus pulchellus ascovirus DNA polymerase among viruses with large double-stranded DNA genomes

Karine Stasiak1,2, Marie-Véronique Demattei1, Brian A. Federici2 and Yves Bigot1,2

IRBI, Groupe d’Etude des Parasites Moléculaires, UPRESA CNRS 6035; Faculté des Sciences, Parc de Grandmont, 37200 Tours, France1
Department of Entomology and Interdepartmental Graduate Program in Genetics, University of California, Riverside, CA 92521, USA2

Author for correspondence: Yves Bigot (at the IRBI). Fax +33 47 36 69 66. e-mail Bigot{at}univ-tours.fr


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The Ascoviridae is a family of large double-stranded (ds) DNA insect viruses that contains four species, the Spodoptera frugiperda (SfAV1), Trichoplusia ni (TnAV2), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses. These are unique among insect viruses in that the primary means of transmission among their lepidopteran hosts is generally by being vectored mechanically by hymenopteran parasitoids. Ascoviruses are similar in virion structure, but their relationships with their parasitoid vectors vary from being opportunistic to obligate. Little is known, however, about the relatedness of these viruses to one another or to other large dsDNA viruses. We therefore cloned and sequenced the {delta} DNA polymerase gene of DpAV4, characterized it and compared it to 59 eukaryotic and viral {delta} and {epsilon} DNA polymerases. Phylogenetic analyses based on these genes revealed that the ascoviruses DpAV4 and SfAV1 formed a group of virus species distinct from, but closely related to, species of the family Iridoviridae. Detailed analyses of the relatedness of ascovirus species based on conserved {delta} DNA polymerase motifs showed two groups within the family Ascoviridae, one containing DpAV4 and the other containing SfAV1, TnAV2 and HvAV3, which was consistent with their host–vector relationships. Despite significant differences in capsid symmetry between ascoviruses and iridoviruses, these results suggest that these viruses may have originated from a common ancestral virus.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The family Ascoviridae is a newly established family of insect viruses characterized by virions that are large, enveloped and typically bacilliform or reniform. They have circular, double-stranded (ds) DNA genomes which, depending on the species, range from 116 to 190 kbp (Bigot et al., 1997a ; Cheng et al., 1999 ; Federici et al., 2000 ). The virions average more than 300 nm in length and contain at least 12 polypeptides varying from 6 to more than 200 kDa (Federici et al., 1990 ). Whereas these virions are similar in structural complexity to those of other large dsDNA viruses such as the entomopoxviruses and iridoviruses, ascoviruses have two properties that differentiate them markedly. Firstly, they induce a significant reorganization of host cell structure that results in lysis of the nucleus, followed by cleavage of the cell into a cluster of virion-containing vesicles (Federici, 1993 ; Govindarajan & Federici, 1990 ), and secondly, ascoviruses are typically transmitted mechanically among their lepidopteran hosts by parasitoid wasps during oviposition (Hamm et al., 1985 ). A corollary to the latter is that ascoviruses are very poorly infectious per os (Govindarajan & Federici, 1990 ). Thus, the maintenance of ascoviruses in host populations is linked closely to the reproductive biology of their parasitoid vectors. In this regard, both opportunistic and obligate virus–vector relationships exist and the consequences of these vary importantly for their wasp vectors. The Spodoptera frugiperda (SfAV1), Trichoplusia ni (TnAV2) and Heliothis virescens (HvAV3) ascoviruses, which attack noctuid larvae, are transmitted horizontally by mechanical contamination of the ovipositor. They replicate in their lepidopteran hosts but not in the wasp vector. Furthermore, they prevent development of parasitoid larvae, such as those of Micropliptis croceipes, that hatch from eggs laid by the vector at the time of infection (Hamm et al., 1985 ). In contrast to this, the Diadromus pulchellus (DpAV4) ascovirus is transmitted vertically through the ovipositor of its solitary endoparasitoid wasp vector, D. pulchellus. It replicates in both its wasp host and its lepidopteran host, the pupa of the leak moth, Acrolepiopsis assectella, though much more extensively in the latter, and enhances development of the larvae of the wasp vector in the lepidopteran host (Bigot et al., 1997a , b ; and unpublished results). Polydnaviruses (family Polydnaviridae) are also transmitted to their insect hosts by parasitoid wasps, but they differ from ascoviruses in that the former do not usually produce progeny virions in their hosts (Stoltz, 1993 ).

As might be anticipated from its general biology, studies of DpAV4 have shown that it shares a much more intimate relationship with its parasitoid and lepidopteran hosts than do the ascoviruses of noctuids. Though not integrated into a wasp chromosome, the DpAV4 genome is carried as an unencapsidated free circular molecule in the nuclei of all wasp cells (Bigot et al., 1997a ). Virions have not been observed in male wasps, or in most tissues of the female, but they do occur in small numbers in the oviducts of mature females. These virions are highly infectious and only a few are required to infect the lepidopteran pupal host when injected with eggs at oviposition (Bigot et al., 1997b ). After infection, the ascovirus develops rapidly and releases progeny virions at about the time of hatching of the parasitoid larvae. Release of the virions is coincident with suppression of melanization during encapsulation.

