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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The unique pathobiology of ascoviruses makes their origin and phylogenetic relationship to other viruses of interest. Comparisons of ascovirus DNA polymerase genes with those of other large DNA viruses suggest that ascoviruses, which have bacilliform to reniform virions, and iridoviruses (family Iridoviridae), which have icosahedral virions, are closely related (Stasiak et al., 2000
). These same studies revealed that viruses of the families Asfaviridae and Phycodnaviridae, which also have icosahedral virions, occur on the same DNA polymerase gene tree branch, but are less related to ascoviruses. Although the overall tree topology provides support for a close relationship between ascoviruses and iridoviruses, the markedly different shapes of their virions make this relationship questionable.
The putative close relationship of the families Ascoviridae and Iridoviridae is intriguing because, if verified, it would be the first case where viruses with different virion symmetries would have been shown to have originated from a common ancestor. Moreover, a better understanding of the relationship between ascoviruses and iridoviruses could provide insights into how viruses evolved as well as clarify the origins and relationships of other large DNA virus families. At best, these remain poorly understood (Van Regenmortel et al., 2000 ; Domingo et al., 1999
). It is difficult to address these problems, especially with respect to the ascoviruses and iridoviruses, because so little is known about the comparative properties of these viruses. For example, the physical configuration of the genome and the sequence of lymphocystis disease virus (LCDV), a vertebrate iridovirus, are known, but comparable knowledge of the invertebrate iridoviruses and ascoviruses is greatly lacking, as summarized in Fig. 1(a)
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Methods |
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Viral DNA purification.
Ascovirus DNAs were purified as described previously (Federici et al., 1990 ). Iridovirus DNAs were purified by using the same procedure except that the sonication step was eliminated and the virions were prepurified by passing samples of crushed infected isopods or mosquito larvae through a nylon membrane filter (10 µm porosity, Millipore). Proteins were removed from the virus samples by treatment in 0·1 M TrisHCl, 50 mM EDTA, pH 9, 0·2% SDS, 200 µg/ml proteinase K for 2 h at 50 °C. During phenol and chloroformisoamyl alcohol extraction, the organic phase was removed by pipetting. The aqueous phase was not pipetted to avoid shearing the DNA.
Wasp and DpAV4 host DNA.
Total genomic DNA from males and females of D. pulchellus and from Acrolepiopsis assectella parasitized by D. pulchellus wasps or artificially infected by stinging with DpAV4 was obtained as described previously (Bigot et al., 1997b ).
Southern blot hybridization.
DNA purification, synthesis of labelled DNA probes and agarose gel electrophoresis followed standard procedures (Ausubel et al., 1994 ). Southern blots were hybridized by using conditions described in the accompanying paper (Stasiak et al., 2000
).
DNA fragment cloning and sequencing.
Fragments and plasmids were purified and then sequenced as described in the accompanying paper (Stasiak et al., 2000 ). Fragments were cloned into the vector pBluescript SK+ (Stratagene). Two clones of each fragment were sequenced, one from each strand. The sequences reported here appear in the DDBJ/EMBL/GenBank sequence database under accession numbers AJ279828 and AJ279829.
Sequence analyses.
The Infobiogen facilities (Dessen et al., 1990 ) were used for database searches, sequence alignments and calculations.
Pulse-field gel electrophoresis (PFGE).
PFGE was done by using a CHEF-DRII PFGE system as described by the manufacturer (Bio-Rad). Gels contained 1% LE agarose and 0·5x TBE (1x TBE is 89 mM TrisHCl, 89 mM boric acid, 25 mM EDTA, pH 8·3). In order to separate fragments of between 5 and 500 kbp, electrophoresis was performed at 120 V, 14 °C, at an angle of 120 ° and a pulse ramping from 0·22 to 44 s for 16 h. A phage DNA ladder (New England Biolabs) was used to estimate DNA sizes. To obtain high resolution of undigested viral DNA, small quantities (12·5 ng) were loaded on the gels. Results were therefore detected by Southern blot hybridization with DNA of the virus species as probes.
CsCl equilibrium density gradient analyses.
