Pandoraviruses are highly derived phycodnaviruses
© Yutin and Koonin; licensee BioMed Central Ltd. 2013
Received: 16 September 2013
Accepted: 16 October 2013
Published: 23 October 2013
The recently discovered Pandoraviruses are by far the largest viruses known, with their 2 megabase genomes exceeding in size the genomes of numerous bacteria and archaea. Pandoraviruses show a distant relationship with other nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes, lack some of the NCLDV core genes and in particular do not appear to be specifically related to the other, better characterized family of giant viruses, the Mimiviridae. Here we report phylogenetic analysis of 6 core NCLDV genes that confidently places Pandoraviruses within the family Phycodnaviridae, with an apparent specific affinity with Coccolithoviruses. We conclude that, despite their many unusual characteristics, Pandoraviruses are highly derived phycodnaviruses. These findings imply that giant viruses have independently evolved from smaller NCLDV on at least two occasions.
This article was reviewed by Patrick Forterre and Lakshminarayan Iyer. For the full reviews, see the Reviewers’ reports section.
The discovery of giant viruses infecting unicellular eukaryotes, in particular amoeba, eliminated the distinction between viruses and cellular life forms in terms of size and genomic complexity . Until very recently, all the discovered true giants of the virus world, with genomes exceeding 1 megabase (Mb) and encompassing more than 1,000 genes, were closely related members of the family Mimiviridae [2–4]. The gap between the members of the Mimiviridae and viruses outside this family was dramatic: apart from the mimiviruses, the largest viral genome, that of Emiliana huxleyi virus 86, was approximately 0.41 Mb in size . The unexpected recent discovery of two strains of Pandoraviruses, Pandoravirus salinus and Pandoravirus dulcis, with genomes of at least 2.5 and 1.9 Mb, respectively, dramatically expanded the range of viral giantry . In addition to being enormous, Pandoravirus genomes turned out to be highly unusual in that they showed little similarity to other viruses, lacked some of the core genes of the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV, or the proposed order Megavirales) of eukaryotes [7–10] and failed to show clear-cut affinities in phylogenetic analysis . We set out to investigate the repertoire of core NCLDV genes in pandoraviruses and their phylogenies in greater detail.
Ancestral NCLDV genes in Pandoraviruses
The ancestral NCLDV genes represented in Pandoraviruses
Presence in the 7 NCLDV familiesa
Best hit for Pandoravirusesb/ % identity/alignment length
YqaJ viral recombinase family (pfam09588)/NCVOG1192
Guillardia theta CCMP2712 (Cryptophyta) /31/212
Bathycoccus sp. RCC1105 virus BpV2 (Phycodnaviridae) /33/579
ribonucleoside diphosphate reductase, alpha subunit/NCVOG1353
Thiothrix flexilis (Proteobacteria) /36/1011
Ribonucleotide reductase small subunit; apparent eukaryotic origin/NCVOG0276
Thalassiosira pseudonana CCMP1335 (Stramenopiles) /75/216
Hordeum vulgare subsp. vulgare (Viridiplantae) /58/153
DNA or RNA helicases of superfamily II (COG1061) (A18hel)/NCVOG0076
Ectocarpus siliculosus virus 1 (Phycodnaviridae) /35/238
pfam04947, Poxvirus Late Transcription Factor VLTF3 like (A2L)/NCVOG0262
Emiliania huxleyi virus 202 (Phycodnaviridae) /34/264
Transcription factor S-II (TFIIS)-domain-containing protein/NCVOG0272
Mus musculus (Opisthokonta) /27/278
Nudix hydrolase (D10 ortholog)/NCVOG0236
Vitis vinifera (Viridiplantae) /28/226
A32-like packaging ATPase/NCVOG0249
516306303, 516303793, 516305958, 516303807, 516305953
Ostreococcus tauri virus 2 (Phycodnaviridae) /45/247
Congregibacter litoralis (Proteobacteria) /50/242
cd00127, DSPc, Dual specificity phosphatases (DSP); Ser/Thr and Tyr protein phosphatases/NCVOG0040
Lausannevirus (Marseillevirus family) /41/149
RING-finger-containing E3 ubiquitin ligase (COG5432: RAD18)/NCVOG0330
Vitis vinifera (Viridiplantae) /43/76
DNA-directed RNA polymerase subunit alpha/NCVOG0274
Cyanidioschyzon merolae strain 10D (Rhodophyta) /35/877
DNA-directed RNA polymerase subunit beta /NCVOG0271
Capsella rubella (Viridiplantae) /38/1283
disulfide (thiol) oxidoreductase; Erv1/Alr family (pfam04777)/NCVOG0052
Setosphaeria turcica Et28A (Opisthokonta) /37/123
Phylogenetic analysis of conserved genes places Pandoraviruses within Phycodnaviridae
The pattern of best database hits in the BLASTP searches for the ancestral gene products of Pandoraviruses yielded a hint of a possible evolutionary relationship between Pandoraviruses and Phycodnaviridae, an expansive family of NCLDV that infect algae and other unicellular eukaryotes . Indeed, among the best hits to homologous proteins from other NCLDV all but one were to homologs from the Phycodnaviridae family (Table 1).
