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Molecular characterisation of a novel cassava associated circular ssDNA virus
Virus Research 166 (2012) 130–135
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Short communication
Molecular characterisation of a novel cassava associated circular ssDNA virus Anisha Dayaram a , Allen Opong b , Anja Jäschke a,c , James Hadfield a , Marianna Baschiera d , Renwick C.J. Dobson a,f , Samuel K. Offei g , Dionne N. Shepherd d , Darren P. Martin e , Arvind Varsani a,f,h,∗ a
School of Biological Sciences, University of Canterbury, Ilam, Christchurch 8140, New Zealand Virology Section, CSIR-Crop Research Institute, Kumasi, Ghana Department of Infectious Diseases, University of Heidelberg, D-69120 Heidelberg, Germany d Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa e Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa f Biomolecular Interaction Centre, University of Canterbury, Ilam, Christchurch 8140, New Zealand g School of Agriculture and Consumer Sciences, University of Ghana, Legon, Ghana h Electron Microscope Unit, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa b c
a r t i c l e
i n f o
Article history: Received 19 January 2012 Received in revised form 10 March 2012 Accepted 14 March 2012 Available online 23 March 2012 Keywords: ssDNA virus Cassava Rolling circle amplification
a b s t r a c t The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus’ likely origin and actual host species. © 2012 Elsevier B.V. All rights reserved.
Our appreciation for the abundance and diversity of known single-stranded DNA (ssDNA) viruses has increased rapidly over the past decade following the development of new molecular techniques. Sequence independent rolling circle amplification (RCA) coupled with viral metagenomic techniques such as shotgun sequencing and viral particle purification, have made it possible to detect and discover a wide variety of previously unknown viral species and genera (Delwart and Li, 2011; Ng et al., 2009, 2011a). In particular, many novel species have been discovered in both the animal-infecting family, Circoviridae (Blinkova et al., 2009, 2010; Ge et al., 2011; Kim et al., 2011; Li et al., 2010, 2011; Phan et al., 2011; Rosario et al., 2011), and the plant-infecting family,
∗ Corresponding author at: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand. Tel.: +64 3 366 7001x4667; fax: +64 3 364 2590. E-mail address: [email protected] (A. Varsani). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2012.03.009
Geminiviridae (Briddon et al., 2010; Martin et al., 2011; Varsani et al., 2009). Primary among the recent advances that have made these discoveries possible is RCA which enables the sequence nonspecific high fidelity amplification of whole circular ssDNA virus genomes using the DNA polymerase of phage phi29 (an enzyme which preferentially amplifies circular DNA molecules; Johne et al., 2009; Rector et al., 2004). In this communication we describe the genome sequence of a probable ssDNA virus with a circular genome sequence similar to those of the geminivirus-related mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1; Yu et al., 2010), a mosquito associated circular ssDNA virus (Mosquito VEM virus SDBVL; Ng et al., 2011b) and a probable virus associated with the faeces of a European badger (called Meles meles faecal virus or MmFV van den Brand et al., 2011). A probable viral genome was isolated from two out of four cassava leaf samples collected from two different plants in Ghana (Kwame Danso, 7◦ 45 36 N; 0◦ 41 20 W) during routine screening
A. Dayaram et al. / Virus Research 166 (2012) 130–135
