# Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes

Sebastien Santini, Sandra Jeudy, Julia Bartoli, Olivier Poirot, Magali Lescot, Chantal Abergel, Valérie Barbe, K. Eric Wommack, Anna a M Noordeloos, Corina P D Brussaard, et al.

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## To cite this version:

Sebastien Santini, Sandra Jeudy, Julia Bartoli, Olivier Poirot, Magali Lescot, et al. Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110 (26), pp.10800-10805. 10.1073/pnas.1303251110. hal-05370967

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# Genome of *Phaeocystis globosa* virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes

Sebastien Santina<sup>a</sup>, Sandra Jeudy<sup>a</sup>, Julia Bartoli<sup>a</sup>, Olivier Poirot<sup>a</sup>, Magali Lescot<sup>a</sup>, Chantal Abergel<sup>a</sup>, Valérie Barbe<sup>b</sup>, K. Eric Wommack<sup>c</sup>, Anna A. M. Noordeloos<sup>d</sup>, Corina P. D. Brussaard<sup>d,e,f</sup>, and Jean-Michel Claverie<sup>a,f,g</sup>

Edited by James L. Van Etten, University of Nebraska, Lincoln, NE, and approved May 1, 2013 (received for review February 22, 2013)

Large dsDNA viruses are involved in the population control of many globally distributed species of eukaryotic phytoplankton and have a prominent role in bloom termination. The genus *Phaeocystis* (*Haphophyta*, *Prymnesiophyceae*) includes several high-biomass-forming phytoplankton species, such as *Phaeocystis globosa*, the blooms of which occur mostly in the coastal zone of the North Atlantic and the North Sea. Here, we report the 459,984-bp-long genome sequence of *P. globosa* virus strain PgV-16T, encoding 434 proteins and eight tRNAs and, thus, the largest fully sequenced genome to date among viruses infecting algae. Surprisingly, PgV-16T exhibits no phylogenetic affinity with other viruses infecting microalgae (e.g., phycodnaviruses), including those infecting *Emiliania huxleyi*, another ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to an emerging clade (the Megaviridae) clustering the viruses endowed with the largest known genomes, including Megavirus, Mimivirus (both infecting acanthamoeba), and a virus infecting the marine microflagellate grazer *Cafeteria roenbergensis*. Seventy-five percent of the best matches of PgV-16T-predicted proteins correspond to two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from a hypersaline lake in Antarctica (Organic Lake), the hosts of which are unknown. As for OLPVs and other Megaviridae, the PgV-16T sequence data revealed the presence of a virophage-like genome. However, no virophage particle was detected in infected *P. globosa* cultures. The presence of many genes found only in Megaviridae in its genome and the presence of an associated virophage strongly suggest that PgV-16T shares a common ancestry with the largest known dsDNA viruses, the host range of which already encompasses the earliest diverging branches of domain Eukarya.

giant virus | core gene | gene duplication | mobile element | horizontal gene transfer

The discovery of the giant *Acanthamoeba polyphaga* Mimivirus in freshwater amoeba and the deciphering of its 1.2-Mb genome sequence initiated a new independent phylogenetic lineage among eukaryotic dsDNA viruses (1, 2), as well as a new chapter in virology. Soon after, it was recognized (3) that the closest homologous sequences to many Mimivirus proteins occurred within metagenomic sequence data from the Global Ocean Survey expedition (4). However, the identity of these—presumably abundant (5)—Mimivirus relatives and the hosts they infect remained elusive. As of today, the only Mimivirus relatives of marine origin for which a complete genome sequence is available are a virus infecting *Cafeteria roenbergensis* (CroV) (6), a major microflagellate grazer, and *Megavirus chilensis*, isolated from near-shore sediments off the coast of central Chile (7). This latter virus is propagated on various species of *Acanthamoeba* in the laboratory, but its environmental host remains unknown. Remarkably, these viruses exhibit the two largest known genomes among marine

viruses: 730 kb and 1.28 Mb for CroV and *Megavirus chilensis*, respectively. Other studies, targeting virus-specific genes [e.g., DNA polymerase B (8) or capsid proteins (9)] have suggested a close phylogenetic relationship between Mimivirus and several giant dsDNA viruses infecting various unicellular algae such as *Pyramimonas orientalis* (*Chlorophyta*, *Prasinophyceae*), *Phaeocystis pouchetii* (*Haptophyta*, *Prymnesiophyceae*), and *Chrysochromulina ericina* (*Haptophyta*, *Prymnesiophyceae*).

