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Provirophages and Transpovirons As the Diverse Mobilome of Giant Viruses

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Provirophages and Transpovirons As the Diverse Mobilome of Giant Viruses Provirophages and transpovirons as the diverse mobilome of giant viruses Christelle Desnuesa,1, Bernard La Scolaa,1, Natalya Yutinb, Ghislain Fournousa, Catherine Roberta, Saïd Azzaa, Priscilla Jardota, Sonia Monteila, Angélique Campocassoa, Eugene V. Kooninb, and Didier Raoulta,2 aAix-Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 7278, Institut de Recherche pour le Développement 198, Institut National de la Santé et de la Recherche Médicale 1095, 13005 Marseille, France; and bNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 Edited by James L. Van Etten, University of Nebraska-Lincoln, Lincoln, NE, and approved September 11, 2012 (received for review May 24, 2012) A distinct class of infectious agents, the virophages that infect giant addition, the Mavirus genome encodes a retroviral-type integrase viruses of the Mimiviridae family, has been recently described. Here and a protein-primed DNA polymerase B; these proteins are ho- we report the simultaneous discovery of a giant virus of Acantha- mologous to the respective proteins of Maverick/polinton DNA moeba polyphaga (Lentille virus) that contains an integrated ge- transposons, which insert into genomes of diverse eukaryotes, nome of a virophage (Sputnik 2), and a member of a previously suggesting an evolutionary link between the Mavirus and the unknown class of mobile genetic elements, the transpovirons. The polintons (15). The third complete virophage genome sequence transpovirons are linear DNA elements of ∼7 kb that encompass six has been identified in the metagenome of the hypersaline Organic to eight protein-coding genes, two of which are homologous to Lake in Antarctica (17). This Organic Lake virophage (OLV) is virophage genes. Fluorescence in situ hybridization showed that thought to parasitize on phycoDNAviruses that infect green algae. the free form of the transpoviron replicates within the giant virus The OLV genome encodes seven proteins with homologs in factory and accumulates in high copy numbers inside giant virus Sputnik (17), including two key proteins, the major capsid protein particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of and the DNA-packaging ATPase, that are shared by all three deep-sequencing data showed that the virophage and the transpo- virophages. Thus, the virophages apparently share a common or- viron can integrate in nearly any place in the chromosome of the igin, although each underwent multiple gene replacements. The giant virus host and that, although less frequently, the transpoviron virophages are likely to be common parasites of nucleocytoplasmic MICROBIOLOGY can also be linked to the virophage chromosome. In addition, inte- large DNA viruses that infect diverse eukaryotes, and show mul- grated fragments of transpoviron DNA were detected in several tiple evolutionary connections to other mobile elements (18). giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains Here we present findings that substantially expand the com- revealed three distinct transpovirons associated with three sub- plexity of the giant virus mobilome through the description of an groups of Mimiviruses. The virophage, the transpoviron, and the integrated form of the virophage and of a distinct class of MGEs, previously identified self-splicing introns and inteins constitute the the transpovirons. complex, interconnected mobilome of the giant viruses and are Results likely to substantially contribute to interviral gene transfer. Isolation of a Distinct Giant Virus and Its (Pro)Virophage. Sputnik 2 is the fourth virophage described thus far; unlike the previously obile genetic elements (MGEs) that are collectively re- identified virophages, Sputnik 2 was isolated from a human-asso- ferred to as the “mobilome” are key players in the genome M ciated sample (19). Sputnik 2 and its virus host, Lentille virus, were evolution of prokaryotes (1) and eukaryotes (2, 3) and are con- isolated from contact lens fluid of a patient with keratitis after sidered “genetic engineers” of biological innovation (1). MGEs treatment of the liquid with a mixture of selected antibiotics to can be roughly grouped into four major classes: transposable prevent bacterial growth. Lentille virus particles were purified from elements (TEs), plasmids, viruses, and self-splicing elements such Sputnik 2 as previously described for Mamavirus and Sputnik (11) as group I and II introns and inteins (4). The mobilomes of many using heat inactivation (68 °C) for 1 h and desiccation for 