for geminiviruses. Total DNA was extracted from each cassava leaf using Extract-N-Amp (Sigma–Aldrich, USA) as previously described (Shepherd et al., 2008), and circular DNA was amplified using TempliPhi (GE Healthcare). Digestion of the rolling circle amplified product with BamHI yielded an approximately 1.5 kb linear DNA fragment, which was ligated with BamHI-digested pUC19 (Fermentas, USA) and sequenced by Macrogen Inc. (South Korea) using primer walking. Preliminary analysis of the ∼1.5 kb sequence indicated that although it had a low degree of sequence similarity to SsHADV-1, it was missing a ∼700 bp fragment. Based on the sequence information, back-to-back primers were designed (G5b forward primer 5 -GAA TGC GGA TGC CTT CAG TCA AGT TG-3 ; G5b reverse primer 5 -TCT ACG TTC CCG TAT TCC TCG TCT G-3 ) to amplify the full circular sequence of this probable viral genome. The full-length sequence was amplified from the crude cassava leaf DNA extracts using AccuzymeTM hi-fidelity DNA polymerase (Bioline, USA) and the following amplification protocol: 95 ◦ C [2 min] (94 ◦ C [30 s], 55 ◦ C [30 s], 72 ◦ C [4 min]) × 30 cycles, 72 ◦ C [5 min], 4 ◦ C [10 min]. The ∼2.2 kb amplicons were ligated to pJET1.2 (Fermentas, USA) and sequenced by Macrogen (Korea) using primer walking. The two amplicons (sharing 99% pairwise identity) contained three large open reading frames (ORFs) with the potential to encode proteins with detectable homology to known ssDNA virus genes. A BLASTn search (Altschul et al., 1990) of the full viral genome revealed that the probable virus isolate is most closely related to SsHADV-1 (GenBank Accession # GQ365709) with maximum identity of 71% (21% coverage; E value – 9 × 10−33 , over a 310 and 271 nucleotide region) of the genome, and is also detectably homologous to (albeit slightly more distantly related to) a circular ssDNA sequence detected during metagenomic analyses of circular DNAs associated with mosquitoes (GenBank Accession # HQ3350861; 7% coverage, maximum identity of 71%, E value – 1 × 10−11 ). SsHADV-1 is the only known ssDNA mycovirus and has a genome organisation similar to that of circoviruses but a replication associated protein (Rep) most closely related to that of geminiviruses (Yu et al., 2010). A NCBI BLASTx search using the largest ORF (945 nt) as a query sequence revealed a significant degree of similarity with the presumed capsid proteins (CP) of SsHADV-1 (GenBank Accession # YP 003104795; 97% coverage, maximum identity of 37%, E value – 4 × 10−53 ), Mosquito VEM virus SDBVL (GenBank Accession # AEF58774; 88% coverage, maximum identity of 34%, E value – 1 × 10−37 ) and MmFV (GenBank Accession # AEF58774; maximum identity of 27%, E value – 4 × 10−24 ) (Ng et al., 2011b; van den Brand et al., 2011; Yu et al., 2010). BLASTx searches of two other large ORFs on the opposite strand to the largest ORF suggested they jointly encoded a homologue of the SsHADV-1 replication associated protein (Rep; GenBank Accession # YP 003104796; 99% coverage, maximum identity of 62%, E value – 1 × 10−147 ): a protein homologous to replication associated proteins found in a wide variety of ssDNA circular replicons including the geminiviruses, nanoviruses, circoviruses, various plasmids and currently unclassified environmental ssDNAs. Interestingly, our BLASTx search also revealed that the Rep is a homologue of a hypothetical protein in the genome of the fungus Serpula lacrymans var lacrymans S7.9 (GenBank Accession # EGO20879; 97% coverage, maximum identity of 32%, E value – 1 × 10−40 ). We propose naming this novel probable ssDNA virus cassava associated circular DNA virus (CasCV). Given that geminiviruses in the genus Mastrevirus have Rep genes expressed from spliced transcripts (Dekker et al., 1991; Mullineaux et al., 1990; Schalk et al., 1989; Wright et al., 1997) we examined alignments of the apparent Rep-encoding ORFs (constructed using MEGA5; Tamura et al., 2011) and identified potential intron acceptor and donor sites that could potentially yield a transcript from which a full length protein might be expressed (Fig. 1). Similar introns were identified in Mosquito VEM virus SDBVL and