*Phaeocystis*, the host for *Phaeocystis globosa* virus (PgV)-16T, has a complex lifecycle that includes a unicellular, motile stage and gelatinous stage that can produce multicellular colonies of several centimeters in diameter. *P. globosa* regularly dominates the phytoplankton community in the coastal zone of the North Atlantic and the North Sea, where its blooms may result in “stinking water” and the production of foam of mucilaginous material that deposits on beaches. The decline of natural blooms was shown to be accompanied by a considerable increase of viruses infecting *P. globosa* (10), and a mesocosm study showed that the abundance of *P. globosa* populations can indeed be controlled by viral lysis (11). The PgV-16T strain reported here belongs to the group I of clonal virus isolates (together with PgV-12T and PgV-14T) obtained from Dutch coastal waters (southern North Sea) and partially characterized previously (12). It is a lytic virus that only infects *Phaeocystis globosa* with a latent period of 8–12 h and a burst size around 300–400 virions (12). Its icosahedral particle is about 150 nm in diameter, and its genome size was estimated at 470 kb (12). Group I viruses are the largest known viruses infecting *P. globosa*, but many others (classified in group II, such as PgV-03T, PgV-04T, PgV-10T) have been isolated that exhibit smaller virions (about 100 nm in diameter) and genome sizes (about 177 ± 3 kb), as well as a different host range (10, 12). Their genomes have not yet been sequenced.

In this study, we used in excess of 20,390,000 Illumina 101-bp reads (paired) for the de novo assembly the PgV-16T genome at very high sequence coverage (average: 4,477 ± 1,624). Unexpectedly, the corresponding predicted proteome was found to be, by far, most similar to the one of two partially sequenced viruses

<footnote><sup>a</sup>Structural and Genomic Information Laboratory, Unité Mixte de Recherche 7256, Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France; <sup>b</sup>Commissariat à l'Energie Atomique-Institut de Génomique, 91057 Evry Cedex, France; <sup>c</sup>Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711; <sup>d</sup>Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, NL-1790 AB Den Burg (Texel), The Netherlands; <sup>e</sup>Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; and <sup>f</sup>Service de Santé Publique et d'Information Médicale, Hôpital de la Timone, Assistance Publique-Hôpitaux de Marseille, FR-13385 Marseille, France</footnote>

<footnote>Author contributions: K.E.W., C.P.D.B., and J.-M.C. designed research; S.S., S.J., J.B., V.B., A.A.M.N., and C.P.D.B. performed research; S.S., S.J., O.P., M.L., C.A., and J.-M.C. analyzed data; and S.S., C.P.D.B., and J.-M.C. wrote the paper.</footnote>

<footnote>The authors declare no conflict of interest.</footnote>

<footnote>This article is a PNAS Direct Submission.</footnote>

<footnote>Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KC662249-KC662250).</footnote>

<footnote n="1">¹To whom correspondence may be addressed. E-mail: corina.brussaard@nioz.nl or Jean-Michel.Claverie@univ-amu.fr.</footnote>

<footnote>This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303251110/-/DCSupplemental.</footnote>
[Organic Lake phycodnavirus (OLPV)1 and OLPV2] infecting an unknown host from a hypersaline lake in Antarctica (Organic Lake) (13). In addition, a subset of data (6,310,000 reads) enabled the assembly of a virophage-like genome, reinforcing the similarity with OLPVs, which also appear to have associated virophages. The PgV-16T genome, furthermore, exhibits the duplication of two types of viral core genes, the packaging ATPase and the second-largest RNA polymerase subunit, genes that have not been reported to occur at more than a single copy in any dsDNA virus.