Within virus families, it is unusual to have species that differ so significantly in their relationships with their hosts. This is especially true for ascoviruses, because the four species mentioned above, SfAV1, TnAV2, HvAV3 and DpAV4, are the only accepted species. While each has distinctive properties, little is known about their relatedness to one another or to other families of large, dsDNA viruses. In the present study, a preliminary determination of the relatedness of DpAV4 and SfAV1, as well as their relatedness to other families of DNA viruses, was made on the basis of an analysis of full-length {delta} DNA polymerase genes. The genomic region encoding the DpAV4 {delta} DNA polymerase was cloned, sequenced, analysed and compared with other DNA polymerase genes. These analyses validated DpAV4 as an ascovirus and revealed that this enzyme is highly similar among viruses of the families Ascoviridae and Iridoviridae. In addition, the analyses permitted degenerate primers to be developed to amplify regions that encode internal conserved DNA motifs from other ascovirus and iridovirus DNA polymerases. Analysis of these motifs confirmed a close relationship between DNA polymerase genes of the families Ascoviridae and Iridoviridae.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses.
The ascoviruses used in this study were SfAV1, TnAV2, HvAV3 and DpAV4 and several variants of these, most of which have been described previously (Bigot et al., 1997a ; Federici et al., 1990 ; Hamm et al., 1998 ). The first three viruses were isolated originally respectively from the noctuid hosts, Spodoptera frugiperda, Trichoplusia ni and Heliothis virescens, whereas DpAV4 was isolated from its wasp host, D. pulchellus (family Ichneumonidae), and pupae of its lepidopteran host, A. assectella (family Yponomeunidae). Noctuid ascoviruses were grown in larvae of host species from which they were isolated or in related species. DpAV4 was grown in pupae of A. assectella. Detailed methods for infecting hosts and rearing healthy and infected hosts and the wasp D. pulchellus have been described previously (Bigot et al., 1997b ; Federici et al., 1990 ; Hamm et al., 1998 ; Lecomte & Thibout, 1984 ). Genomic DNA of all ascoviruses was isolated from virions purified from infected lepidopteran hosts (Bigot et al., 1997a ; Federici et al., 1990 ).

Four iridoviruses (family Iridoviridae) were used in this study: Chilo iridovirus (CIV, iridovirus type 6), isolated from the lepidopteran Chilo suppressalis (Fukaya & Nasy, 1966 ); isopod iridovirus (IV31, iridovirus type 31), isolated from the terrestrial isopod Armadillidium vulgare (Federici, 1980 ; Cole & Morris, 1980 ); and two mosquito iridoviruses, regular mosquito iridovirus (R-MIV, iridovirus type 3 regular strain) and turquoise mosquito iridovirus (T-MIV, iridovirus type 3 T strain), isolated from larvae of Aedes taeniorhynchus (Clark et al., 1965 ). CIV was kindly provided by C. J. Funk (USDA–ARS, Phoenix, AZ 85040, USA) and R-MIV and T-MIV were provided by J. J. Becnel (USDA, Gainesville, FL 32604, USA). Genomic DNA was isolated from purified iridovirus virions by using the same procedures used to isolate DNA from the ascoviruses.

{blacksquare} DNA manipulations.
Viral DNA purification, synthesis of labelled DNA probes and agarose gel electrophoresis were carried out by using standard procedures (Ausubel et al., 1994 ). For Southern blots, probes were hybridized in 0·1% SDS, 0·5 M Na2HPO4–NaH2PO4 buffer, pH 7 at 65 °C. The stringency of washing at 65 °C was either high (0·5x SSC; 1x SSC is 0·15 M NaCl/0·015 M trisodium citrate) or low (2x SSC). Exposure times were short (4 h) or long (24 h).

{blacksquare} DNA amplification
Amplification of the PolIII–PolI fragment from the DpAV4 genome.
Taking into account previous studies on eukaryotic and viral {delta} and {epsilon} DNA polymerases belonging to the B family (Braithwaite & Ito, 1993 ; Pellock et al., 1996 ; Knopf, 1998 ), two primers were designed from two conserved amino acid domains, Pol II and Pol III, located in the carboxy-terminal region containing the polymerase activity. These degenerate primers, pPol3 (5'-AARRTNWCNGCNAAYTC-3') and pPol1r (3'-TCNGTRTCRCYRTANA-3'), were used to amplify a 165 bp internal fragment from the DpAV4 {delta} DNA polymerase gene by PCR. Amplification was performed on 10 ng of DpAV4 genomic DNA in 50 µl of 10 mM Tris–HCl, pH 9, 1·5 or 2·5 mM MgCl2, 50 mM KCl, 0·1% Triton X-100, 150 µM each of dATP, dCTP, dGTP and dTTP, 0·8 µM of each oligonucleotide and 1 U Taq polymerase (Promega). A programmable thermal controller (Perkin-Elmer) was used for 30 cycles of 94 °C for 30 s, 39 or 45 °C for 1 min and 72 °C for 30 s. At the end of the 30th cycle, extension was allowed to proceed at 72 °C for 2 min. This 165 bp fragment was then used to probe a {lambda} phage EMBL4 library containing the complete DpAV4 genome.