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MmFV (Fig. 1). In mastreviruses, whereas full-length Rep proteins are expressed from spliced transcripts, it appears that shorter Rep-like proteins with unique C-termini, called RepA proteins, are expressed from unspliced transcripts. Since in mastreviruses RepA seems to play a pivotal role in inducing a cellular environment conducive to virus replication, in these viruses the relative amounts of Rep and RepA are a major determinant of how effectively they replicate (Heyraud-Nitschke et al., 1995; Wright et al., 1997). The spliced CasCV Rep shares 62.7% amino acid identity with the Rep of SsHADV-1, and 45.7% and 42.9% identity with the spliced Reps of Mosquito VEM virus SDBVL and MmFV respectively (Fig. 1). The spliced Rep amino acid sequence of the novel CasCV replicon was aligned together with other Reps of ssDNA viruses, phytoplasma plasmids and fungal integrons with similarity to ssDNA Reps, using Clustal X (Larkin et al., 2007). Maximum likelihood (ML) phylogenetic trees were constructed using PhyML (Guindon et al., 2010) with the JTT+g4 substitution model with chi-squared approximate likelihood ratio test used to determine degrees of branch support (Anisimova and Gascuel, 2006; Fig. 2). The Rep ML phylogenetic tree revealed that that the CasCV replicon is likely the genome of a virus within the same family as SsHADV-1 and a previously described Mosquito VEM virus SDBVL. Interestingly, based on degrees of sequence similarity the CPs of these viruses are possibly more closely related to circo/cycloviruses, whereas the Reps are most closely related to probable Rep integrons within the genomes of various fungi (including those of Laccaria bicolor, Serpula lacrymans var lacrymans and Aspergillus fumigates), geminiviruses and various possible ssDNA viruses detected during metagenomic surveys of soil, marine and freshwater environments. It must be stressed however that the extremely low degrees of sequence similarity between the CP sequences of the major ssDNA virus groups and the lack of a suitable outgroup makes it difficult to come to a firm conclusion as to whether the fungus associated ssDNA virus CP sequences share a more recent common ancestor with the circoviruses and nanoviruses than they do with the geminiviruses. Located between the Rep and the CP ORFs is a 136-nt intergenic region which we call here the long intergenic region (LIR). The LIR has similar characteristics to analogous intergenic regions found in geminiviruses, nanoviruses and circoviruses, with a geminivirus-like nonanucleotide (TAATATTAT in the new sequence and SsHADV-1 as opposed to a TAATATTAC sequence in geminiviruses) at the presumed (by analogy with the geminiviruses) origin of virion-strand replication (ori; Fig. 1). We were, however, unable to find a similarly conserved ori nonanucleotide in the previously described Mosquito VEM virus SDBVL sequence that closely resembles the novel CasCV sequence described here. As in SsHADV1 and the geminiviruses, the ori nonanucleotide resides within the loop of a probable hairpin structure. We additionally identified a probable TATA box between this hairpin structure and the CP start codon that is in a similar position to an analogous sequence found in the geminiviruses. Also in the LIR downstream of the hairpin is a GC-rich region in a similar position to a G-Box element of geminiviruses that is known to be involved in their transcriptional regulation (Eagle and Hanley-Bowdoin, 1997; Fenoll et al., 1988, 1990). The Rep protein is highly conserved amongst ssDNA viruses and plasmids that replicate by a rolling circle mechanism (Ilyina and Koonin, 1992; Koonin and Ilyina, 1992). In geminiviruses and probably other ssDNA replicons it recognises, binds and cleaves the origin via its N-terminal ∼100 amino acids (Desbiez et al., 1995; Nash et al., 2011). A search of the NCBI’s Conserved Domain Database (MarchlerBauer et al., 2009) with the probable Rep sequence of CasCV revealed that it contains both a N-terminal geminivirus-like Rep catalytic domain and a geminivirus-like Rep protein central domain. The NMR structure of the Rep catalytic domain from the
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Fig. 1. Predicted stem-loop structure in the origin of virion-strand replication of cassava associated circular DNA virus (CasCV) (A), two-dimensional graphical representation of pairwise amino acid sequence identities (calculated with pairwise deletion of gaps; scale represents percentage identity) of the predicted Rep and CP of CasCV with those of SsHADV-1, Mosquito VEM virus SDBVL and MmFV (B). Genome organisation of CasCV (C), Mosquito VEM Gemini Fungi virus-SDBVL G (D), MmFV (E), SsHADV-1 (F) and the predicted introns (G).