## Results and Discussion

**General Genome Features.** The genome of PgV-16T was fully assembled as a linear DNA sequence of 459,984 bp, making it the third-largest described genome among marine viruses behind CroV and Megavirus. Its overall nucleotide composition (A+T content: 68%) is less A+T-biased than that of CroV and Megavirus (A+T: 77% and 75%, respectively), although it reaches 87 ± 8% A+T between genes. Using a conservative annotation protocol (SI Materials and Methods), we identified 434 putative protein-coding sequences with an average length of 320 aa (ranging from 45 to 3,739 aa) and eight tRNAs (three tRNA<sub>Leu</sub>, two tRNA<sub>Asn</sub>, one tRNA<sub>Arg</sub>, one tRNA<sub>Gln</sub>, one tRNA<sub>Ile</sub>). With an average intergenic distance of 104 nt (summing to 42,717 noncoding base pairs) the PgV-16T genome reaches a coding density of 90.7%. Unexpectedly, the partially assembled genome sequences of two other PgV strains deposited directly in the GenBank database (PgV 12-T, accession no. HQ634147.1; PgV 14-T, accession no. HQ634144.1) were found to be nearly identical to PgV-16T in terms of gene content and percentage of identity (98.1% of identity; 449 and 444 genes). We searched the final genome sequence assembly for the presence of large (direct or inverted) repeated regions of >5 kb. No evidence of block duplication involving a string of several genes was found (Fig. S1), indicating that the large genome size of PgV is not the result of segmental duplications. A finer look at these data also showed that unchecked short-repeat expansions do not contribute to the large size of the PgV genome. As with previously characterized dsDNA viruses infecting unicellular eukaryotes, most of the genes within the PgV-16T genome were vertically inherited from their common ancestor, and recent horizontal gene transfers do not account for a significant fraction of the gene content. Finally, unlike the Poxviruses, Asfarviruses, or Chlorovirus, there is no sequence similarity (direct or inverted) between the two extremities (telomeres) of the PgV genome.

Applying a BlastP E-value conservative cutoff of 10⁻⁵ (and disregarding the matches with the nearly identical partial PgV-12T and PgV-14T genome sequences), 245 coding DNA sequences (CDSs) (56.5%) exhibited a similarity to protein sequences in the National Center for Biotechnology Information (NCBI) nonredundant (NR) databank (14). Among those, 178 (73%) had their closest homolog in a large dsDNA virus, 35 in bacteria, 25 in eukaryotes, and 2 in archaebacteria. Unexpectedly, among the viral best matches, 138 (77.5%) were found in OLPV1 and/or OLPV2 (the DNA polymerase sequences of PgV-16T and the OLPVs share about 48% identical residues) and the only four described genes of *P. pouchetii* virus (sharing 85% identical residues in average) (Fig. 1). Only four PgV-predicted proteins (PgV140, PgV143, PgV411, PgV423) were found to be most similar to their homolog in *Emiliania huxleyi* virus (EhV)86 (15) (or related strains), the only other previously sequenced giant viruses known to infect a haptophyte.

**Identification of a Virophage-Like Sequence.** Virophages are a class of satellite virus that have now been found in association with giant viruses such Mimivirus/Megavirus (16, 17), as well as with CroV (18) and OLPV (13). Virophages presumably use the characteristic replicative cytoplasmic virion factories of these viruses for their own replication (19). In addition to the single-scaffold PgV-16T genome sequence, the assembly process generated a 19,527-bp-long sequence scaffold, indicating the presence of an unsuspected

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Global database similarity of the PgV-16T predicted proteome. (A) Close to 50% of PgV predicted proteins do not exhibit a significant match (E < 10⁻⁵) in the NCBI NR protein database, as is typical for viruses belonging to a newly explored lineage with no other sequenced representatives (2, 6). (B) Surprisingly, for 77% of the PgV predicted proteins exhibiting a viral protein as their best match, this homolog is found in the partially sequenced OLPV1 or -2. This finding indicates strong global phylogenetic affinities between PgV (infecting a haptophyte) and the OLPVs infecting an unknown host.</figcaption></figure>