Amplification of PolII–PolIII and PolIII–PolI fragments from ascovirus and iridovirus genomes.
Three amino acid domains, PolII, PolIII and PolI, are known to be conserved among viral {delta} DNA polymerases (Braithwaite & Ito, 1993 ; Knopf, 1998 ). Preliminary analyses of the corresponding genes of SfAV1, DpAV4 and the insect iridoviruses revealed that these domains were also highly conserved between ascoviruses and iridoviruses. This permitted the following series of eight degenerate primers to be developed for PCR amplification of fragments containing these domains: Pol2a, 5'-TAYCCNWCNHTNATGGC-3'; Pol2b, 5'-TAYCCNWCNHTNATHGC-3'; Pol3ra, 5'-GARTTNGCNGWNAYYTT-3'; Pol3rb, 5'-TTNGCNGWNAYYTTRTA-3'; Pol3a, 5'-CNTAYAARRTNACNGCN-3'; Pol3b, 5'-CNTAYAARRTNACNGCNAA-3'; Pol1ra, 5'-SWRTCNGTRTCNCYRTA-3'; and Pol1rb, 5'-RTCNGTRTCNCYRTANAA-3'. Specific fragments containing these domains were amplified by using the following primer mixes: M1 (Pol2a+Pol3ra), M2 (Pol2a+Pol3rb), M3 (Pol2b+Pol3ra), M4 (Pol2b+Pol3rb), M5 (Pol3a+Pol1ra), M6 (Pol3a+Pol1rb), M7 (Pol3b+Pol1ra), M8 (Pol3b+Pol1rb), M9 (Pol2a+Pol1ra), M10 (Pol2a+Pol1rb), M11 (Pol2b+Pol1ra) and M12 (Pol2b +Pol1rb). The expected sizes of the amplified fragments with ascovirus or iridovirus as the DNA source were about 450–600 bp with M1–M4, 170 bp with M5–M8 and 620–800 bp with M9 and M10.

In general, amplifications were performed with 1–10 ng of genomic viral DNA according to the PCR procedure and conditions described above. A marked improvement in amplification was obtained by modification of the ramping between the annealing and extension steps, from 1 to 2 s per °C. In addition, although there were some variations depending on the DNA source, the specificity of fragment amplification was improved by adding 5 or 10% deionized formamide to the amplification solution.

Amplifications by inverse PCR (IPCR).
Each IPCR was performed by using standard procedures and conditions employing specific nucleotide primers (25-mer) and restriction enzymes (Ausubel et al., 1994 ). Briefly, viral DNA was digested with a specific restriction enzyme. After dilution (final DNA concentration of less than 1 ng/µl), the restriction fragments were circularized by incubation for 12 h with T4 DNA ligase at 16 °C. The fragments of interest were amplified by PCR by using circularized DNA, specific primers and a Long Expand System kit (Boehringer Mannheim) under the conditions described by the manufacturer.

{blacksquare} Cloning of PCR fragments.
PCR fragments were purified after separation by agarose gel electrophoresis and eluted by using Qiagen or Geneclean kits according to the conditions described by the manufacturers. PCR fragments were then cloned by using a pGEM-T Easy kit (Promega).

{blacksquare} Genome cloning.
Genomic DNA isolated from DpAV4 virions (Bigot et al., 1997a ) was partially digested with Sau3AI, selected for fragments about 20 kb in length on a sucrose gradient and ligated into the BamHI site of {lambda} phage EMBL4. Phage were packaged and plated by using E. coli host strain K802 (Stratagene). Positive clones were screened and purified by hybridization of DNA blots from plated phages by using the radiolabelled 165 bp internal fragment of the DpAV4 DNA polymerase. Fragments from positive clones were sub-cloned into pBluescript SK+.

{blacksquare} DNA sequencing.
Plasmids were isolated and purified by alkaline lysis followed by precipitation with LiCl or PEG (Ausubel et al., 1994 ). Double-stranded templates were used for dideoxy-nucleotide sequencing reactions with the Sequitherm long-read cycle sequencing kit (Epicentre Technologies) and labelled by using universal and reverse IRD800 primers. DNA was amplified by 25 cycles of PCR (denaturation at 94 °C for 30 s, annealing at 50 °C for 15 s and elongation at 70 °C for 1 min). Sequencing was performed by using a LI-COR 4000 sequencer. Both strands were sequenced for each clone and two clones of each fragment were sequenced. The sequences reported here have been deposited in the DDBJ/EMBL/GenBank sequence database under accession numbers AJ279812AJ279828.