begomovirus Tomato yellow leaf curl virus has indicated that this domain is related to a large group of proteins that bind nucleic acid sequences. In CasCV this is therefore also likely a crucial component of the replisome (Campos-Olivas et al., 2002; Desbiez et al., 1995). Within the two geminivirus-like domains of the CasCV Rep are five conserved rolling circle replication (RCR) related motifs that have been identified in geminivirus, circovirus and nanovirus Reps (Ilyina and Koonin, 1992; Koonin and Ilyina, 1992, 1993), (Fig. 3, Supplementary Fig. 1): from the N- to C-terminus these are RCR motif I, RCR motif II, the Geminivirus Rep sequence motif (GRS), RCR motif III, and RCR motif IV. While highly conserved amongst all RCR replicons, the specific function of RCR motif I is unknown. It is, however, reasonably likely that it may be involved in the sequence specific recognition of tandemly repeated nucleotide sequences (called iterons) near the ori (Arguello-Astorga and Ruiz-Medrano, 2001). During origin cleavage RCR motif II (HLHxxxQ) is believed to coordinate the binding of metal ions via its two histidine residues (Ilyina and Koonin, 1992). The GRS motif has been implicated in the ssDNA cleavage reaction that occurs during the initiation of rolling circle replication (Nash et al., 2011) and while less conserved that the other four motifs, has been found in the Rep proteins of geminiviruses and some phytoplasmal and algal plasmids. The actual catalytic site for DNA cleavage is RCR motif III (YxxKD/E)
within which the conserved tyrosine residue is believed to covalently bind to DNA after cleavage (Heyraud-Nitschke et al., 1995; Laufs et al., 1995). RCR motif IV (EGX4 GKTX32 DD) is believed to encode a NTP-binding domain (Gorbalenya et al., 1989), such as that found in proteins with helicase, protease and kinase activity, and exhibits ATPase activity that is required for replication (Desbiez et al., 1995). It has also been suggested that RCR motif IV might confer Rep with a helicase-like activity that is crucial during RCR (Bisaro, 1996; Gutierrez, 1999). Given the high degree of sequence conservation between the Reps of CasCV and geminiviruses we used the computer program MODELLER (Eswar et al., 2006) and the 3D nuclear magnetic resonance (NMR) determined structure of the geminivirus tomato yellow leaf curl virus (TYLCV) Rep N-terminal region (PDB ID 1L2M; Campos-Olivas et al., 2002) to infer the likely structure of the homologous Rep region of CasCV. This structure was visualised with PyMOL Ver 0.99 (www.pymol.org). In support of the hypothesis that the CaSCV Rep homologue is a functional Rep protein, the structural arrangements of the four RCR related motifs within the examined region (RCR motifs I, II and III and the GRS motif) were also highly conserved (Fig. 3c) and are therefore likely to be functionally very similar to their geminivirus homologues.
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Fig. 2. Maximum likelihood phylogenetic tree of the predicted amino acid alignments of the Rep of CasCV with those of representative ssDNA viruses, phytoplasma plasmids and fungal integrons. Closed circles indicate branches supported of greater than 90% whereas open circles indicate branch support of 50–89%.
Noting that the CasCV Rep and CP respectively share 62.7% and 39.8% similarity to those of SsHADV-1, we assumed that CasCV could potentially be a mycovirus-infecting a fungus associated with the cassava leaf samples that we examined. We therefore screened total DNA extracts of the cassava leaf samples from which CasCV was derived, using the fungal 18S rRNA specific PCR primers – 18S forward primer 5 -CAA GGT CTC TGT TGG TGA ACC AGC GG-3 , 18S reverse primer 5 -TCC TCC GCT TAT TGA TAT GCT TAA GTT-3 (White et al., 1990. We identified two fungal pathogens, one Collectotrichum-like and the other
Plectosphaerella cucumerina. Whereas Collectotrichum gloeosporioides is a fungus known to cause damaging serious anthracnose disease in cassava and yams (Amusa, 2001), we were unable to find any documented association of P. cucumerina with cassava. We were also unable to culture fungal material from the cassava leaf samples. Therefore, although it is quite probable that CasCV is a mycovirus that should be classified within the same taxonomic family as SsHADV-1, it remains for proper host range studies using infectious clones to demonstrate its likely origin.
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Fig. 3. Sequence motifs found near the N-terminus of predicted CasCV and other representative ssDNA virus Rep proteins (A). Alignment of selected Rep sequences with identified rolling circle replication motifs I, II, III and IV, and the GRS highlighted (B). Homology modelling of the N terminal portion of CasCV Rep, with the models drawn using PyMOL (C).