virophage (denoted PgVV) associated with the PgV-16T particles of *Phaeocystis globosa*. However, no small viral particle (typically less than 75 nm in diameter) characteristic of previously described virophages were observed in infected *P. globosa* cells. Upon assembly, the PgVV genome sequence exhibited a much higher coverage (e.g., density of reads mapped to the sequence) than the PgV-16T genome (33,151 ± 14,153 vs. 4,477 ± 1,624), despite a similar G+C content (36% vs. 32%). This suggests that the PgVV genome was in a higher copy number (by a factor of ~7.5) than the PgV-16T virus in the initial DNA sample. The central PgVV genome sequence is flanked by two 1-kb-long, nearly identical telomeric sequences complementary to each other (Fig. 2). Such a feature, not previously described for virophages, is common among poxviruses, chloroviruses, and asfarviruses and was also encountered in the plasmid prophage N15 (20). In all these cases, this genome feature was associated with covalently closed hairpin ends (telomeres). The PgVV genome was predicted to encode 16 proteins (numbered pgw01 to pgw16), all of them transcribed from the same DNA strand (Fig. 2), another unique characteristic. However, as for the previously described virophages, most of these protein sequences (10/16; 62.5%) had no significant matches or were remotely similar to hypothetical proteins of unknown function (2/16; 12.5%). Three of them, including an endonuclease (pgv02) and the predicted DNAprimase/polymerase (pgv04) have a homolog in the virophage infecting CroV (Mavirus) and the one associated with OLPVs (OLV1) (Fig. 2). Because no PgVV genes appeared to encode a product even remotely similar to a capsid protein, we suspected that the PgVV genome could either be carried as a linear plasmid within the PgV-16T particle or could integrate within the PgV genome, as described previously for the Sputnik virophage (17). We explored this eventuality by generating a map of the “broken” paired-end reads perfectly matching on the PgVV genome on one end and on the PgV-16T genome on the other end (Fig. S2). A total of 796 pair-end reads were found to meet these criteria, suggesting a multiplicity of integration sites.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Map of the PgV-16T-associated virophage-like genome. Predicted ORFs are indicated by boxes colored according to their annotation status: with a predicted function (green), with a significant (E < 10-5) match in the NR database (yellow), and no match (white). The flanking arrows represent two 1-kb-long, nearly identical sequences complementary to each other.</figcaption></figure>

A possible integration "hot spot" defined by 28 read pairs was found between positions 47,500 and 50,000 in the PgV genome sequence (ORF no. 42), and positions 3200 and 4200 in the PgVV genome (ORF nos. 3 and 4). The presence of PgVV DNA packed in the PgV-16T virions was directly validated by PCR on DNA extracted from particles produced from an independent culture and purified by centrifugation on a cesium chloride gradient (SI Materials and Methods). Finally, pgw14 is similar to a protein found in several copies in the PgV genome, supporting the possible role of virophages as gene shuttles between large DNA viruses.

**Gene Content.** Using a BlastP with a conservative E-value threshold of 10⁻⁵, we found 48 genes shared by Megavirus, Mimivirus, CroV, and PgV, the four largest fully sequenced, viral genomes known (Fig. 3 A and B). Among those, only 18 correspond to genes defined previously as "core genes," that is, genes found in a majority of large dsDNA viruses and presumed to encode functions essential for the replication of these viruses (Table S1) (2, 21). This finding suggests that these four viruses share these 48 genes as a result of a common ancestry, rather than functional requirements imposed on all large DNA viruses, in particular, those replicating in the cytoplasm of their host cell. Similar three-way comparisons with the *Emiliania huxleyi* virus (EhV) (having the fifth largest viral genome at 407 kb) (15) resulted in a very different picture, with only 14 genes in common, 11 of which are bona fide core genes (Fig. 3 C and D) encoding the fundamental components of the DNA replication and transcription machineries and a major capsid protein. Thus, even though EhV and PgV are two large DNA viruses infecting two plankton species belonging to the haptophytes, they do not show extensive evidence of common ancestry and clearly belong to different virus families.

The 48 genes (Table S1) shared between PgV and the three other largest sequenced viral genomes [but also found in the draft genome sequences of other Megavirus relatives (17) and, for most of them, within the partial OLPV1 and OLPV2 sequences] are devoted to essential functions such as deoxynucleotide synthesis (ribonucleotide reductase, deoxycytidine deaminase, thymidine kinase, thymidylate synthase, Nudix-like hydrolase); DNA replication [primase, ribonuclease H1, DNA polymerase B, PCNA clamp, three clamp loaders (replication factor C), NAD-dependent DNA ligase, topoisomerase IA and II]; DNA repair (Lambda-type exonuclease, MutS7, Holliday junction resolvase, XRN 5'-3' exonuclease); DNA transcription (Ribonuclease III, VV A18-like and VV D11-like helicases, TATA-box-binding protein, polyadenylate polymerase, transcription factors TFIIB-like, VLTF3-like, and TFIIS-like); protein synthesis and processing (mRNA-capping enzyme, translation initiation factor eIF4E-like, DNAJ, thiol oxidoreductase, cysteine protease); and virion structure (capsid protein and DNA packaging ATPase). However, seven of