{blacksquare} Sequence analyses.
The Infobiogen facilities were used for database searches, extractions, sequence alignments and calculations (Dessen et al., 1990 ). Four programs were used to perform the protein sequence designations and alignments: tBlastN, BlastP, Search of conserved motif in Prosite and the Identify website (http://motif.stanford.edu/identify/; Nevill-Manning et al., 1998 ). Phylogenetic analyses were performed by using the Parsimony and Neighbor-Joining programs of the PHYLIP package, version 3.5c (Felsenstein, 1993 ). The neighbour-joining trees were derived from matrices of distances based on the category distance model proposed by George et al. (1988) . To check the alignment quality of amino acid sequences, the data file was divided into three sub-files of about 610 aligned positions each, the first containing the amino-terminal third, the second containing the central third and the third containing the carboxy-terminal third. All of the trees calculated with these three different files had identical topologies and bootstrap values at nodes, thus confirming that the tree obtained with the full-length sequences was not due to large misalignment problems. Alignments of the {delta} DNA polymerase amino acid sequences used for phylogenetic calculations have been deposited in the DDBJ/EMBL/GenBank sequence database under the accession number DS41569.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Sequence of the DpAV4 {delta} DNA polymerase gene
By using primers pPol3 and pPol1r, a 165 bp fragment of the DpAV4 {delta} DNA polymerase gene was amplified from DpAV4 genomic DNA. This fragment was cloned and sequenced and analysis of the sequence revealed that it encoded a 55 amino acid fragment that was 55% similar to the PolIII–PolI fragment of the SfAV1 {delta} DNA polymerase described previously (Pellock et al., 1996 ). The DpAV4 genomic library was screened by colony hybridization using the 165 bp fragment as a probe and a single clone (K8) of 17·9 kb was identified that hybridized with this probe. This fragment was recovered and mapped with the restriction enzymes BamHI, EcoRI and HindIII (Fig. 1). With the location of the 165 bp fragment as a guide, a 12255 bp fragment of this clone extending approximately from position 5200 to 17700 was sequenced (accession no. AJ279812). Analysis of this fragment identified an open reading frame (ORF) of 956 amino acids extending from position 10519 to 7652 (Fig. 1; 1737 to 4604 in frame 3 of the minus strand in Fig. 2). Depending on the domains compared, the amino acid sequence of this protein was 25–45% similar to the SfAV1 {delta} DNA polymerase. Moreover, it contained the 10 conserved motifs of the B family of DNA polymerases (Fig. 2). Taken together, these results indicated that this ORF was the {delta} DNA polymerase of DpAV4.



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Fig. 1. Restriction map of the 17·8 kb DpAV4 genome fragment cloned into phage K8. The dashed area represents the 12255 bp region that was sequenced. The black and grey arrows below the map indicate the locations and orientations of the {delta} DNA polymerase and the putative DNA helicase. B, BamHI; E, EcoRI; H, HindIII.

 



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Fig. 2. Nucleotide sequence of the region containing the DpAV4 {delta} DNA polymerase ORF (Fig. 1). The sequence illustrated corresponds to the minus strand of the gene. The deduced amino acid sequence is shown above the nucleotide sequence. The ExoI–ExoIII and PolI–PolVII motifs are boxed, respectively, in grey and black. The putative polyadenylation signal in the 3' region is boxed in grey and underlined with two arrows indicating the palindromic motif.

 
Analysis of the region extending 200 bp upstream from the DpAV4 DNA polymerase ORF did not reveal any known transcription motifs such as a CAAT box, TATA box or Kozak box. Analysis of the 500 bp downstream of this ORF identified one putative polyadenylation signal (ATCAAA) at position 4653 and an 8 bp palindromic motif at positions 4698–4721 (Fig. 2).

Relatedness of large dsDNA viruses based on {delta} DNA polymerase genes
By using the amino acid sequences of the SfAV1 and DpAV4 {delta} DNA polymerases, tblastN searches of the database identified 58 DNA polymerases of the B family. These sequences were aligned by using CLUSTAL V. The alignment was improved by making adjustments based on data for viral DNA polymerases published previously (Braithwaite & Ito, 1993 ; Pellock et al., 1996 ; Knopf, 1998 ). The final alignment was composed of 60 sequences containing 1836 positions. The relationships of the viruses based on the polymerase genes were determined by two methods, parsimony and neighbour-joining, with a sampling of 1000 bootstraps. Unrooted trees obtained with the two methods were similar, but the bootstrap values obtained with the parsimony analysis were more consistent (Fig. 3). The final consensus tree was highly consistent, with the node values separating the branches of the different virus families as high as 80% except for three nodes located in the group of herpesviruses and one in the group of vertebrate poxviruses (Fig. 3).