Within the Phycodnaviridae, the preferred grouping of Pandoraviruses was with Emiliana huxlei virus (the type member of the genus Coccolithovirus ) as exemplified by the phylogenetic tree of the DNA polymerase, one of the most highly conserved genes of the NCLDV for which a reliable phylogeny can be obtained (Figure 2A). The highly conservative Approximately Unbiased (AU) test rejected all tested tree topologies with Pandoraviruses placed outside the Phycodnaviridae branch for the D5-like helicase-primase; for the other genes, some of the alternative topologies were not rejected by the AU test but all were assigned lower likelihood values (Additional file 2). Perhaps the strongest evidence of an evolutionary link between Pandoraviruses and Coccolithoviruses comes from the phylogenetic trees of two RNA polymerase (RNAP) subunits in which the two confidently grouped together as indicated by the bootstrap support value of 0.99 (Figure 3). Coccolithoviruses are the only genus of phycodnaviruses that encode the RNAP subunits; the rest of the phycodnaviruses have lost the ancestral RNAP genes, presumably because these viruses employ the host RNAP during a nuclear phase of their reproduction cycle [11, 12]. Thus, the shared presence of the two monophyletic RNAP subunit genes in Pandoraviruses and Coccolithoviruses is a shared derived character that supports the common origin of these viruses.
Taken together, the phylogenetic analysis results indicate that the ancestral NCLDV genes in Pandoraviruses largely share the evolutionary history with the homologous genes of Phycodnaviruses, and more specifically, appear to have evolved from a common ancestor with Coccolithoviruses.
Implications for the evolution of giant viruses
Despite their enormous size, Pandoraviruses show no evolutionary connection with the other family of giant viruses, the Mimiviridae. Instead, phylogenetic analysis of the ancestral NCLDV genes points to an affinity between Pandoraviruses and Phycodnaviruses. Moreover, Pandoraviruses appear to belong within the Phycodnavirus branch, being a sister group of Coccolithoviruses. Certainly, the phylogenomic analysis that leads to this conclusion involves a proverbial “tree of 1%” . Indeed, the entire evidence hinges on the topologies of 6 phylogenetic trees, albeit those for key NLCDV genes, and on the finding that two RNAP subunits genes are shared between Pandoraviruses and Coccolithoviruses, to the exclusion of other Phycodnaviruses. However, given that altogether Pandoraviruses retain only 17 of the 49 inferred ancestral NCLDV genes, there is not much potential for obtaining additional evidence on the relationship between these viruses and the other NCLDV although, as noticed above, some interviral gene exchanges within amoeba might have occurred.
Thus, it appears that, despite their extremely unusual gene repertoires, Pandoraviruses are highly derived Phycodnaviruses. This conclusion implies that giant viruses have evolved independently from less complex NCLDV on at least two independent occasions, within the families Mimiviridae and Phycodnaviridae (Figure 2A). Given the much smaller genomes of the other NCLDV and the lack of substantial similarity between the gene repertoires of Pandoraviruses and Mimiviruses, the scenario of independent gain of numerous genes in two lineages of NCLDV appears much more plausible than the alternative that would involve extensive degradation of extremely complex ancestors in multiple lineages. The discovery of additional, perhaps independently evolving giant viruses appears likely, and identification of the aspects of virus biology that favor such dramatic genome expansions is of major interest.
Phylogenomic analysis indicates that the giant Pandoraviruses, by far the largest viruses discovered to date, are highly derived Phycodnaviruses, most likely, the sister group of Coccolithoviruses. The more general implication of these findings is that giant viruses independently evolved in at least two lineages of the NCLDV.
