Giant Marseillevirus Highlights the Role of Amoebae As a Melting Pot in Emergence of Chimeric Microorganisms
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Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms Mickae¨ l Boyera,1, Natalya Yutinb,1, Isabelle Pagniera, Lina Barrassia, Ghislain Fournousa, Leon Espinosaa, Catherine Roberta, Saïd Azzaa, Siyang Sunc, Michael G. Rossmannc,2, Marie Suzan-Montia,3, Bernard La Scolaa, Eugene V. Kooninb, and Didier Raoulta,2 aUnite´de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche, Institut de Recherche pour le De´veloppement 6236, Faculte´deMe´ decine, Universite´ delaMe´ diterrane´e, 13385 Marseille Cedex 5, France; bNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894; and cDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907 Contributed by Michael G. Rossmann, October 22, 2009 (sent for review September 1, 2009) Giant viruses such as Mimivirus isolated from amoeba found in neously (Fig. 1C), leading to the formation of immature and mature aquatic habitats show biological sophistication comparable to that of viral particles (Fig. 1D). The Marseillevirus replication cycle was simple cellular life forms and seem to evolve by similar mechanisms, complete at5hp.i., an unusually rapid course of virus reproduction. including extensive gene duplication and horizontal gene transfer Kinetics and quantification of the Marseillevirus replication cycle (HGT), possibly in part through a viral parasite, the virophage. We are presented in SI Text. A preliminary cryo-electron microscopy report here the isolation of ‘‘Marseille’’ virus, a previously uncharac- (cryo-EM) 3D reconstruction using images of purified virus showed terized giant virus of amoeba. The virions of Marseillevirus encom- that the virus has a roughly icosahedral shape with a diameter of pass a 368-kb genome, a minimum of 49 proteins, and some messen- about 250 nm. In addition, the virus possesses 12-nm-long fibers ger RNAs. Phylogenetic analysis of core genes indicates that with globular ends on the surface (Fig. 1 E and F). The capsid shell Marseillevirus is the prototype of a family of nucleocytoplasmic large is Ϸ10 nm thick and is separated from the internal nucleocapsid by DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the a gap of Ϸ5 nm. The nucleocapsid has a shape that roughly matches virus is composed of typical NCLDV core genes and genes apparently the external capsid structure and might be surrounded by a mem- obtained from eukaryotic hosts and their parasites or symbionts, both brane (Fig. 1G). bacterial and viral. We propose that amoebae are ‘‘melting pots’’ of Using 2D gel electrophoresis followed by matrix-assisted laser microbial evolution where diverse forms emerge, including giant desorption/ionization time-of-flight (MALDI-TOF) mass spec- viruses with complex gene repertoires of various origins. trometry (Table S1), we identified 49 proteins in purified Marseil- levirus virions (Fig. S1). The proteins detected in the virion giant virus ͉ horizontal gene transfer ͉ nucleocytoplasmic large DNA virus ͉ represent diverse predicted functions, including bona fide structural viral evolution proteins (e.g., capsid proteins) and some proteins potentially in- volved in the early stage of the virus cycle (e.g., an early transcrip- efinitions of viruses are commonly based on size criteria (1), tion factor, a protein kinase, and an ankyrin repeat-containing Dand fine filters are routinely used for virus isolation. For this protein). The detected virion proteins included products of some of reason and also because virus research heavily focused on viruses the (nearly) universal nucleocytoplasmic large DNA virus infecting animals and plants, giant viruses have not been discovered (NCLDV) genes (8, 9), the most abundant ones being the capsid until recently. Accordingly, viruses were generally regarded as protein, a D6R-type helicase, and a S/T protein kinase, as well as small, specialized complexes of biomolecules rather than complex products of genes that are conserved in subsets of the NCLDV, such organisms (2). The concept of ‘‘giant virus’’ emerged with the as thioredoxin/glutaredoxin, RNase III, papain-like cysteine pro- discovery of phycodnaviruses, whose particle size is between 160 tease, and an ankyrin-repeat protein (Table S1). Western blot and 200 nm (i.e., Paramecium bursaria Chlorella virus) (3). Amoe- analysis with a mouse polyclonal antiserum against purified viral bae, as wild phagocytes, ingest any particles larger than 0.2 m (4) particles identified antigenic properties for 11 viral proteins, in- and are therefore a potential source of giant viruses. Previous cluding products of four genes without detectable homologs (OR- findings indicate that amoebae of the genus Acanthamoeba support Fans) (Fig. S1 and Table S1). Extensive posttranslational modifi- multiplication of giant viruses such as Mimivirus and Mamavirus (5, cation occurred during Marseillevirus protein synthesis: 10 of the 49 6) as well as the virophage Sputnik, a small virus parasite of the identified virion proteins were glycosylated and 19 were phosphor- giant Mamavirus (7). Here we describe Marseillevirus, a giant virus ylated (Fig. S1 and Table S1). The virion also encapsidates some isolated from the same host. viral messenger RNAs similarly to Mimivirus (SI Text). Results and Discussion Structural Characterization of a Large Icosahedral Virus Isolated from Author contributions: B.L.S. and D.R. designed research; M.B., N.Y., I.P., L.B., G.F., L.E., C.R., S.A., S.S., M.G.R., M.S.