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Fig. 3. Unrooted consensus tree illustrating phylogenetic relationships of the DNA polymerase B family. The tree is based on sequences available in the database and was developed by using the protein parsimony procedure. Numbers at each node correspond to bootstrap values (percentages of 1000 repetitions). Abbreviations for the organisms or viruses from which the DNA polymerase genes originated are as follows: {delta} proto, protozoon; {delta} s.c, Saccharomyces cerevisiae; {delta} Ss.c, Schizosaccharomyces sp.; {delta} D.m, Drosophila melanogaster; {delta} M.m, Mus musculus; {delta} B.t, Bos taurus; {delta} H.m, Homo sapiens; {delta} archeoB, Pyrococcus furiosus (archebacterium); {delta} E. coli, Escherichia coli; SfAV1, Spodoptera frugiperda ascovirus; DpAV4, Diadromus pulchellus ascovirus; LCDV, lymphocystis disease virus; RSBIV, Red Sea bream iridovirus; ADE G1, 40, 12, 07, 05 and 02 are six different adenovirus strains; AcNPV, Autographa californica nucleopolyhedrovirus; BmNPV, Bombyx mori NPV; CfNPV, Choristoneura fumiferana NPV; OpNPV, Orgyia pseudotsugata NPV; HzNPV, Heliothis zea NPV; LdNPV, Lymantria dispar NPV; FPV, fowlpox virus; MCV, Molluscum contagiosum virus; OVU, parapoxvirus E10R homologue gene; VarV, variola major virus; VV, variola virus; GHV, gallid herpesvirus; FHV1, feline herpesvirus-1; BHV1, bovine herpesvirus-1; EHV1, equine herpesvirus-1; EHV2, equine herpesvirus-2; HHV1, 2, 6, 7, human herpesviruses 1, 2, 6 and 7; MHV, murine herpesvirus; HVS, herpesvirus saimiri; KSV, Kaposi’s sarcoma-associated herpesvirus; RRV, rhesus monkey herpesvirus; EBV, Epstein–Barr virus; CbPV, Choristoneura biennis poxvirus; MsPV, Melonoplus sanguinipes poxvirus; XcGV, Xestia c-nigrum granulovirus; ASFV, African swine fever virus; HCMV, human cytomegalovirus; RCV, rhesus CMV; GPCV, guinea pig CMV; FV, Feldmannia sp. virus; CHV, chlorella virus; PBCV, Paramecium bursaria chlorella virus; GA1, bacteriophage GA1; Phi 29, Bacillus subtilis phage {phi}29; Cp1, Streptococcus pneumoniae bacteriophage; PRD1, bacteriophage PRD1; T4, E. coli phage T4; RB69, Neurospora crassa mitochondrial DNA polymerase.

 
The comparative analysis of these {delta} DNA polymerases revealed several key findings regarding the relatedness and putative phylogenetics of these viruses. (i) The {delta} DNA polymerase most closely related to that of DpAV4 is that of SfAV1. This provides molecular evidence, previously lacking, for classification of DpAV4 as a member of the family Ascoviridae. (ii) The DNA polymerase genes most closely related to those of the family Ascoviridae are those of the family Iridoviridae. Significantly, the branch lengths indicative of genetic distance in the consensus tree suggest that the genes of these two families are more closely related than those of subgroups A (nucleopolyhedroviruses) and B (granuloviruses) of the family Baculoviridae or those of the vertebrate and invertebrate subgroups of the family Poxviridae. (iii) The {delta} DNA polymerases of the Ascoviridae and Iridoviridae are closely related to those of two other families of eukaryotic viruses, the Phycodnaviridae [chlorella virus (CHV) and Paramecium bursaria chlorella virus (PBCV)] and the Asfaviridae (African swine fever virus, ASFV). Interestingly, prior to 1990, the latter three families were classified together as ‘iridescent viruses’ or iridoviruses (Matthews, 1982 ). Several DNA polymerases of bacterial phages, GA1, Phi29, Cp1 and PRD1, were also found to be closely related to those of these four families of eukaryotic viruses, thus suggesting that part of the genome of these bacterial viruses might have a common origin with the ‘iridescent viruses’. (iv) The eukaryotic viruses are generally associated in groups in which the virions are similar in symmetry. Viruses with icosahedral virions, i.e. the Herpesviridae, Iridoviridae, Phycodnaviridae, Asfaviridae and Adenoviridae, cluster in the lower part of the tree, whereas those having bacilliform or more complex virion structures, the Baculoviridae and Poxviridae, respectively, are located in the upper part of the tree. However, a unique feature of this analysis is that the Ascoviridae and Iridoviridae, viruses with virion structures that differ markedly, cluster together.