Viral DNA samples purified as described above were analysed by equilibrium density gradient centrifugation in CsCl gradients to determine whether genomic DNA occurred at different densities, indicative of different physical conformations from linear DNA molecules to supercoiled and relaxed circular DNA molecules. Standard conditions used for plasmid purification were followed (Ausubel et al., 1994 ). Briefly, 13·5 ml ultraclear ultracentrifugation tubes (Beckman) containing 200 µg viral DNA in a 1·6 g/cm3 CsCl gradient and 100 µg/cm3 ethidium bromide (EtBr) were centrifuged in a Ti 55.2 rotor (Beckman) at 38000 r.p.m. for 30, 38 or 48 h at 25 °C. Bands containing DNA were visualized under UV light (312 nm) at the end of the ultracentrifugation.
Detection of methylated bases.
DNA was hydrolysed by the four-hour-two-enzymes technique (Gehrke et al., 1984 ), which treats denatured nucleic acid successively with nuclease P1 and bacterial alkaline phosphatase for 4 h. The nucleosides were separated by high-speed HPLC with a Colochrom Spherisorb 80ODS2 column (25 cm long, 5 µm porosity). Elution was performed with 0·05 M K2HPO4, pH 4·4, 16% methanol at 20 °C at a flow rate of 1·2 ml/min. Detection was done at 254 nm with a Waters 996 photodiode array detector. Nucleosides were identified by comparing their retention times with those of standard synthetic nucleosides (Sigma). Amounts of deoxycytidine and 5-methyldeoxycytidine were calculated by using the Millennium software (Waters).
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Results |
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CsCl ultracentrifugation.
When measured by CsCl gradients, the density of double-stranded molecules of DNA depends on their tertiary structure. Linear or relaxed circular molecules have a mean density of 1·59 g/cm3, whereas superhelical circular molecules are of higher density. The increase in density depends upon the number of turns present in the circular molecule. The genome of AcMNPV has a superhelical circular configuration (Summers & Anderson, 1972 ). However, in CsCl equilibrium gradients of AcMNPV genomic DNA, relaxed circular and linear molecules occur in addition to superhelical circular molecules. The relaxed circular and linear forms result respectively from nicking and shearing of the superhelical molecules (Britten et al., 1974
; Summers & Anderson, 1972
). In our experiments, the linear and relaxed circular forms were separated easily from the superhelical forms in a CsClEtBr gradient (Fig. 3
, left tube). As observed in previous studies of baculovirus DNA (Summers & Anderson, 1972
), the band containing the superhelical DNA was less dense than that containing linear and relaxed circular molecules, indicating that the large superhelical circular molecules were fragile and frequently nicked during DNA purification.
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Characteristics of repetitive interspersed DNA
In previous studies, repetitive regions of DNA were reported in ascovirus genomes (Bigot et al., 1997a ; Federici et al., 1990
), but such regions have not been reported in iridoviruses. In the present study, therefore, a comparative search for the presence and location of repeated interspersed regions of DNA was undertaken with the genomes of SfAV1a, HvAV3a, IV31 and T-MIV by restriction enzyme analysis and Southern hybridization. The regions identified were then characterized.
In the SfAV1 genome, repeated fragments were found in HpaII (1 and 1·2 kbp), SspI (1·1 and 1·5 kbp) and XhoI (0·85 and 2·5 kbp) digests. Of these, the two HpaII and two SspI fragments were cloned and sequenced. Comparison of the sequences of the four fragments revealed that they were 98100% identical, permitting construction of a 3·1 kbp consensus fragment and its restriction map (Fig. 4a; accession no. AJ279828). In hybridization experiments, the two cloned HpaII fragments hybridized to two XhoI fragments in a Southern blot containing EcoRI, XbaI and XhoI DNA digests from three SfAV1 isolates (Fig. 4b
, c
). In conjunction with the restriction map and hybridization data, these results indicated that the repeated motif in the SfAV1 genome was at least 3·8 kbp (the sum of the fragments produced by XhoI digest being at least 0·85+2·5 kbp, plus the 0·4 kbp fragment that originated from the 3' end of the XhoISspI fragment; Fig. 4a
). Moreover, the two XhoI fragments of about 2·65 and 2·90 kbp that hybridized with the probe indicated that the locations of the XhoI site in the 5' region of the repeats were variable (Fig. 4
). This was probably due to size differences among the repeats. Differences in intensity of the 0·85, 2·5, 2·65 and 2·90 kbp XhoI fragments in the SfAV1a, SfAV1b and SfAV1c XhoI digests confirmed that the repeats were variable in size. These results also showed that the repeats varied in size among the different SfAV1 isolates (Fig. 4b
, lanes 3, 6 and 9; arrows in the right margins). Some of the differences observed between the EcoRI and XbaI digests of the three SfAV1 isolates were due to differences in the size of these repeats (Fig. 4b
, lanes 12, 45 and 78). Other restriction digests with enzymes unable to cut within the repeats and dot-blot hybridization experiments indicated that five or six repeats were present in the SfAV1a genome. There appeared to be fewer repeats in the SfAV1b and SfAV1c genomes (data not shown).