these genes uniquely conserved among these giant viruses are also predicted to encode proteins of unknown function ("hypothetical proteins": PgV50, -58, -134, -183, -259, -260, -298) or were similar to enzymes performing seemingly insignificant roles such as a phospholipase (PgV117) or a methyltransferase (PgV234). Most curious among these well-conserved homologs was asparagine synthetase (>38% identical residues), which occurred in PgV-16T, Megavirus, and Mimivirus isolates, in OLPV1 and OLPV2, and *Micromonas* and *Ostreococcus* viruses. A phylogenetic tree built from these viral sequences was amazingly congruent with the global similarity of these viruses and clearly suggests that all known viral asparagine synthetase genes derived from a common ancestor that diverged from the cellular version quite a long time ago and were since vertically inherited within two distinct viral lineages (Fig. S3). The rationale behind the conserved occurrence of the asparagine synthetase gene in the genome of this subset of dsDNA viruses, or the role that asparagine might specifically play in their lifecycle, is unknown.

Of the 434 putative CDS mapped in the PgV genome, only 150 (34.6%) could be assigned a putative function based on homology, a percentage typical of large eukaryotic viruses. This small fraction of known genes was nevertheless sufficient to draw a number of conclusions.

For instance, the PgV genome encodes a high number of RNA polymerase II subunit homologs (RPB1, RPB2, RPB5, RPB6, RPB7, RPB9, and RPB10), a complete set of transcription initiation (PgV113), elongation (PgV146, PgV158), and termination factors (PgV321), together with a polyA polymerase (PgV 73), a TATA-box-like-binding protein (PgV48), and a mRNA-capping enzyme (PgV235). This impressive complement of transcription-related genes suggests that PgV most likely replicates in its host's cytoplasm, even though its infectious cycle has not yet been studied at the cellular level.

In addition to the previously cited DNA manipulating enzymes common to the four largest known viral genomes, PgV possesses DNA-repair enzymes encountered in a more sporadic manner in a variety of other viruses: an ERCC4-type nuclease (PgV318, with homologs in OLPV and Asfarviruses), an apurinic-apyrimidinic

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Fraction of protein-coding genes shared by the largest known viral genomes at increasing evolutionary distance. (A and B) Venn diagrams indicating the global proximity in gene content of the four large genomes of Mimivirus, Megavirus, CroV, and PgV. (C and D) Venn diagrams indicating that EhV and PgV exhibit a markedly different gene content (they only share "core" genes conserved in most large eukaryotic dsDNA virus, irrespective of family; Table S1), even though they both infect a haptophyte.</figcaption></figure>
exoDNase (PgV94, with a homolog in CroV), and a mismatch repair enzyme (PgV283) of a type (MutS8) uniquely found in OLPV1 and -2, *Phaeocystis poucheti* virus, and *Pyramimonas orientalis* virus (22). The PgV genome is also rich in putative ribonucleases such as a bacterial ribonuclease HII-like (PgV121, with homologs in CroV and EhV), a RNase T-like (PgV123, with homologs found in many viruses and phage but very divergent in Megavirus and Mimivirus), and a PIN-domain containing RNase H (PgV303, with homologs in CroV and phages).

The PgV genome also contains a large number of putative endonucleases of the HNH-type (PgV60, -72, -243, -438) and GYI-YIG type (PgV69, -216, -359, -429), but none of them was found associated to an intein, or a type I intron (although such introns cannot be reliably identified when they occur within a protein without a significant homolog in the databases). A single intein was identified within the coding region of the VV A18-like helicase (PgV190). The closest relative of the predicted mature PgV190 gene product was found in OLPV1, but this homolog exhibits no trace of intein. The intein was likely inserted into the PgV190-coding sequence after PgV diverged from the OLPV lineage. Two predicted transposases with a strong similarity (77% identical residues) were also identified (PgV137, -217), the sole viral homologs of which could be found in OLPV1 (also in two copies). The presence of several DNA methylases and type II restriction endonucleases (with their best viral homologs in CroV or *Ostreococcus* viruses) suggests that the PgV genome contains modified nucleotides (such as 5mC or 6mA), as already well documented for *Paramecium bursaria Chlorella* virus 1 (23).