Development of more-specific {delta} DNA polymerase primers
Our analysis indicated that the {delta} DNA polymerases from the Iridoviridae, Phycodnaviridae and Asfaviridae were the most closely related to those of the Ascoviridae (Fig. 3). To facilitate the identification of new ascoviruses and to study their phylogenetic relatedness, the coding regions of the 10 conserved motifs, ExoI to ExoIII and PolI to PolVII, of the DNA polymerase sequences from key viruses were analysed. This enabled the development of primers that could amplify one or two fragments of this gene selectively. Motifs PolII, PolIII and PolI showed a high degree of conservation (Fig. 4). Each of these motifs contained specific amino acid positions that appeared to be unique for ascoviruses and iridoviruses. This permitted the development of eight degenerate oligonucleotide primers specific for ascoviruses and iridoviruses (two for PolII, four for PolIII and two for PolI). By using these in different combinations, it was theoretically possible to prepare 12 primer combinations that would amplify either a 450–600 bp PolII–PolIII fragment (M1–M4), a 160–175 bp PolIII–PolI fragment (M5–M8) or a 610–775 bp PolII–PolI fragment (M9–M12).



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Fig. 4. Amino acid sequence alignments of the conserved PolII, PolIII and PolI motifs of selected large, dsDNA viruses. Motifs are from the ascoviruses SfAV1 and DpAV4, the iridoviruses LCGV and RSBIV, the phycodnavirus CHV and the asfavirus ASFV. Identical positions are highlighted in black and other major conserved or identical positions are highlighted in grey. The amino acid equivalents used in the analysis are based on those given by Nevill-Manning et al. (1998) .

 
These primers were used to amplify PolII–PolIII and PolIII–PolI fragments from ascovirus DNAs corresponding to those described by Federici et al. (1990) (referred to here as SfAV1a, HvAV3a and HvAV3c) and Hamm et al. (1998) (referred to here as HvAV3b and HvAV3d). More recent ascovirus isolates were also examined, including two SfAV1 isolates originating from Mexico (Mex1 and Mex2, referred to as SfAV1b and SfAV1c) and two from an ascovirus referred to here as TnAV2b. To determine the specificity and efficiency of the primers, four iridoviruses were also examined: CIV, IV31, R-MIV and T-MIV (accession nos AJ279816–AJ279827). As the gene sequences encoding the DNA polymerases of the vertebrate iridoviruses lymphocystis disease virus (LCDV) and frog virus 3 (FV3) were available in the database, this virus sample covered all four genera of the Iridoviridae (Williams & Cory, 1994 ; Williams, 1994 ; Webby & Kalmakoff, 1998 ).

Use of two sets of primer combinations, M1–M4 and M5–M8, allowed the amplification of each of the two expected fragments in at least one of the four combinations used for most of the viruses. However, use of the M1–M4 combinations failed to amplify the PolII–PolIII fragment from IV31. This region was amplified by IPCR by using the nucleotide sequence obtained from the cloned PolIII–PolI fragment. Translation of the resulting sequence showed that the amino acid sequence of the IV31 PolII motif was YPSLIQA. The original Pol2a and Pol2b primers were designed based on the consensus motif YP(S/T)LI(M/I)A. The presence of an unexpected Q in the IV31 PolII motif explained the failure of the M1–M4 combination to amplify the IV31 PolII–PolIII fragment.

With respect to the M9–M12 combinations, although PCR products were obtained with these primers, most of the sequences were shown to be artifacts and thus these combinations were ineffective for amplification.

Relatedness of the Ascoviridae and the Iridoviridae
By using the above primer sets, {delta} DNA polymerase PolII–PolI regions were cloned, sequenced and reconstructed for all 12 virus samples. Analysis of nucleic acid and deduced amino acid sequences of these viruses revealed that the three SfAV1 isolates were very similar (Fig. 5). The sequences of the two TnAV2 isolates and four HvAV3 isolates were respectively identical. However, although the PolII–PolIII regions of the TnAV2 and HvAV3 isolates were very similar (approximately 95%), the PolIII–PolI regions were very divergent (less than 50% similar). This allowed us to separate these viruses into two groups within the family Ascoviridae (Fig. 6).



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Fig. 5. Amino acid sequence alignments of the PolII–PolI regions from selected large dsDNA viruses. Identical and 100% similar positions are highlighted in black. Major conserved or identical positions are in grey. For ascoviruses, taxa are in accordance with current guidelines for naming species and their variants (Federici et al., 2000 ).

 


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Fig. 6. Phylogenetic analysis of the PolII–PolI region from {delta} DNA polymerases of one phycodnavirus, CHV, six iridoviruses, LCDV, FV3, CIV, IV31, T-MIV and R-MIV, and 10 isolates of four ascovirus species, SfAV1a, SfAV1b, SfAV1c, HzAV2a, HzAV2b, HvAV3a, HvAV3b, HvAV3c, HvAV3d and DpAV4. The consensus tree is rooted with CHV and was obtained by using the protein neighbour-joining procedure. Numbers given at each node correspond to the percentage bootstrap values (for 1000 repetitions). Branches relating iridoviruses are in dark grey and those for the ascoviruses are in black.

