IRBI, Groupe dEtude 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
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Abstract |
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Introduction |
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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 DNA polymerase genes. The genomic region encoding the DpAV4
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.
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Methods |
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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 (USDAARS, 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.
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 Na2HPO4NaH2PO4 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).
DNA amplification
Amplification of the PolIIIPolI fragment from the DpAV4 genome.
Taking into account previous studies on eukaryotic and viral and
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
DNA polymerase gene by PCR. Amplification was performed on 10 ng of DpAV4 genomic DNA in 50 µl of 10 mM TrisHCl, 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
phage EMBL4 library containing the complete DpAV4 genome.
Amplification of PolIIPolIII and PolIIIPolI fragments from ascovirus and iridovirus genomes.
Three amino acid domains, PolII, PolIII and PolI, are known to be conserved among viral 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 450600 bp with M1M4, 170 bp with M5M8 and 620800 bp with M9 and M10.
In general, amplifications were performed with 110 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.
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).
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
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+.
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.
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
DNA polymerase amino acid sequences used for phylogenetic calculations have been deposited in the DDBJ/EMBL/GenBank sequence database under the accession number DS41569.
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Results |
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Relatedness of large dsDNA viruses based on DNA polymerase genes
By using the amino acid sequences of the SfAV1 and DpAV4 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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Development of more-specific DNA polymerase primers
Our analysis indicated that the 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 450600 bp PolIIPolIII fragment (M1M4), a 160175 bp PolIIIPolI fragment (M5M8) or a 610775 bp PolIIPolI fragment (M9M12).
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Use of two sets of primer combinations, M1M4 and M5M8, 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 M1M4 combinations failed to amplify the PolIIPolIII fragment from IV31. This region was amplified by IPCR by using the nucleotide sequence obtained from the cloned PolIIIPolI 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 M1M4 combination to amplify the IV31 PolIIPolIII fragment.
With respect to the M9M12 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, DNA polymerase PolIIPolI 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 PolIIPolIII regions of the TnAV2 and HvAV3 isolates were very similar (approximately 95%), the PolIIIPolI 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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Phylogenetic relatedness of ascoviruses and iridoviruses
The 16 aligned PolIIPolI 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 PolIIIPolI 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 5065 to 110130 and the insertions occurring at positions 198 to 225 (Fig. 5
).
Genes flanking the DpAV4 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 52906250) 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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Discussion |
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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 PolIIPolI 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 PolIIPolI 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 PolIIPolI region of a
DNA polymerase.
Although the focus of the present study was an analysis of 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 AJ279813AJ279815) 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 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.
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Acknowledgments |
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Footnotes |
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References |
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Received 31 March 2000;
accepted 16 August 2000.