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In order to characterize further the 980 bp repeats identified previously in the DpAV4 genome (Bigot et al., 1997a ), these repeats and their flanking regions were cloned and sequenced. Analysis of these sequences showed insertions of sequences that varied in size and sequence (Fig. 6
). These insertions resulted in variation among the DpAV4 repeats that yielded sequence identities ranging from 72 to 94%.
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Presence of 5-methyldeoxycytidine in ascovirus DNA
Cytosine methylation of genomic DNA is a characteristic of several vertebrate iridoviruses (Willis & Granoff, 1980 ; Wagner et al., 1985
). In order to determine whether ascovirus and invertebrate iridovirus genomes are also methylated, hydrolysed genomic DNAs from several representatives of these viruses were examined for the presence of 5-methyldeoxycytidine by HPLC with synthetic nucleosides as controls (Fig. 7
). A very high level of 5-methyldeoxycytidine was observed in DpAV4 DNA extracted from A. assectella, with approximately 76% of the deoxycytidines being methylated (Fig. 7b
). Because DpAV4 virions are difficult to purify due to their fragility, the possibility existed that the methylated DNA originated from contamination with pupal host DNA during the virion purification process. Previous studies indicated that approximately 5% of the DNA in DpAV4 DNA preparations was of host origin (Bigot et al., 1997a
, b
). Therefore, DNA isolated from non-parasitized, uninfected pupae of the lepidopteran host, A. assectella, was analysed. High levels of 5-methyldeoxycytidine were observed in these extracts, 36% in male DNA and 56% in female DNA (Fig. 7c
). However, given the relatively low level of host DNA (5%) in DpAV4 DNA preparations, the data indicate that most of the 5-methyldeoxycytidine observed in the DNA isolated from infected hosts originated from DpAV4 DNA. In DNA isolated from DpAV4 and A. assectella, a sixth peak was present corresponding to an unidentified non-nucleoside molecule.
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Because methylation of ascovirus DNA is a potentially important aspect of ascovirus biology, the pattern of virus cytosine methylation in different DpAV4 hosts was examined. This was done by using restriction enzymes that are sensitive to the presence of 5-methyldeoxycytidine. Four types of DpAV4 DNA were examined, each corresponding to a different host or developmental stage of the viral genome. These were DpAV4 DNA from (i) D. pulchellus males, (ii) D. pulchellus females, (iii) 4-day-old A. assectella pupae parasitized by D. pulchellus and (iv) 4-day-old A. assectella pupae infected artificially by injecting them with DpAV4 virions (Bigot et al., 1997a , b
). In males of D. pulchellus, where no virus replication has been detected, the DpAV4 genome is present in nuclei as an unencapsidated circular molecule (Bigot et al., 1997a
). In D. pulchellus females, the virus replicates but the number of virions produced is small. In the two types of infected pupae, virions levels are high. Because these different sources of viral DNA produce highly variable amounts of DNA, the DNA extracts were examined by Southern blot hybridization with specific DpAV4 probes. The viral DNA used for the blots was digested with EcoRI and ClaI (Fig. 8a
) and HpaII or MspI (Fig. 8b
). Activity of ClaI and HpaII is inhibited when 5-methyldeoxycytidine is present in their restriction sites.