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Short communication
Molecular characterisation of a novel cassava associated circular ssDNA virus Anisha Dayaram a , Allen Opong b , Anja Jäschke a,c , James Hadfield a , Marianna Baschiera d , Renwick C.J. Dobson a,f , Samuel K. Offei g , Dionne N. Shepherd d , Darren P. Martin e , Arvind Varsani a,f,h,∗ a
School of Biological Sciences, University of Canterbury, Ilam, Christchurch 8140, New Zealand Virology Section, CSIR-Crop Research Institute, Kumasi, Ghana Department of Infectious Diseases, University of Heidelberg, D-69120 Heidelberg, Germany d Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa e Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa f Biomolecular Interaction Centre, University of Canterbury, Ilam, Christchurch 8140, New Zealand g School of Agriculture and Consumer Sciences, University of Ghana, Legon, Ghana h Electron Microscope Unit, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa b c
a r t i c l e
i n f o
Article history: Received 19 January 2012 Received in revised form 10 March 2012 Accepted 14 March 2012 Available online 23 March 2012 Keywords: ssDNA virus Cassava Rolling circle amplification
a b s t r a c t The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus’ likely origin and actual host species. © 2012 Elsevier B.V. All rights reserved.
Our appreciation for the abundance and diversity of known single-stranded DNA (ssDNA) viruses has increased rapidly over the past decade following the development of new molecular techniques. Sequence independent rolling circle amplification (RCA) coupled with viral metagenomic techniques such as shotgun sequencing and viral particle purification, have made it possible to detect and discover a wide variety of previously unknown viral species and genera (Delwart and Li, 2011; Ng et al., 2009, 2011a). In particular, many novel species have been discovered in both the animal-infecting family, Circoviridae (Blinkova et al., 2009, 2010; Ge et al., 2011; Kim et al., 2011; Li et al., 2010, 2011; Phan et al., 2011; Rosario et al., 2011), and the plant-infecting family,
∗ Corresponding author at: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand. Tel.: +64 3 366 7001x4667; fax: +64 3 364 2590. E-mail address: [email protected] (A. Varsani). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2012.03.009
Geminiviridae (Briddon et al., 2010; Martin et al., 2011; Varsani et al., 2009). Primary among the recent advances that have made these discoveries possible is RCA which enables the sequence nonspecific high fidelity amplification of whole circular ssDNA virus genomes using the DNA polymerase of phage phi29 (an enzyme which preferentially amplifies circular DNA molecules; Johne et al., 2009; Rector et al., 2004). In this communication we describe the genome sequence of a probable ssDNA virus with a circular genome sequence similar to those of the geminivirus-related mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1; Yu et al., 2010), a mosquito associated circular ssDNA virus (Mosquito VEM virus SDBVL; Ng et al., 2011b) and a probable virus associated with the faeces of a European badger (called Meles meles faecal virus or MmFV van den Brand et al., 2011). A probable viral genome was isolated from two out of four cassava leaf samples collected from two different plants in Ghana (Kwame Danso, 7◦ 45 36 N; 0◦ 41 20 W) during routine screening
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for geminiviruses. Total DNA was extracted from each cassava leaf using Extract-N-Amp (Sigma–Aldrich, USA) as previously described (Shepherd et al., 2008), and circular DNA was amplified using TempliPhi (GE Healthcare). Digestion of the rolling circle amplified product with BamHI yielded an approximately 1.5 kb linear DNA fragment, which was ligated with BamHI-digested pUC19 (Fermentas, USA) and sequenced by Macrogen Inc. (South Korea) using primer walking. Preliminary analysis of the ∼1.5 kb sequence indicated that although it had a low degree of sequence similarity to SsHADV-1, it was missing a ∼700 bp fragment. Based on the sequence information, back-to-back primers were designed (G5b forward primer 5 -GAA TGC GGA TGC CTT CAG TCA AGT TG-3 ; G5b reverse primer 5 -TCT ACG TTC CCG TAT TCC TCG TCT G-3 ) to amplify the full circular sequence of this probable viral genome. The full-length sequence was amplified from the crude cassava leaf DNA extracts using AccuzymeTM hi-fidelity DNA polymerase (Bioline, USA) and the following amplification protocol: 95 ◦ C [2 min] (94 ◦ C [30 s], 55 ◦ C [30 s], 72 ◦ C [4 min]) × 30 cycles, 72 ◦ C [5 min], 4 ◦ C [10 min]. The ∼2.2 kb