Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes Sebastien Santinia, Sandra Jeudya, Julia Bartolia, Olivier Poirota, Magali Lescota, Chantal Abergela, Valérie Barbeb, K. Eric Wommackc, Anna A. M. Noordeloosd, Corina P. D. Brussaardd,e,1, and Jean-Michel Claveriea,f,1 aStructural and Genomic Information Laboratory, Unité Mixte de Recherche 7256, Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France; bCommissariat à l’Energie Atomique–Institut de Génomique, 91057 Evry Cedex, France; cDepartment of Plant and Soil Sciences, University of Delaware, Newark, DE 19711; dDepartment of Biological Oceanography, Royal Netherlands Institute for Sea Research, NL-1790 AB Den Burg (Texel), The Netherlands; eAquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; and fService de Santé Publique et d’Information Médicale, Hôpital de la Timone, Assistance Publique–Hôpitaux de Marseille, FR-13385 Marseille, France 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 viruses: 730 kb and 1.28 Mb for CroV and Megavirus chilensis, globally distributed species of eukaryotic phytoplankton and have respectively. Other studies, targeting virus-specific genes [e.g., a prominent role in bloom termination. The genus Phaeocystis (Hap- DNA polymerase B (8) or capsid proteins (9)] have suggested tophyta, Prymnesiophyceae) includes several high-biomass-forming a close phylogenetic relationship between Mimivirus and several phytoplankton species, such as Phaeocystis globosa, the blooms of giant dsDNA viruses infecting various unicellular algae such as which occur mostly in the coastal zone of the North Atlantic and the Pyramimonas orientalis (Chlorophyta, Prasinophyceae), Phaeocys- North Sea. Here, we report the 459,984-bp-long genome sequence of tis pouchetii (Haptophyta, Prymnesiophyceae), and Chrysochromulina P. globosa virus strain PgV-16T, encoding 434 proteins and eight ericina (Haptophyta, Prymnesiophyceae). tRNAs and, thus, the largest fully sequenced genome to date among Phaeocystis, the host for Phaeocystis globosa virus (PgV)-16T, viruses infecting algae. Surprisingly, PgV-16T exhibits no phyloge- has a complex lifecycle that includes a unicellular, motile stage and MICROBIOLOGY netic affinity with other viruses infecting microalgae (e.g., phycod- gelatinous stage that can produce multicellular colonies of several naviruses), including those infecting Emiliania huxleyi,another centimeters in diameter. P. globosa regularly dominates the phy- ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to toplankton community in the coastal zone of the North Atlantic an emerging clade (the Megaviridae) clustering the viruses endowed and the North Sea, where its blooms may result in “stinking water” with the largest known genomes, including Megavirus, Mimivirus and the production of foam of mucilaginous material that deposits (both infecting acanthamoeba), and a virus infecting the marine on beaches. The decline of natural blooms was shown to be ac- fl fi micro agellate grazer Cafeteria roenbergensis. Seventy- ve percent companied by a considerable increase of viruses infecting P. glo- – of the best matches of PgV-16T predicted proteins correspond to bosa (10), and a mesocosm study showed that the abundance of two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from P. globosa populations can indeed be controlled by viral lysis (11). a hypersaline lake in Antarctica (Organic Lake), the hosts of which The PgV-16T strain reported here belongs to the group I of clonal are unknown. As for OLPVs and other Megaviridae, the PgV-16T virus isolates (together with PgV-12T and PgV-14T) obtained from sequence data revealed the presence of a virophage-like genome. Dutch coastal waters (southern North Sea) and partially charac- However, no virophage particle was detected in infected P. globosa terized previously (12). It is a lytic virus that only infects Phaeocystis cultures. The presence of many genes found only in Megaviridae in globosa with a latent period of 8–12 h and a burst size around 300– its genome and the presence of an associated virophage strongly 400 virions (12). Its icosahedral particle is about 150 nm in di- suggest that PgV-16T shares a common ancestry with the largest ameter, and its genome size was estimated at 470 kb (12). Group I known dsDNA viruses, the host range of which already encompasses viruses are the largest known viruses infecting P. globosa, but many the earliest diverging branches of domain Eukarya. others (classified in group II, such as PgV-03T, PgV-04T, PgV-10T) have been isolated that exhibit smaller virions (about 100 nm in giant virus | core gene | gene duplication | mobile element | diameter) and genome