Surprisingly, given the high A+T content of their genomes, both Megavirus and Mimivirus lack deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an enzyme crucial to preventing the misincorporation of deoxyuridine instead of thymidine. This enzyme is present in most large eukaryotic DNA viruses (including CroV and EhV), as well as in phages. Thus, the most parsimonious scenario is that the dUTPase gene, present in the common ancestor of all these viruses, was uniquely lost in the branch leading to Megavirus and Mimivirus. The presence of a dUTPase homolog in PgV (PgV389, most similar to the one of OLPV1) adds support to this hypothesis.

**Large dsDNA Virus Core Gene Duplication.** After the first round of gene annotation, a number of redundancies were found within the subset of 150 PgV-16T genes with putative functional assignments. Multiple copies of genes encoding a similar function could arise in three ways: (i) vertical ancestral inheritance; (ii) acquisition of additional copies by horizontal transfer; and (iii) in situ gene

duplication. Molecular phylogenetic approaches were of little help in deciphering these mechanisms, given the small number of available viral homologs for these genes. Therefore, we used the following simple heuristics to determine which mechanisms were most likely responsible for the duplications: when the gene copies were found to be most similar to each other (relative to a whole NR database search), they were classified as "recent duplication"; if they were separately found to be most similar to their homologs in OLPV (currently the closest known relative of PgV-16T), the duplication was classified as "vertically inherited"; all other cases were presumed to indicate an acquisition by horizontal gene transfer. Table 1 summarizes the results of this analysis, pointing out eight cases of probable duplication since the divergence of the OLPV and PgV lineages, including that of a transposase that might itself be involved in the duplication process. This analysis led us to unravel two unprecedented cases of "core gene" duplication within giant viruses. The first one involves the A32-like packaging ATPase (NCVOG0249) that is uniquely found in two tandem copies in PgV-16T (PgV64, PgV65), most likely the result of a recent event. The second case involves the DNA-directed RNA polymerase second largest subunit (PgV208, PgV228) (NCVOG0271). In this case, the duplication was most likely inherited from the PgV-OLPV common ancestor, because two copies of this RNA polymerase subunit are also present in OLPV. Phylogenetic analyses are consistent with these interpretations (Fig. S4 A and B). The packaging ATPase and the RNA polymerase are both "viral hallmark genes" encoding key functions that are shared by a diverse subset of viruses (24) and are usually found in a single copy per viral genome. However, nothing in these sequences suggests that one of the two copies is a defective, nonfunctioning gene.

**Discovery of a Putative Mobile Element: PgV-MIGE.** The above systematic search for genes individually duplicated in the PgV-16T genome led us to identify 12 copies of a PgV-specific protein (e.g., without significant similarity in the NR database) for which no function could be predicted. These repeated ORFans are evenly dispersed throughout the genome, as indicated by their gene numbers (9, 29, 45, 78, 139, 220, 273, 297, 322, 333, 370, 433) and transcribed from both strands (six from each). These ORFans averaged 320 aa in length (range 270 to 355 aa), with an average pairwise similarity of about 58% (range, 42-65%). The N-terminal halves of the predicted proteins exhibited lower conservation than the C-terminal halves. An exception was PgV333, the most divergent (and shortest) paralog from which the last 50 residues were missing. In a multiple alignment of the 11 full-length homologs, 95 residues are strictly conserved among the 170 C-terminal positions

<table><caption>**Table 1. Duplications events in the PgV-16T genome**</caption><thead><tr><th>Gene pair</th><th>ID aa, %</th><th>Predicted function</th><th>Origin of the duplication, comment</th></tr></thead><tbody><tr><td>137, 217</td><td>77</td><td>Transposase</td><td>Recent duplication</td></tr><tr><td>60, 72</td><td>76</td><td>Homing endonuclease, HNH-type</td><td>Recent duplication</td></tr><tr><td>64-65</td><td>72</td><td>Packaging ATPase (VV32-like)</td><td>Recent tandem duplication, usually a single copy core gene</td></tr><tr><td>96-97</td><td>57</td><td>Phage tail collar domain family protein</td><td>Recent tandem duplication</td></tr><tr><td>142,149</td><td>41</td><td>Deoxynucleoside kinase (DNK)</td><td>Recent duplication, usually a single copy gene</td></tr><tr><td>208,228</td><td>35</td><td>DNA-dependent RNA Pol second largest subunit (Rpb2)</td><td>Vertically inherited, usually a single copy core gene (also duplicated in OLPV)</td></tr><tr><td>7-15 (16)</td><td>35 (14)</td><td>Methyltransferase fkbm family</td><td>7-15: recent duplication; 16: independent HGT</td></tr><tr><td>424-425</td><td>29</td><td>2OG-Fe(II) oxygenase family protein</td><td>Recent tandem duplication</td></tr><tr><td>34, 427</td><td>23</td><td>DNA-J domain containing protein</td><td>One copy common to all Megaviridae, one (old) HGT</td></tr><tr><td>301,414</td><td>17</td><td>2-Polyprenylphenol 6-hydroxylase</td><td>HGT, ubiquinone synthesis</td></tr><tr><td>118,313</td><td>18</td><td>Disulfide-isomerase</td><td>HGT, protein folding</td></tr><tr><td>240,411</td><td>17</td><td>NUDIX-like hydrolase</td><td>HGT, nucleotide metabolism</td></tr></tbody></table>