 
Analyses of the PolII–PolI region of invertebrate iridoviruses showed that these sequences were more variable in length (450–500 bp) than those from vertebrate iridoviruses and ascoviruses (Fig. 5). These analyses also revealed that the genes encoding the R-MIV and T-MIV {delta} DNA polymerases were similar, although these two viruses differ significantly in capsid diameter and genome size (Anthony & Compans, 1991 ).

Phylogenetic relatedness of ascoviruses and iridoviruses
The 16 aligned PolII–PolI regions for the various ascovirus and iridovirus isolates were analysed by using the phycodnavirus CHV as an outgroup and unrooted consensus trees were developed based on a sampling of 1000 bootstraps and using two types of analysis, parsimony and neighbour-joining. The results obtained with each were similar. Ascovirus isolates described previously (Federici et al., 1990 ; Hamm et al., 1998 ) clustered as a single taxon containing the three species SfAV1, TnAV2 and HvAV3 (Fig. 6). This taxon appeared to be more related to iridoviruses of invertebrate origin than to those from vertebrates.

In these trees, DpAV4 was not associated with the main ascovirus taxon, but rather was located at the base of the taxon containing the invertebrate iridoviruses (Fig. 6). This position, very close to the root of these two virus families, was probably an artifact due to deletions or insertions observed in the PolIII–PolI regions. However, importantly, these deletions and insertions differed specifically between the two virus types, with the deletions in DpAV4 occurring at positions 61 to 121, the deletions in the iridoviruses occurring at positions 50–65 to 110–130 and the insertions occurring at positions 198 to 225 (Fig. 5).

Genes flanking the DpAV4 {delta} DNA polymerase
Further analysis of both strands of the 12255 bp DpAV4 sequence identified 25 other ORFs encoding up to 70 amino acids (Table 1). On the basis of comparisons with other proteins in the database, putative functions were assigned to these proteins or, in cases where the function was unknown, relatedness to other proteins was designated (Table 1). Analysis of the upstream regions of these ORFs showed a putative CAAT box in most, frequently degenerate at one position, but no TATA box. A putative polyadenylation signal was found in the 3' region of most (21/26) of these ORFs, as well as inverted repeats (14/26). In addition, a 960 bp repeat (positions 5290–6250) that occurs several times in the DpAV4 genome (Bigot et al., 1997a , 2000 ) was identified just downstream from the DNA polymerase gene.


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Table 1. Features of ORFs identified in the 12255 bp sequence obtained from phage K8

 
Among the 25 ORFs, there was a gene encoding a putative DNA helicase (positions 10570–12048) adjacent and in the inverse orientation to the {delta} DNA polymerase gene. The helicase encoded by this gene was identified as a ‘DEAD’ helicase, in that it contained the six conserved domains associated with this type of enzyme (Gorbalenya et al., 1988 , 1989 ; Hodgman, 1988 ; Bideshi et al., 1998 ). This suggests that genes involved in DNA replication might be linked in other ascovirus genomes. To test this possibility, a circularized XhoI digest of the SfAV1 genome was used as a DNA source to amplify the region immediately upstream from the {delta} DNA polymerase of SfAV1 by IPCR. Two primers in the inverse orientation were designed (5'-ATGTCGTTGCAGTCGTGATG-3' and 5'-GCCTTTCAGACATAGACCTT-3') in the 5' region of the published sequence 177 bp upstream of an XhoI site (Pellock et al., 1996 ; accession no. U35732). PCR amplified a 3543 bp (3366+177 bp) fragment that was cloned and sequenced (accession no. AJ279830). The complete region of 8917 bp was reconstructed and 18 ORFs of 70 amino acids or greater were identified. None of these encoded a DNA helicase (Table 2). Analysis of the 5' and 3' regions of the SfAV1 ORF for transcriptional motifs showed the same features as those observed for the DpAV4 genes. Considered together, these results suggest that clustering of enzyme genes involved in replication is not a feature of these ascoviruses, although the 5' and 3' motifs of these genes did appear to have similar features.


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Table 2. Features of ORFs in the 8917 bp sequenced region of the SfAV1 genome containing the gene encoding {delta} DNA polymerase

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In the present study, we have provided additional evidence that DpAV4 is an ascovirus through phylogenetic analyses in which we compared the full length of the DpAV4 {delta} DNA polymerase gene with the sequences of the corresponding genes from a wide variety of other large DNA viruses. Previous studies have grouped together the four ascoviruses, SfAV1, TnAV2, HvAV3 and DpAV4, on the basis of structural features of the virions and their associations with endoparasitic wasps (Federici et al., 1990 , 2000 ; Bigot et al., 1997a ). Thus, molecular data presented here for DpAV4 combined with previous data on the SfAV1 {delta} DNA polymerase gene (Pellock et al., 1996 ) and our phylogenetic analyses support this grouping. In addition, our analysis of the PolII–PolI regions of the ascovirus species shows two distinct groups, suggesting that the family Ascoviridae is composed of two genera, one consisting of the ascoviruses attacking noctuids, SfAV1, TnAV2 and HvAV3, and the other, at this point, containing only a single species, DpAV4. These distinct groupings are consistent with the different biological relationships that these viruses have with their wasp vectors, the ascoviruses attacking noctuids being opportunistic (Hamm et al., 1985 ; Govindarajan & Federici, 1990 ) and DpAV4 being obligately associated with its vector, D. pulchellus (Bigot et al., 1997a ).