P. dulcis and P. salinus protein sequences were retrieved from the non-redundant database at the National Center for Biotechnology Information (NIH, Bethesda). The non-redundant protein sequence database was searched using the PSI-BLAST program , with default parameters and the predicted Pandoravirus protein sequences used as queries. The reported results reflect searchers performed in August, 2013. The sequences for phylogenetic analysis were collected using (i) BLAST searches against nr and environmental (env_nr) databases initiated by Pandoravirus protein sequences; (ii) the corresponding NCVOG sequences ; and (iii) the corresponding mimiCOG sequences . Nearly identical sequences were eliminated using BLASTCLUST (http://www.ncbi.nlm.nih.gov/Web/Newsltr/Spring04/blastlab.html). Protein sequences were aligned using the MUSCLE program with default parameters ; columns containing a large fraction of gaps (greater than 30%) and non-homogenous columns defined as described previously  were removed from the alignment prior to phylogenetic analysis. A preliminary maximum-likelihood tree was constructed using the FastTree program with default parameters (JTT evolutionary model, discrete gamma model with 20 rate categories ) . The preliminary tree and the alignment were then used to determine the best substitution matrix using Prottest . Best matrices found by Prottest were as follows: LG+G (NCVOG0052, NCVOG1068, NCVOG0236, NCVOG0276, NCVOG0330, NCVOG1115), LG+G+F (NCVOG0249, NCVOG0040, NCVOG0023, NCVOG0076, NCVOG0038, NCVOG0274, NCVOG0271, NCVOG0262, NCVOG1353, NCVOG0272), and WAG+G+F (NCVOG1192). The final maximum-likelihood trees were constructed using TreeFinder (1,000 replicates, Search Depth 2), with the substitution matrix that was found to be the best for a given alignment . The Expected-Likelihood Weights (ELW) of 1,000 local rearrangements were used as confidence values of TreeFinder tree branches . For tree topology testing, whenever applicable, alternative (constrained) topologies were constructed and compared to the initial trees using TreeFinder . Approximately unbiased (AU) test P value cutoff 0.05 was used for rejecting tree topologies .
Reviewer 1: Patrick Forterre (Institut Pasteur)
Pandoraviruses are fascinating new organisms, which illustrates the capacity of viruses to produce drastically different types of virions, with strikingly different structures and genomes encoding from 2 genes up to 2500 genes . In this paper, Yutin and Koonin have revisited the genomes of the two isolated Pandoraviruses and identified 6 of the 17 core NCLDV genes which consistently group within Phycodnaviridae (one of the NCLDV – or Megavirales – families) in phylogenetic analyses. They concluded that Pandoraviruses evolved from smaller Phycodnaviridae. The implication is that giant viruses (Mimiviridae and Pandoviruses) evolved twice independently from smaller viruses and not from cellular organisms.
The authors did not discuss the possibility that some Pandoravirus ancestor captured these 6 genes as an operon from a Phycodnavirus. We know that LGT can indeed occur between viruses co-infecting the same hosts. The authors state that: “in none of the trees pandoraviruses would cluster with any viruses outside the family Phycodnaviridae”. However, it seems that the dual specificity phosphatase NCVOG0040 branch with Mimiviridae (Lausannevirus and Marseillevirus) suggesting that LGT have indeed occurred between Pandoraviruses and Mimiviridae. In their paper, Philippe and co-workers mentioned the existence of 17 genes of P. salinus that have their closest homolog (34% identical residues in average) within the Megaviridae . This seems in contradiction with the results reported here.
Authors’ response: The exceptional case of the dual specificity phosphatase was overlooked in the original submission (although the tree was included in Additional file 2 ), and we appreciate the reviewer pointing out this omission. Indeed, this case of apparent phylogenetic affinity between ancestral genes of Pandoraviruses and Marseilleviruses (sic! not Mimiviridae) is likely to originate from intervirus gene exchange within amoeba, and so do the non-ancestral genes apparently shared between Pandoraviruses and Mimiviruses. This aspect of the evolution of the giant viruses is briefly discussed in the revised manuscript. The full characterization of such gene exchanges requires a comprehensive phylogenomic analysis of giant viruses that is currently underway in our group. It should be noted, however, that ancestral genes of the NCLDV do not form operons or clusters, so the scenario under which pandoraviruses acquired the ancestral genes from Phycodnaviruses “as an operon” is hardly justified. More importantly, there is no contradiction between the conclusions of this work and the possibility of horizontal gene transfer between Pandoraviruses and Mimiviruses (and/or other viruses of amoeba) as the latter involved non-ancestral genes.
Pandoraviruses do encode an MCP that share ancestry with that of Phycodnaviruses, but is highly divergent and cannot be detected by sequence similarity.
The structural proteins of Pandoraviruses are unrelated to those of NCLDV, but the detected ATPase is involved in packaging.
The structural proteins of Pandoraviruses involved in formation of the virion are unrelated to those of megavirales and the detected ATPase is not involved in packaging.