sizes (about 177 ± 3 kb), as well as a dif- horizontal gene transfer ferent host range (10, 12). Their genomes have not yet been sequenced. he discovery of the giant Acanthamoeba polyphaga Mimivirus In this study, we used in excess of 20,390,000 Illumina 101-bp Tin freshwater amoeba and the deciphering of its 1.2-Mb ge- reads (paired) for the de novo assembly the PgV-16T genome at nome sequence initiated a new independent phylogenetic lineage very high sequence coverage (average: 4,477 ± 1,624). Un- among eukaryotic dsDNA viruses (1, 2), as well as a new chapter in expectedly, the corresponding predicted proteome was found to be, virology. Soon after, it was recognized (3) that the closest homol- by far, most similar to the one of two partially sequenced viruses ogous sequences to many Mimivirus proteins occurred within metagenomic sequence data from the Global Ocean Survey ex- pedition (4). However, the identity of these—presumably abun- Author contributions: K.E.W., C.P.D.B., and J.-M.C. designed research; S.S., S.J., J.B., V.B., dant (5)—Mimivirus relatives and the hosts they infect remained 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 elusive. As of today, the only Mimivirus relatives of marine origin data; and S.S., C.P.D.B., and J.-M.C. wrote the paper. fl for which a complete genome sequence is available are a virus The authors declare no con ict of interest. infecting Cafeteria roenbergensis (CroV) (6), a major micro- This article is a PNAS Direct Submission. flagellate grazer, and Megavirus chilensis, isolated from near-shore Data deposition: The sequences reported in this paper have been deposited in the Gen- Bank database (accession nos. KC662249–KC662250). sediments off the coast of central Chile (7). This latter virus is 1To whom correspondence may be addressed. E-mail: [email protected] or Jean- propagated on various species of Acanthamoeba in the laboratory, [email protected]. but its environmental host remains unknown. Remarkably, these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. viruses exhibit the two largest known genomes among marine 1073/pnas.1303251110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303251110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 [Organic Lake phycodnavirus (OLPV)1 and OLPV2] infecting an A unknown host from a hypersaline lake in Antarctica (Organic Lake) Eukaryote 25 Archaea 2 (13). In addition, a subset of data (6,310,000 reads) enabled the as- sembly of a virophage-like genome, reinforcing the similarity with Bacteria 34 OLPVs, which also appear to have associated virophages. The PgV-16T genome, furthermore, exhibits the duplication of two types of No Match 188 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. Virus 179 Results and Discussion General Genome Features. The genome of PgV-16T was fully as- B sembled as a linear DNA sequence of 459,984 bp, making it the EhV 4 2 Other 9 third-largest described genome among marine viruses behind 4 OLV CroV and Megavirus. Its overall nucleotide composition (A+T PpV content: 68%) is less A+T-biased than that of CroV and Mega- Megavirus 6 + ± virus (A T: 77% and 75%, respectively), although it reaches 87 6 8% A+T between genes. Using a conservative annotation protocol CroV OLPV 138 (SI Materials and Methods), we identified 434 putative protein- OsV 10 coding sequences with an average length of 320 aa (ranging from 45 to 3,739 aa) and eight tRNAs (three tRNALeu, two tRNAAsn, one tRNAArg, one tRNAGln, one tRNAIle). With an average inter- genic distance of 104 nt (summing to 42,717 noncoding base pairs) the PgV-16T genome reaches a coding density of 90.7%. Un- expectedly, the partially assembled genome sequences of two other Fig. 1. Global database similarity of the PgV-16T predicted proteome. (A) PgV strains deposited directly in the GenBank database (PgV 12-T, Close to 50% of PgV predicted proteins do not exhibit a significant match < −5 accession no. HQ634147.1; PgV 14-T, accession no. HQ634144.1) (E 10 ) in the NCBI NR protein database, as is typical for viruses belonging to a newly explored lineage with no other sequenced representatives (2, 6). were found to be nearly identical to PgV-16T in terms of gene (B) Surprisingly, for 77% of the PgV predicted proteins exhibiting a viral protein content and percentage of identity (98.1% of identity; 449 and as their best match, this homolog is found in the partially sequenced OLPV1 444 genes). We searched the final genome sequence assembly for or -2. This finding indicates strong global phylogenetic affinities between the presence of large (direct or inverted) repeated regions of >5 PgV (infecting a haptophyte) and the OLPVs infecting an unknown host.