amplicons were ligated to pJET1.2 (Fermentas, USA) and sequenced by Macrogen (Korea) using primer walking. The two amplicons (sharing 99% pairwise identity) contained three large open reading frames (ORFs) with the potential to encode proteins with detectable homology to known ssDNA virus genes. A BLASTn search (Altschul et al., 1990) of the full viral genome revealed that the probable virus isolate is most closely related to SsHADV-1 (GenBank Accession # GQ365709) with maximum identity of 71% (21% coverage; E value – 9 × 10−33 , over a 310 and 271 nucleotide region) of the genome, and is also detectably homologous to (albeit slightly more distantly related to) a circular ssDNA sequence detected during metagenomic analyses of circular DNAs associated with mosquitoes (GenBank Accession # HQ3350861; 7% coverage, maximum identity of 71%, E value – 1 × 10−11 ). SsHADV-1 is the only known ssDNA mycovirus and has a genome organisation similar to that of circoviruses but a replication associated protein (Rep) most closely related to that of geminiviruses (Yu et al., 2010). A NCBI BLASTx search using the largest ORF (945 nt) as a query sequence revealed a significant degree of similarity with the presumed capsid proteins (CP) of SsHADV-1 (GenBank Accession # YP 003104795; 97% coverage, maximum identity of 37%, E value – 4 × 10−53 ), Mosquito VEM virus SDBVL (GenBank Accession # AEF58774; 88% coverage, maximum identity of 34%, E value – 1 × 10−37 ) and MmFV (GenBank Accession # AEF58774; maximum identity of 27%, E value – 4 × 10−24 ) (Ng et al., 2011b; van den Brand et al., 2011; Yu et al., 2010). BLASTx searches of two other large ORFs on the opposite strand to the largest ORF suggested they jointly encoded a homologue of the SsHADV-1 replication associated protein (Rep; GenBank Accession # YP 003104796; 99% coverage, maximum identity of 62%, E value – 1 × 10−147 ): a protein homologous to replication associated proteins found in a wide variety of ssDNA circular replicons including the geminiviruses, nanoviruses, circoviruses, various plasmids and currently unclassified environmental ssDNAs. Interestingly, our BLASTx search also revealed that the Rep is a homologue of a hypothetical protein in the genome of the fungus Serpula lacrymans var lacrymans S7.9 (GenBank Accession # EGO20879; 97% coverage, maximum identity of 32%, E value – 1 × 10−40 ). We propose naming this novel probable ssDNA virus cassava associated circular DNA virus (CasCV). Given that geminiviruses in the genus Mastrevirus have Rep genes expressed from spliced transcripts (Dekker et al., 1991; Mullineaux et al., 1990; Schalk et al., 1989; Wright et al., 1997) we examined alignments of the apparent Rep-encoding ORFs (constructed using MEGA5; Tamura et al., 2011) and identified potential intron acceptor and donor sites that could potentially yield a transcript from which a full length protein might be expressed (Fig. 1). Similar introns were identified in Mosquito VEM virus SDBVL and
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MmFV (Fig. 1). In mastreviruses, whereas full-length Rep proteins are expressed from spliced transcripts, it appears that shorter Rep-like proteins with unique C-termini, called RepA proteins, are expressed from unspliced transcripts. Since in mastreviruses RepA seems to play a pivotal role in inducing a cellular environment conducive to virus replication, in these viruses the relative amounts of Rep and RepA are a major determinant of how effectively they replicate (Heyraud-Nitschke et al., 1995; Wright et al., 1997). The spliced CasCV Rep shares 62.7% amino acid identity with the Rep of SsHADV-1, and 45.7% and 42.9% identity with the spliced Reps of Mosquito VEM virus SDBVL and MmFV respectively (Fig. 1). The spliced Rep amino acid sequence of the novel CasCV replicon was aligned together with other Reps of ssDNA viruses, phytoplasma plasmids and fungal integrons with similarity to ssDNA Reps, using Clustal X (Larkin et al., 2007). Maximum likelihood (ML) phylogenetic trees were constructed using PhyML (Guindon et al., 2010) with the JTT+g4 substitution model with chi-squared approximate likelihood ratio test used to determine degrees of branch support (Anisimova and Gascuel, 2006; Fig. 2). The Rep ML phylogenetic tree revealed that that the CasCV replicon is likely the genome of a virus within the same family as SsHADV-1 and a previously described Mosquito VEM virus SDBVL. Interestingly, based on degrees of sequence similarity the CPs of these viruses are possibly more closely related to circo/cycloviruses, whereas the Reps are most closely related to probable Rep integrons within the genomes of various