Could the authors discuss these different possibilities? Did they use sensitive methods to specifically search for MCP? Philippe et al. identified two abundant proteins that could be involved in formation of the virion. Did the authors analyse these proteins?
Authors’ response: indeed, the absence of detectable capsid proteins in Pandoraviruses is most intriguing and is emphasized in the revised manuscript. Of the three hypotheses brought up in this comment, (1) and (2) appear to be most plausible. We did employ a sensitive search strategy to detect possible diverged capsid proteins homologous to those of other NCLDV as pointed out in the revised manuscript. With regard to the abundant virion proteins of pandoraviruses, we prefer to cite the original publication. An exhaustive analysis of the sequences and predicted structures of these and other proteins of Pandoraviruses is a separate undertaking that will be published in due course.
Viral lineages are better defined by their capsid proteins, because these proteins are hallmarks of viruses (I use here capsid in a broad definition, including all type of structural assemblage involved in the formation of a virion) . It has been shown that viruses producing homologous capsids can use different types of replicons, and that exchanges of replicons cassette genes have rather frequently occurred between viruses . At the moment, it is therefore a bit premature to definitely classify Pandoraviruses as an NCLDV, because we know nothing about their virion structural proteins. One could thus imagine that Pandoraviruses belong to a novel major viral lineage and recruited in the past a cassette of replication/transcription genes from a Phycodnavirus. However, this scenario, gene cassette shuffling. is especially prevalent in viruses with small DNA genomes and has never or rarely been observed in large DNA viruses. Could the authors comment on this last point?
Authors’ response: The nature of viral “lineages” and the comparative utility of structural and replicative proteins for reconstructions of virus evolution are matters of a long, storied debate[23–27]. Probably, the key message is that viral evolution is a complex network of relationship that involves both numerous gene exchanges and intervals of vertical evolution of gene modules[28, 29]. Accordingly, both structural proteins and replicative proteins are important for evolutionary reconstructions. As repeatedly argued, replicative proteins are more informative because they retain more sequence conservation, show a strong tendency to come in coevolving modules, and most crucially, provide the potential for reconstructing evolutionary relationships between viruses and capsid-less selfish elements. As demonstrated in detail elsewhere, such relationships are pervasive in the evolution of different classes of selfish agents and essential for understanding the routes of their evolution. Under the weight of all these considerations, we stick to our classification of Pandoraviruses as bona fide members of the NCLDV (Megavirales). As for the transfer of cassettes of replicative genes, we are indeed unaware of such events in the evolution of NCLDV.
My feeling is that the authors’s interpretation (independent evolution of “giant” viruses from “big” viruses) is the correct one, in agreement with previous suggestion that NCLDV originated from smaller viruses predating LUCA  and the recent accordion model for genome evolution of Megavirales proposed by Jonathan Filée . However, it will be important to obtain more insights into the origin and history of other genes of Pandoraviruses, especially those involved in the formation of the virion.
Authors’ response: we could not agree more.
Anticipating criticisms, Yutin and Koonin remark that their analysis is a case of “tree of 1%” or less, since it is based on 7 genes only, out of 2500. However, one should not forget that the rRNA tree (0.1%) was sufficient to identify the three domains structure of the universal tree of life.
Philippe N, Legendre M, Doutre G, Couté Y, Poirot O, Lescot M, Arslan D, Seltzer V, Bertaux L, Bruley C, Garin J, Claverie JM, Abergel C. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science. 2013, 341:281-286.
Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nat Rev Microbiol. 2008, 6:315-319
Krupovic M, Bamford DH. Does the evolution of viral polymerases reflect the origin and evolution of viruses? Nat Rev Microbiol. 2009, 7:250;
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Filée J. Route of NCLDV evolution: the genomic accordion. Curr Opin Virol. 2013 Jul 26. doi:pii: S1879-6257(13)00115-6. 10.1016/j.coviro.2013.07.00
Reviewer 2: Lakshminarayan Iyer (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health).
The giant Pandoraviruses are the largest dsDNA viruses sequenced to date with over 2000 genes. Although the initial sequencing effort recognized the relationship of the Pandoraviruses to the NCLDV, it did not clarify their precise affinities to other viruses within this group. Yutin and Koonin convincingly demonstrate that the Pandoraviruses are divergent Phycodnaviruses, and with the existing data posit a special relationship to Coccolithoviruses. The observations are independently reproducible and the conclusions justified given the data.
NY collected the data; NY and EVK analyzed the data; EVK wrote the manuscript which was read and approved by both authors.
The authors thank members of the Koonin group for useful discussions. The authors’ research is supported by the US Department of Health and Human Services intramural funds (to National Library of Medicine).
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