Universität Hohenheim, Institut für Genetik, FG Allgemeine Virologie, Emil-Wolff-Str. 14, D-70599 Stuttgart, Germany
Correspondence
Artur J. P. Pfitzner
pfitzner{at}uni-hohenheim.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY971002.
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INTRODUCTION |
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Endosymbiotic Chlorella algae have been found in association with many organisms from different classes of the animal kingdom like Rhizopoda, Ciliata, Hydrozoa and Turbellaria. However, so far, viruses have only been isolated from symbiotic Chlorella strains of P. bursaria (Ciliata) and H. viridis (Hydrozoa). Some viruses infecting P. bursaria Chlorella strains have been characterized in detail. Paramecium bursaria Chlorella virus (PBCV-1) is the prototype of this virus group and the complete nucleotide sequence of the viral genome is available.
In this study, a new Chlorovirus from an endosymbiotic Chlorella strain of the heliozoon Acanthocystis turfacea (Rhizopoda), designated Acanthocystis turfacea Chlorella virus (ATCV), was identified. It was isolated from a freshwater pond in Stuttgart, Germany. The virus was purified and characterized with respect to its structural, physical and biochemical properties. In addition, a conserved region of the DNA polymerase gene and random parts of the DNA genome of ATCV were sequenced. Data were compared to the sequence of PBCV-1, the prototype of the genus Chlorovirus, and to other DNA viruses.
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METHODS |
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Isolation of virus particles.
Water samples were collected from freshwater ponds around the University of Hohenheim, Stuttgart, Germany. Water samples were filtered through a nitrocellulose membrane filter (0·45 µm pore size) and 1 ml of each water sample was used to infect an exponentially growing culture of Chlorella SAG 3.83. After 2 weeks cultivation, algae were pelleted by centrifugation at 5000 g for 10 min. The culture supernatants were used for virus plaque assays. To this end, 1·2x108 Chlorella cells were mixed with 100 µl samples of each culture supernatant and 2·5 ml soft agar and the mixtures were plated on MBBM. Plates were incubated at 25 °C in continuous light. After 35 days, plaque formation could be observed.
For large-scale virus purification, a single plaque was picked from the algal lawn and transferred to an exponentially growing culture of Chlorella SAG 3.83 (2x108 cells ml1) in 800 ml MBBM in a 2 litre Erlenmeyer flask. The culture was propagated for 5 days at 25 °C in continuous light on a rotating shaker at 200 r.p.m. until cell lysis was observed. The lysate was centrifuged at 5000 g for 5 min to remove cell debris. Triton X-100 was added to the supernatant to a final concentration of 1 % and virus particles were precipitated by centrifugation at 43 000 g for 1 h at 4 °C. Virus particles were resuspended in 4 ml 50 mM Tris/HCl, pH 7·8, and were further purified on a 1040 % sucrose gradient by centrifugation for 20 min at 72 000 g. Purified virus particles formed a major band in the middle of the sucrose gradient. The virus band was removed from the gradient with a sterile needle and diluted to 30 ml with 50 mM Tris/HCl, pH 7·8. The particles were pelleted by centrifugation for 3 h at 75 000 g and resuspended in 8 ml 50 mM Tris/HCl, pH 7·8. The concentration of the purified virus particles (p.f.u. ml1) was determined on a lawn of Chlorella SAG 3.83.
Electron microscopy.
For electron microscopic studies, purified virus particles were treated with 2 % phosphotungstic acid and analysed using an LEO EM 912 AB transmission electron microscope (LEO Oberkochen).
Analysis of viral proteins.
Protein analysis of ATCV-1, ATCV-2 and PBCV-1 was carried out using 15 % SDS-PAGE with 2 µl (4·0x1011 p.f.u. ml1) purified virus particles as described previously (Laemmli, 1970). Proteins were visualized by Coomassie blue staining (Ausubel et al., 1987
).
Preparation of viral DNAs and construction of a genomic library.
DNA was isolated from purified ATCV-1, ATCV-2 and PBCV-1 by phenol extraction after treatment of virus isolates with proteinase K (0·1 mg ml1) for 8 h at 55 °C. Viral DNAs were digested with EcoRI, BamHI or HindIII and samples were separated overnight on a 1 % agarose gel. DNA was visualized by ethidium bromide staining. For construction of a genomic library, 15 µg extracted viral DNA was digested overnight with BamHI. Reaction products were extracted with phenol, precipitated with ethanol, resuspended in TE (10 mM Tris/HCl, pH 8·0, 1 mM EDTA) and ligated into the BamHI site of pUC18. The ligation was transformed into Escherichia coli DH5 competent cells and plasmid DNA from minipreparations was used for sequencing viral fragments (Sambrook et al., 1989
; Sanger et al., 1977
).
PCR amplification of DNA polymerase sequences.
For amplification of a conserved region of the DNA polymerase gene of ATCV, degenerate primers were designed according to Chen & Suttle (1995). Using the upstream primer pol1 [5'-GA(A/G)GGIGCIACIGTI(T/C)TIGA(T/C)GC-3'] and the downstream primer pol2 [5'-(G/C)(A/T)(A/G)TCIGT(A/G)TCICC(A/G)TA-3'], a 440 bp fragment could be amplified from 100 ng ATCV-1 or ATCV-2 DNA under the conditions given by Chen & Suttle (1995)
. PBCV-1 DNA was used as a positive control. The amplified DNA polymerase fragments were inserted into pUC18 by TA cloning and sequenced.