fungi (including those of Laccaria bicolor, Serpula lacrymans var lacrymans and Aspergillus fumigates), geminiviruses and various possible ssDNA viruses detected during metagenomic surveys of soil, marine and freshwater environments. It must be stressed however that the extremely low degrees of sequence similarity between the CP sequences of the major ssDNA virus groups and the lack of a suitable outgroup makes it difficult to come to a firm conclusion as to whether the fungus associated ssDNA virus CP sequences share a more recent common ancestor with the circoviruses and nanoviruses than they do with the geminiviruses. Located between the Rep and the CP ORFs is a 136-nt intergenic region which we call here the long intergenic region (LIR). The LIR has similar characteristics to analogous intergenic regions found in geminiviruses, nanoviruses and circoviruses, with a geminivirus-like nonanucleotide (TAATATTAT in the new sequence and SsHADV-1 as opposed to a TAATATTAC sequence in geminiviruses) at the presumed (by analogy with the geminiviruses) origin of virion-strand replication (ori; Fig. 1). We were, however, unable to find a similarly conserved ori nonanucleotide in the previously described Mosquito VEM virus SDBVL sequence that closely resembles the novel CasCV sequence described here. As in SsHADV1 and the geminiviruses, the ori nonanucleotide resides within the loop of a probable hairpin structure. We additionally identified a probable TATA box between this hairpin structure and the CP start codon that is in a similar position to an analogous sequence found in the geminiviruses. Also in the LIR downstream of the hairpin is a GC-rich region in a similar position to a G-Box element of geminiviruses that is known to be involved in their transcriptional regulation (Eagle and Hanley-Bowdoin, 1997; Fenoll et al., 1988, 1990). The Rep protein is highly conserved amongst ssDNA viruses and plasmids that replicate by a rolling circle mechanism (Ilyina and Koonin, 1992; Koonin and Ilyina, 1992). In geminiviruses and probably other ssDNA replicons it recognises, binds and cleaves the origin via its N-terminal ∼100 amino acids (Desbiez et al., 1995; Nash et al., 2011). A search of the NCBI’s Conserved Domain Database (MarchlerBauer et al., 2009) with the probable Rep sequence of CasCV revealed that it contains both a N-terminal geminivirus-like Rep catalytic domain and a geminivirus-like Rep protein central domain. The NMR structure of the Rep catalytic domain from the
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Fig. 1. Predicted stem-loop structure in the origin of virion-strand replication of cassava associated circular DNA virus (CasCV) (A), two-dimensional graphical representation of pairwise amino acid sequence identities (calculated with pairwise deletion of gaps; scale represents percentage identity) of the predicted Rep and CP of CasCV with those of SsHADV-1, Mosquito VEM virus SDBVL and MmFV (B). Genome organisation of CasCV (C), Mosquito VEM Gemini Fungi virus-SDBVL G (D), MmFV (E), SsHADV-1 (F) and the predicted introns (G).
begomovirus Tomato yellow leaf curl virus has indicated that this domain is related to a large group of proteins that bind nucleic acid sequences. In CasCV this is therefore also likely a crucial component of the replisome (Campos-Olivas et al., 2002; Desbiez et al., 1995). Within the two geminivirus-like domains of the CasCV Rep are five conserved rolling circle replication (RCR) related motifs that have been identified in geminivirus, circovirus and nanovirus Reps (Ilyina and Koonin, 1992; Koonin and Ilyina, 1992, 1993), (Fig. 3, Supplementary Fig. 1): from the N- to C-terminus these are RCR motif I, RCR motif II, the Geminivirus Rep sequence motif (GRS), RCR motif III, and RCR motif IV. While highly conserved amongst all RCR replicons, the specific function of RCR motif I is unknown. It is, however, reasonably likely that it may be involved in the sequence specific recognition of tandemly repeated nucleotide sequences (called iterons) near the ori (Arguello-Astorga and Ruiz-Medrano, 2001). During origin cleavage RCR motif II (HLHxxxQ) is believed to coordinate the binding of metal ions via its two histidine residues (Ilyina and Koonin, 1992). The GRS motif has been implicated in the ssDNA cleavage reaction that occurs during the initiation of rolling circle replication (Nash et al., 2011) and while less conserved that the other four motifs, has been found in the Rep proteins of geminiviruses and some phytoplasmal and algal plasmids. The actual catalytic site for DNA cleavage is RCR motif III (YxxKD/E)