Multiple occurrence of genes encoding protein with identical predicted functions are interpreted as horizontal gene transfer (HGT) or tandem duplications based on their percentage of identical residues (ID aa%). A third copy of the fkbm methyltransferase (pgvv16), is only 14% identical (in parenthesis) to the two other copies.
(56%), suggestive of a constrained functional signature. However, submitting this multiple alignment to the sensitive 3D structure homology search program Fugue (25) failed to return any significant match or functional clue. To further analyze the primary structure of the DNA segments encompassing and flanking these multiple ORF copies, we focused on the eight most similar ones (average pairwise identity of 63%). A local alignment of their nucleotide sequences identified a highly conserved DNA segment of about 300 nt, beginning immediately upstream of the ORF N terminus (Fig. S5). The nucleotide conservation of this noncoding segment contrasts with the flanking N-terminal part of the ORFan coding region that is not well-conserved (Fig. S5). A huge diversity of mobile elements has been described in the three domains of life (26–28), and the list is still growing (29). However, the PgV-16T repeats do not resemble any of them. For instance, examining the sequences and putative secondary structures flanking the 5' and 3' boundaries of these repeated segments failed to reveal any direct or inverted repeats, or putative target sequences shared by the various repeated elements (Fig. S5). The conserved predicted protein associated with these repeats is also puzzling. To our knowledge, the coding regions associated with large mobile genetic elements (even those using a rolling-circle replication process) always encode proteins bearing a recognizable similarity with DNA-processing enzymes (e.g., homing endonucleases, transposases, integrases, reverse transcriptase, etc.). This is not the case here. The presence of a highly conserved noncoding region within the repeat is also a remarkable feature (Fig. S6). We can only hypothesize that these two conserved elements are involved in the molecular mechanism leading to their

duplication. Given their lack of resemblance to any previously described mobile genetic element, we named these PgV-specific repeats: "PgV major interspersed genomic element" (PgV-MIGE).

**Estimating the Actual Genome Sizes of OLPV1 and OLPV2, the Closest Relatives to PgV.** The PgV-16T genome can be used to estimate the level of completion of the OLPV1 and OLPV2 partial genomes assembled from metagenomic data. Fig. S6 indicates that a total of 145 predicted proteins are shared between OLPV1 or OLPV2 and PgV. Given that OLPV1 and OLPV2 are much closer to each other (their shared predicted proteins are about 80% identical) than they are from PgV (OLPV-PgV orthologous protein share 50% identical residues in average), it is reasonable to assume that most genes shared between PgV and OLPV1 or OLPV2 should also be present in both OLPVs. Following this hypothesis, we can now perform a simple statistical computation: out of the 145 "conserved genes," only 121 are retrieved in the available OLPV2 draft genome. This might be taken to indicate that this genome is only 121/145 (83.4%) complete. Reciprocally, only 125 of these "conserved" genes occur in the draft OLPV1 genome, which, in turn, suggests that it is 125/145 (86%) complete. Applying these proportions, we can now estimate the total number of genes for these two viruses as 401/0.86 = 466 genes for OLPV1, and 326/0.834 = 390 genes for OLPV2. According to this extrapolation, OLPV1, OLPV2, and PgV-16T would possess numbers of genes falling within the variation range exhibited by close members from the same virus family, a result consistent with the fact that PgV proteins have OLPV proteins as their closest homologs.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Phylogenetic position of PgV-16T among dsDNA viruses. Neighbor-joining (midpoint-rooted) tree computed from a multiple alignment of 49 viral DNA polymerase B sequences (405 ungapped sites) using the default options of the MAFFT server (30) and drawn using Mega (31). The tree was collapsed for bootstrap values of <50. The tree clearly suggests that the viruses with the largest known genome form a well-supported cluster, irrespective of their hosts: phagotrophic protozoans (in red) or photosynthetic unicellular algae (in green). Viruses presently classified as "phycodnaviruses" (e.g., "algal viruses") (green and blue) do not form a monophyletic lineages.</figcaption></figure>
## Conclusion