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RESULTS |
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Morphology of ATCV
Chlorella viruses are known to be large icosahedral particles. Depending on the microscopic technique used, diameters of 140190 nm have been reported for these viruses (Van Etten et al., 1991). Electron microscopic analysis of ATCV-1 and ATCV-2 with negative staining revealed that their capsids also have a distinct icosahedral shape with a diameter of 160190 nm (Fig. 3
). There are no apparent structural dissimilarities between ATCV-1 and ATCV-2 (data not shown). The particles of both viruses have filamentous structures extending from the vertices (Fig. 3b
). Earlier investigations on the structure of PBCV-1 had demonstrated similar extensions and it has been suggested that the filamentous structures may be involved in attachment of the virus to the host cell wall (Van Etten et al., 1991
).
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Analysis of ATCV genomic DNA
To analyse the relatedness between ATCV and PBCV-1, and, in particular, between ATCV-1 and ATCV-2 at the genomic level, purified viral DNAs were incubated with the restriction endonucleases EcoRI, BamHI and HindIII. Digested DNAs were separated on a 0·8 % agarose gel and the DNA patterns were compared to each other (Fig. 5). Whereas the restriction enzyme fragmentation patterns of ATCV-1 and ATCV-2 DNAs were identical with the three endonucleases, the restriction pattern of PBCV-1 DNA was completely different from the ATCV pattern. This result may suggest a rather low overall similarity between ATCV and PBCV-1 at the DNA level. On the other hand, the number and size of the restriction fragments produced by different restriction enzymes indicate that ATCV has a large DNA genome of about 350 kb, like many phycodnaviruses, including PBCV-1.
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DISCUSSION |
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Even more interestingly, some ATCV proteins are more similar to proteins from distant species than to the homologous PBCV-1 proteins (Table 1). The GDP-mannose dehydratase of ATCV (ORF3) exhibits 66 % identity to the GDP-mannose dehydratase of Y. enterocolitica, whereas identity to the PBCV-1 protein A118R is only 42 %. ORF6 of ATCV shows 27 % identity to the prokaryotic-type RB ribonucleotide reductase of Roseophage SIO1, a T7-like virus infecting marine prokaryotic host organisms. At the same genome position, PBCV-1 contains a gene for the small subunit of ribonucleotide reductase, A476R. The PBCV-1 gene exhibits no similarity to the ribonucleotide reductase of ATCV. On the other hand, A476R displays a high identity (58 %) to ribonucleotide reductase from tobacco and thus is of eukaryotic origin. Together, these data suggest that ATCV and PBCV-1 contain genes with corresponding functions that have been obtained from different sources. Since ATCV and PBCV-1 are clearly structurally related, they most likely developed from a common ancestor virus. During evolution, however, the two viruses appear to have acquired genes with equivalent functions from quite diverse sources.
Evolutionary relationship of ATCV to Chlorella viruses and other DNA viruses
Phylogenetic trees based on the amino acid sequences of the main capsid proteins have demonstrated that the family Phycodnaviridae are related to the family Iridoviridae, members of which commonly infect insects and some vertebrates (Van Etten & Meints, 1999). Iridoviruses also share some other properties with phycodnaviruses, e.g. icosahedral capsid and a large dsDNA genome. In addition, cladistic analysis with genes shared by different virus groups supports the monophyly of poxviruses, African swine fever virus and phycodnaviruses (Iyer et al., 2001
). Phylogenetic analysis based on sequences of the DNA polymerase conserved deoxynucleotide-binding domain from several algal viruses places the Pbi and NC64A Chlorella viruses in separate, albeit closely related groups (Chen & Suttle, 1996
). Furthermore, these data suggest that phycodnaviruses are more closely related to herpesviruses than to other DNA viruses. To clarify the relatedness of ATCV to Chlorella viruses and to other viral groups, the conserved deoxynucleotide-binding domain of DNA polymerase from ATCV-1 and ATCV-2 was cloned. Degenerate primers were designed and a 440 bp fragment was amplified via PCR from the viral DNAs. Sequence analysis showed that there are no differences in the nucleotide sequence of this domain between ATCV-1 and ATCV-2 (data not shown), which supports our previous conclusion that ATCV-1 and ATCV-2 are two strains of the same virus.
The derived amino acid sequence was compared to the DNA polymerase domains from Pbi and NC64A viruses and, using CLUSTALW, to those from other DNA viruses. Fig. 6 shows an alignment of the amino acid sequence of the ATCV DNA polymerase domain with the corresponding sequences of the Pbi virus Chlorella virus A-1 (CVA-1) and the NC64A virus PBCV-1. The identity of the ATCV sequence to the CVA-1 sequence is 89 %, whereas identity to the PBCV-1 sequence is only 82 %. The main difference between the ATCV, CVA-1 and PBCV-1 sequences is a sequence of six additional amino acids in the PBCV-1 DNA polymerase domain. The significance of these additional amino acids in the PBCV-1 DNA polymerase gene is not clear. However, the insertion might indicate the presence of a so far undetected intron in the PBCV-1 sequence. Introns have been found in other parts of the DNA polymerase gene of PBCV-1 and the positions and sequences of the introns are conserved in many NC64A viruses, but not in Pbi viruses (Zhang et al., 2001
). The absence of the six amino acids in the DNA polymerase sequences of ATCV and CVA-1 indicates that ATCV is more closely related to Pbi viruses than to PBCV-1 and other NC64A viruses. This view is also supported by the phylogenetic tree generated from multiple alignment of the DNA polymerase deoxynucleotide-binding domain sequences from different DNA viruses by CLUSTALW (Fig. 7
). Although ATCV is closely related to PBCV-1, the neighbour-joining algorithm puts ATCV on a separate branch together with the Pbi Chlorella viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 March 2005;
accepted 20 June 2005.
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