within which the conserved tyrosine residue is believed to covalently bind to DNA after cleavage (Heyraud-Nitschke et al., 1995; Laufs et al., 1995). RCR motif IV (EGX4 GKTX32 DD) is believed to encode a NTP-binding domain (Gorbalenya et al., 1989), such as that found in proteins with helicase, protease and kinase activity, and exhibits ATPase activity that is required for replication (Desbiez et al., 1995). It has also been suggested that RCR motif IV might confer Rep with a helicase-like activity that is crucial during RCR (Bisaro, 1996; Gutierrez, 1999). Given the high degree of sequence conservation between the Reps of CasCV and geminiviruses we used the computer program MODELLER (Eswar et al., 2006) and the 3D nuclear magnetic resonance (NMR) determined structure of the geminivirus tomato yellow leaf curl virus (TYLCV) Rep N-terminal region (PDB ID 1L2M; Campos-Olivas et al., 2002) to infer the likely structure of the homologous Rep region of CasCV. This structure was visualised with PyMOL Ver 0.99 (www.pymol.org). In support of the hypothesis that the CaSCV Rep homologue is a functional Rep protein, the structural arrangements of the four RCR related motifs within the examined region (RCR motifs I, II and III and the GRS motif) were also highly conserved (Fig. 3c) and are therefore likely to be functionally very similar to their geminivirus homologues.
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Fig. 2. Maximum likelihood phylogenetic tree of the predicted amino acid alignments of the Rep of CasCV with those of representative ssDNA viruses, phytoplasma plasmids and fungal integrons. Closed circles indicate branches supported of greater than 90% whereas open circles indicate branch support of 50–89%.
Noting that the CasCV Rep and CP respectively share 62.7% and 39.8% similarity to those of SsHADV-1, we assumed that CasCV could potentially be a mycovirus-infecting a fungus associated with the cassava leaf samples that we examined. We therefore screened total DNA extracts of the cassava leaf samples from which CasCV was derived, using the fungal 18S rRNA specific PCR primers – 18S forward primer 5 -CAA GGT CTC TGT TGG TGA ACC AGC GG-3 , 18S reverse primer 5 -TCC TCC GCT TAT TGA TAT GCT TAA GTT-3 (White et al., 1990. We identified two fungal pathogens, one Collectotrichum-like and the other
Plectosphaerella cucumerina. Whereas Collectotrichum gloeosporioides is a fungus known to cause damaging serious anthracnose disease in cassava and yams (Amusa, 2001), we were unable to find any documented association of P. cucumerina with cassava. We were also unable to culture fungal material from the cassava leaf samples. Therefore, although it is quite probable that CasCV is a mycovirus that should be classified within the same taxonomic family as SsHADV-1, it remains for proper host range studies using infectious clones to demonstrate its likely origin.
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Fig. 3. Sequence motifs found near the N-terminus of predicted CasCV and other representative ssDNA virus Rep proteins (A). Alignment of selected Rep sequences with identified rolling circle replication motifs I, II, III and IV, and the GRS highlighted (B). Homology modelling of the N terminal portion of CasCV Rep, with the models drawn using PyMOL (C).
In summary, we describe a novel ssDNA virus, at a molecular level, which we name cassava associated circular DNA virus (CasCV), which has a genome architecture similar to that of circoviruses, but which likely expresses a geminivirus-like Rep that, like some geminiviruses, is translated from a spliced mRNA. However, amongst known virus species the nearest relative of CasCV is clearly the mycovirus SsHADV-1 and it is likely that these two species as well as Mosquito VEM virus SDBVL and MmFV will ultimately be classified within a new, fungus infecting ssDNA virus family. GenBank Accession #: JQ412056 and JQ412057. Acknowledgements This work was supported by a Biolmolecular Interaction Centre (University of Canterbury) seed grant awarded to Arvind Varsani and Renwick C.J. Dobson. We also thank the Alliance for Green Revolution in Africa (AGRA) through the West Africa Centre for Crop Improvement (WACCI), University of Ghana for their support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2012.03.009. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology 215 (3), 403–410. Amusa, N., 2001. Screening of cassava and yam cultivars for resistance to anthracnose using toxic metabolites of Colletotrichum species. Mycopathologia 150 (3), 137–142. Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Systematic Biology 55 (4), 539–552.
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