The genetic analysis of the complete genome of *P. globosa* virus PgV-16T revealed a number of specific features only found to be associated with the group of viruses exhibiting the largest and most complex viral genomes known to date, in coherence with the molecular phylogeny of its DNA polymerase (Fig. 4). Although group I *P. globosa*-infecting viruses cluster with the Megaviridae (re-grouping the viruses with the largest known genomes), group II PgVs belong to a very different clade yet apart from other algae-infecting viruses (Fig. S7). Most unexpected was the discovery of a 19,527-bp virophage genome-like DNA molecule that appears to be part of the DNA content of the PgV-16T virion. PgVV exists either as a linear plasmid-like molecule or as a randomly integrated provirus occurring at multiple sites within the PgV-16T genome, as recently described for Sputnik 2, a virophage associated with the Mimiviridae Lentille virus (17). The PgV-associated virophage genome shares only three homologous genes (including a primase, pgvv04) with the two previously described OLPV and Mavirus virophages. Because none of the other PgVV predicted gene products resembled a capsid protein, we hypothesize that PgVV cannot exist as a free virus particle, a situation akin to another mobile element associated with the various Mimiviridae mobile genetic elements, the transpovirons (17). Given its predicted linear genome, its lack of capsid-like protein, and its minimal resemblance with Sputnik, Mavirus, and OLV1, PgVV might represent the first example of a virophage/transpoviron intermediate mobile element.

In addition to its association with a virophage-like sequence, the PgV-16T genome (as well as draft genomes of PgV 12-T and PgV 14-T strains) shares many of the hallmark features of the largest known viral genomes such as a MutS7-type mismatch DNA-repair enzyme, a large complement of DNA-directed RNA polymerase subunit and transcription factors, a TATA-box-like-binding pro-

tein, a mRNA-capping enzyme, DNA topoisomerases of the two types (I and II), a poly-A polymerase, and the mysteriously conserved Asparagine synthetase (Fig. S3). Thus, PgV-16T belongs to the recently proposed Megaviridae family (7, 32), within which DNA viruses exhibiting the largest known genomes appear to cluster, although they infect hosts distributed among a wide taxonomic range of unicellular eukaryotes, from unikonts to bikonts (33).

## Materials and Methods

**Phaeocystis Globosa Culture and Virus Purification.** Exponential axenic cultures of *Phaeocystis globosa* Pg-G (Groningen University Culture Collection, The Netherlands), cultured according to ref. 34 with 40 µmol quanta-m⁻²·s⁻¹ illumination, were infected with axenic PgV-16T at a virus to algal host ratio of 10 allowing a one-step virus growth cycle (Fig. S8). Upon full lysis of the host, viruses were concentrated by ultrafiltration on Vivaflow 200 (molecular mass cutoff, 30 kDa; Vivascience), after which the viral concentrate was clarified of cell debris by low speed centrifugation at 7,500 × g for 30 min at 4 °C. The supernatant was decanted, and viral particles were concentrated by ultracentrifugation at 141,000 × g for 2 h at 8 °C using a TFF55.38 rotor. Viral pellets were resuspended in TE buffer (pH 8.0) and stored frozen until further use.

**Genome Sequencing and Assembly.** Around 1 µg of phenol-chloroform-extracted PgV-16T virus DNA was used to construct a paired-end library, with ~280-bp insert size, as described by the manufacturer's (Illumina) protocol. Around 60 million paired-end reads were produced on an Illumina HiSeq2000 corresponding to ~6 Gb of raw nucleotide sequence. *SI Materials and Methods* contains further details and the bioinformatic procedures of genome assembly and annotation.

**ACKNOWLEDGMENTS.** We thank Harry Witte for practical support. We are glad to acknowledge the use of the electron microscopy platform of the Mediterranean Institute of Microbiology. This work was partially supported by Centre National de la Recherche Scientifique and a grant from the French National Genomic Center-Genoscope.

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