-M., and E.V.K. performed research; M.B., N.Y., I.P., L.B., G.F., L.E., C.R., Amoeba. Cocultivation experiments were performed between A. S.A., S.S., M.G.R., M.S.-M., B.L.S., E.V.K., and D.R. analyzed data; and M.B., N.Y., S.S., M.G.R., polyphaga and samples of water from a cooling tower located in M.S.-M., B.L.S., E.V.K., and D.R. wrote the paper. Paris and monitored during 52 weeks, as previously described for The authors declare no conflict of interest. Mamavirus isolation (7). Cell lysis was observed at 19 weeks of Data deposition: The Marseillevirus genome reported in this paper has been deposited in monitoring, and transmission electron microscopy showed the the GenBank database (accession no. GU071086). presence of virus particles of about 250 nm in diameter with an 1M.B. and N.Y. contributed equally to this work. icosahedral capsid morphology (Fig. 1). Between 30 min and 1 h 2To whom correspondence may be addressed. E-mail: [email protected] or didier.raoult@ postinfection (p.i.), viruses were shown entering the amoeba (Fig. gmail.com. 1A); at later times p.i., a virus factory (VF) with a diffuse aspect was 3Present address: INSERM U912, 23 Rue Stanislas Torrents, 13006 Marseille, France. observed close to the amoeba nucleus (Fig. 1B), where both capsid This article contains supporting information online at www.pnas.org/cgi/content/full/ assembly and viral DNA encapsidation seemed to occur simulta- 0911354106/DCSupplemental. 21848–21853 ͉ PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911354106 Downloaded by guest on September 29, 2021 A B C VF D N E F G Fig. 1. Ultrastructure of Marseillevirus. Transmission electron microscopy images were taken at 30 min p.i. (A) and at 6 h p.i. (B). (A) Marseillevirus particles being phagocytosed by an amoeba. (Scale bar: 2 m.) (B) A virus factory (VF) developed through the cell cytoplasm, near the nucleus (N). (Scale bar: 2 m.) (C) Different stages of Marseillevirus assembly. (D) Complete immature and mature virus particles. (E-G) Cryo-EM 3D reconstruction using images of purified Marseillevirus. (E) Shaded-surface representation of the Marseilles virus 3D density map at contour level ϭ 0.5 viewed along an icosahedral twofold axis. (F) Same density map as (E) at a higher contour level ( ϭ 1.75). The density of the fibers is lower than that of the capsid and is not visible at this contour level. (G) A central sliced view of the Marseillevirus 3D density map at contour level ϭ 1.2. Only the globular ends of the fibers are visible as an outer layer of density (white arrow). The stems of the fibers are not visible. However, the fibers can be seen in the original micrographs. The absence of the fibers in the reconstruction is a result of low resolution and/or the fibers MICROBIOLOGY being flexible. Marseillevirus Represents a Unique NCLDV Family. The genome of putative virus family, although none of these sequences appeared Marseillevirus is a circular double-stranded DNA molecule of to originate from close relatives (other strains) of Marseillevirus 368,454 bp with a GϩC content of 44.73%, which makes Marseil- (Table S1 and Fig. S4). levirus the fifth largest viral genome sequenced so far, after Comparative analysis of the protein sequences encoded by the Mimivirus (6), Mamavirus (7), Emiliania huxleyi virus 86 (10), and Marseillevirus genome identified 28 protein families (Table S3). Paramecium bursaria Chlorella virus NY2A (11). A total of 457 The largest family consists of 20 proteins containing bacterial- ORFs were predicted to encode proteins ranging from 50 to 1,537 like membrane occupation and recognition nexus (MORN) aa residues (Fig. 2 and Table S1). The coding sequences represent repeat domains that typically mediate membrane-membrane or 89.33% of the genome, with Ϸ1.2 genes per kilobase, a tight gene membrane-cytoskeleton interactions (12). Marseillevirus is un- arrangement typical of NCLDV genomes. The ORFs were equally usually rich in serine/threonine protein kinases, with two distinct distributed on both strands (233 and 224 ORFs on negative and clusters of 11 and three kinases, respectively, and a unique kinase positive strand, respectively). Sequence similarity and conserved shared by Marseillevirus, Iridoviruses, and Ascoviruses (Tables domain searches against the respective NCBI databases identified S3 and S4).