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These results indicated that the degree of DpAV4 genome methylation was dependent upon the source. Supernumerary fragments were identified in the MspI/HpaII digests and the intensity of hybridization to these was lower than to other fragments present in the digests, indicating that not all of the cytosine positions were methylated in the DpAV4 genome populations present in each of the extracts. However, we were unable to determine the relative levels of cytosine methylation in each source because it was not possible to determine whether the supernumerary fragments were generated from methylated or unmethylated ClaI and HpaII sites.
Methylation patterns were also examined by using HvAV3c and HvAV3d DNA extracts purified from infected larva of four different hosts: Heliothis zea, H. virescens, Trichoplusia ni and Spodoptera exigua. No differences were found among these four samples, indicating that there was no significant degree of methylation or differences in methylation among HvAV3 isotypes from those that replicated in different infected hosts (data not shown).
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Discussion |
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The low level of superhelicity observed in ascovirus genomes in the present study (Fig. 3) was not detected in previous studies. This is probably because the method used to purify viral DNA in previous studies included sonication and substantial pipetting (Federici et al., 1990
). Because proteins have a lower density (about 1·3 g/cm3) than DNA, it is unlikely that the second, upper layer we observed was due to proteinase K-resistant proteins binding to the DNA. Had this occurred, it would have resulted in a second proteinDNA layer with a density of less than 1·59 g/cm3. In addition, superhelicity was not detected in the genome of DpAV4, in this case probably because the tubes used for equilibrium sedimentation (3 ml) were small (Bigot et al., 1997a
). This apparently prevented the two molecule classes from separating from one another to an extent that they could be resolved. A possible explanation for the low level of supercoiling in ascoviruses is that the genome, although circular, does not have the same packing restrictions as, for example, baculovirus genomes. Many of the latter viruses, such as Trichoplusia ni granulovirus, assemble a DNA genome of 130160 kbp into a narrow, rod-shaped nucleocapsid about 300 nm in length by 80 nm in diameter (Summers & Anderson, 1972
). In contrast, the ascoviruses assemble a DNA genome of about the same size or slightly larger (130180 kbp) in an inner particle that measures 350 nm in length by 130 nm at its maximum width (Federici et al., 1990
).
Interspersed DNA repeats were previously reported in the genomes of SfAV1a (Federici et al., 1990 ) and DpAV4 (Bigot et al., 1997a
) but were only partially characterized. In the present study, we found four to eight interspersed repeats in all ascovirus genomes, ranging from 960980 bp to more than 3·8 kbp. None of these DNA repeats contained ORFs encoding more than 70 amino acids. Also, of the small ORFs detected, none shared any sequence similarity with repeats from other ascovirus species, beyond what would be expected randomly. This lack of sequence similarity among the DNA repeats of the different ascovirus species indicates that they do not correspond to a family of small repeated genes, such as those described for the ALI genes in the genome of the Melanoplus sanguinipes entomopoxvirus (Afonso et al., 1999
).
Although their function remains unknown, the DNA repeats we have characterized might be useful as a taxonomic tool for ascoviruses. Characteristics we observed, such as their presence or absence as well as their length, position and polymorphism, especially in the SfAV1 and HvAV3 repeats, could be used for the rapid characterization of ascovirus isolates and determination of whether an isolate represents a new species or is affiliated to a known species. For example, analysis of the ascovirus DNA polymerase genes has shown that TnAV2 and HvAV3 are closely related and may possibly represent members of a large complex of variants of a single species. However, by using the HvAV3c 1·186 kbp AluI repeat as a probe, we were readily able to distinguish several HvAV3 variants from one another and from variants of TnAV2 (Fig. 5
). Differences in length of the repeats in comparison with those reported previously (Bigot et al., 1997a
) and internal polymorphisms ranging from 72 to 94% were also found in the DpAV4 genome (Fig. 6
).
In the iridoviruses, terminal repeats at the 5' and 3' ends of each permuted genomic molecule have been described in LCDV, frog virus 3 and CIV, suggesting that this is a general feature of iridovirus genomes (Schnitzler et al., 1987a , b
). However, we found no evidence of large interspersed repeats, such as those that occur in ascoviruses, in the invertebrate iridovirus genomes we examined. Aside from physical configuration, i.e. linear versus circular, this would appear to be another major difference between these two virus types.