The observation that ascovirus and iridovirus DNA polymerase genes are closely related has also been made recently by Knopf (1998) . However, his finding was based on an analysis restricted to sequences for the 10 conserved motifs of the DNA polymerases of the B family. By including the full-length sequences in our analyses and analysing the sequences as segments composed of thirds, we were able to show not only the relationship of the ascovirus and iridovirus genes, but their close relationship to those of the Phycodnaviridae and Asfaviridae. Viruses of the latter families, like members of the Iridoviridae, have virions with an icosahedral capsid.

An interesting observation derived from analyses of the PolII–PolI region was that the DNA polymerase genes of invertebrate iridoviruses were more closely related to those of ascoviruses than to those of the vertebrate iridoviruses. The extent to which this putative relationship is valid awaits the cloning, sequencing and analysis of a larger number of full-length DNA polymerase sequences, as well as other genes from ascoviruses and iridoviruses. This relationship may be an artifact of the use of short sequences for the analyses. Alternatively, it could indicate that the origin of DpAV4 occurred close to the time when ascoviruses and iridoviruses diverged and, moreover, that once the iridoviruses were established in vertebrates they diverged more rapidly than their relatives in invertebrates.

Previously, it has been suggested that ascoviruses and ichnoviruses may be related on the basis of similarities between the structure and size of their virions (Federici, 1991 ). In an attempt to test this hypothesis, we tried to amplify the viral PolII–PolI region by using DNA samples extracted from virus and/or ichneumonid female wasps (using DNA from Cardiochiles nigriceps, Campoletis sonorensis, Hyposoter exigua, Meteorus leviventris, Olesiacampe benefactor and Tranosema rostrale kindly provided by Peter Krell, University of Guelph, Canada; Donald Stoltz, Dalhousie University, Halifax, Nova Scotia, Canada; and Bruce Webb, University of Kentucky, Lexington, USA). Although PCR assays were performed using a variety of amplification conditions (number of cycles, 30/35/40; annealing temperatures, 39, 42 and 45 °C), we were unable to detect any fragments encoding the PolII–PolI region of a {delta} DNA polymerase.

Although the focus of the present study was an analysis of {delta} DNA polymerase genes, preliminary analyses of other DpAV4 and SfAV1 genes support the close relationship of ascoviruses and iridoviruses. For example, database searches suggest that genes such as the SfAV1 P2 gene and genes for DpAV4 P5, P20 and P23 listed in Table 1 are more closely related to iridovirus genes than to those from other virus groups. This relationship, however, is not without contradictions. For example, the complete genomic sequence of the iridovirus LCDV is known (Tidona & Darai, 1997 ), but, according to our unpublished data, only a few of the proteins encoded by SfAV1 and DpAV4 genes (Tables 1 and 2; and accession nos AJ279813–AJ279815) appear to be related to known iridovirus proteins. Moreover, the DNA helicase of DpAV4 presents an even more ambiguous situation. The most closely related genes of virus origin are the ‘DEAD’ DNA helicases of iridoviruses (Sonntag et al., 1994 ). However, other unpublished preliminary results we have indicate that there are at least 10 other DNA helicases from different eukaryotic organisms that are more similar to the DpAV4 helicase than those of iridovirus origin. This suggests that, if the iridovirus and ascovirus ‘DEAD’ DNA helicases are related, they do not originate directly from a common virus gene ancestor. Their similarities suggest that they originate from different eukaryotic genes that encoded related DNA helicases.

In summary, despite differences in virion symmetry and genome structure, our data and analyses of {delta} DNA polymerase genes suggest that the families Ascoviridae and Iridoviridae may have had a common origin. In order to determine whether these families are indeed so related, the comparative analysis of a larger number of genes encoding enzymatic and structural proteins is required.


   Acknowledgments
 
We thank Dr C. Augé-Gouillou, Professor G. Periquet, Dr D. Bideshi, M. H. Hamelin and J. J. Johnson for their assistance throughout our investigations. This work was supported by grants from the CNRS (UPRES-A 6035) and NATO to Y. Bigot, US National Science Foundation Grant INT-9726818 to B. Federici and the Ministère de l’Education Nationale, de la Recherche et de la Technologie.


   Footnotes
 
The DDBJ/EMBL/GenBank accession numbers of the sequences reported here are AJ279812AJ279828.


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Received 31 March 2000; accepted 16 August 2000.