For the third major characteristic we examined, cytosine methylation, we found significant variation within the ascoviruses and iridoviruses. In the invertebrate iridoviruses we studied, and most of the ascoviruses, we found only a low level of cytosine methylation. However, a very high level of methylation (76%) was detected in DpAV4, in fact the highest for any known virus. From previous reports, it is known that certain vertebrate viruses have a high level of cytosine methylation, in the range of 20% (Willis & Granoff, 1980 ; Wagner et al., 1985
). In eukaryotic species in which chromosomal DNA is methylated, viruses attacking them are known to have their genomes methylated (Karlin & Burge, 1995
; Regev et al., 1998
). The cytosine methylation in these viruses is due to the presence of a virus-encoded enzyme (Kaur et al., 1995
) and it is therefore not surprising to find 5-methyldeoxycytidine in the genomes of these viruses. The function of genome methylation in these viruses is not certain but it is proposed to play a role in the regulation of gene expression (Goorha et al., 1984
). Thus, it is possible that genome methylation is involved in regulating ascovirus gene expression, especially in DpAV4, which has a high level of methylation and varies in its pattern of replication depending on the host in which replication occurs.
In vertebrate genomes, the spontaneous deamination of 5-methyldeoxycytidine induces more frequent CT transitions than other point mutations. As 95% of methylation occurs on CpG, this results in vertebrate genomes in an observed to expected CpG frequency ratio with a mean value of 0·20·5 (Jabbari et al., 1997 ). This phenomenon, the so-called CpG shortage, is also observed in most vertebrate viruses (Karlin & Burge, 1995
). Dinucleotide data available from analysis of DpAV4 DNA (more than 35 kbp) does not reveal any CpG shortage. However, it does reveal a significant CpC and CpT shortage in eight sequences analysed (ranging from 1·5 to 12·2 kbp; accession nos AJ279812AJ279815). Moreover, similar results were obtained with four SfAV1a sequences (3·1 and 8·9 kbp; accession nos AJ279828 and AJ279830; and two unregistered sequences of 1·1 and 1·2 kbp). Interestingly, although no CpG and CpC shortage is found in the available sequences of invertebrate iridoviruses like CIV, IV31 and T-MIV, a weak but significant CpT shortage is found (accession nos L22300, M81388, AF003534 and AF83915 for CIV; unregistered sequences for IV31 and T-MIV). Overall, these data suggest that cytosine methylation occurs in all ascovirus and iridovirus genomes. However, it appears that the specificity of dinucleotide methylation varies among different groups within a family. Thus, it appears that CpG is the most methylated dinucleotide in the vertebrate iridoviruses, whereas it is CpT in the invertebrate iridoviruses and CpC and the CpT in the ascoviruses.
Our purpose in undertaking this study was to characterize some of the key physical and biochemical properties of invertebrate ascovirus and iridovirus genomes in order to provide a firmer foundation for comparisons of their genomes. The results of the present study show that, for the properties we examined, these viruses are very different, whereas the accompanying data on the phylogenetic relatedness of several of the genes they have in common suggest that they are closely related (Stasiak et al., 2000 ). If these two virus types are closely related, this apparent paradox may be due to host shifts during evolution, after which there was rapid evolution that facilitated adaptation of the viruses to the biologies of their new hosts. Some evidence in support of this hypothesis comes from our accompanying study of ascovirus phylogenetics (Stasiak et al., 2000
). In this study, based on an analysis of
DNA polymerase genes, we showed that the family Ascoviridae contains two different types, with one species, DpAV4, being clearly distinguished from the other three, SfAV1, TnAV2 and HvAV3. This clear phylogenetic separation of these ascoviruses based on the
DNA polymerase gene corresponded with their biologies, an important trait being that the DpAV4 genome is capable of existing as an unencapsidated molecule in nuclei of its parasitoid vector, whereas this trait is not known for the other ascoviruses. Thus, further studies of these viruses, including studies based on the phylogenetic relatedness of structural protein genes, are now of even greater interest due to the results obtained in the present study. These studies have the potential to resolve the relatedness of these virus families and to contribute to our knowledge of the evolution of large dsDNA viruses.
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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.