48 h. This bacteria, archaea, and unicellular eukaryotes include all of these virus isolate was then cloned and used to reinfect A. polyphaga. elements in a free or integrated form. Given that viruses consti- Spontaneous release of Sputnik 2 particles was observed in the tute a part of the mobilome, they are not normally considered to infected amoebae, indicating that, although not detected by possess mobilomes of their own. However, some large viruses contain retrovirus sequences integrated into their genomes (5, 6), whereas others, including members of the Mimiviridae family, – Author contributions: C.D., B.L.S., and D.R. designed research; C.D., N.Y., G.F., C.R., S.A., harbor self-splicing introns and/or inteins (7 9). Furthermore, P.J., S.M., A.C., and E.V.K. performed research; C.D., B.L.S., N.Y., G.F., S.A., E.V.K., and D.R. many viruses support the reproduction of satellite viruses (10). analyzed data; and C.D., B.L.S., E.V.K., and D.R. wrote the paper. The discovery of the Sputnik virophage in 2008 added a new The authors declare no conflict of interest. twist to the existing understanding of the relationships between This article is a PNAS Direct Submission. different mobile elements by demonstrating for the first time that a giant virus could be infected by another, much smaller virus in Freely available online through the PNAS open access option. a manner similar to the viral infection of cells (11). The Sputnik Data deposition: The Lentille Virus Whole Genome Shotgun Project reported in this paper has been deposited in the DNA Data Base in Japan/European Molecular Biology Labora- virophage is a small icosahedral virus (74 nm in diameter) that tory/GenBank database (accession no. AFYC00000000). The Sputnik 2 genome reported in parasitizes on Mamavirus, a member of the Mimiviridae family (12, this paper has been deposited in the GenBank database (accession no. JN603369.1). The 13). Sputnik replicates inside Mamavirus or Mimivirus viral fac- transpovirons reported in this paper have been deposited in the GenBank database tories when the host giant virus is grown in amoebae such as [accession no. JQ063126 (Megavirus Courdo7 transpoviron Courdo7), accession no. JQ063127 (Mimivirus Lentille transpoviron Lentille), accession no. JQ063128 (Acantha- Acanthamoeba castellanii or A. polyphaga (11). An in-depth anal- moeba castellanii Mamavirus transpoviron Mamavirus), and accession no. JQ063129 ysis of the Sputnik proteins has suggested an evolutionary con- (Moumouvirus Monve transpoviron Monve)]. Accession numbers for the sequences used nection between this virophage and a distinct class of TEs (14). The in Fig. 3 are indicated in SI Appendix, Table S8. second virophage, the Mavirus (15), was isolated as a parasite of 1C.D. and B.L.S. contributed equally to this work. a distinct member of the Mimiviridae family, Cafeteria roenbergensis 2To whom correspondence should be addressed. E-mail: [email protected]. virus (CroV) (16). At least four Mavirus proteins, including the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. major capsid protein, are homologous to proteins of Sputnik. In 1073/pnas.1208835109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208835109 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 electron microscopy (SI Appendix,Fig.S1), Sputnik 2 or its DNA Virophage Integration in the Giant Viral Host Genome. Virus DNA remained associated with Lentille virus particles. isolated from cloned Lentille virus particles was pyrosequenced Fluorescence in situ hybridization (FISH) was performed using with the 454/Roche GS FLX Kit using shotgun technology and Lentille virus and Sputnik 2-specificprobes(Fig.1A and SI Ap- the 454/Roche GS FLX Titanium Kit using paired-end tech- pendix,Fig.S2for combined immunofluorescence-FISH). At 7 h nology (SI Appendix, Table S1). The results of the de novo as- postinfection (p.i.), a strong DAPI signal was detected in the viral sembly of the combined runs are shown in SI Appendix, Table S4 factories (VFs). Both the host virus (red) and the virophage (green) (SI Appendix, Tables S2 and S3 for separate assembly of shotgun reproduce within the VF (Fig. 1A). In addition, colocalization of and paired-end pyrosequencing data, respectively). The Lentille the Lentille virus signal and the Sputnik 2 signal (Fig. 1A,white virus genome was further sequenced using SOLiD technology, arrow) was observed for several viral particles already released into which gives higher coverage and allows closing gaps and cor- the cytoplasm of the amoeba, an observation that is compatible recting sequencing errors (SI Appendix, Table S1). with virophage localization inside the host virus particles. We fur- After finishing, the Lentille virus genome consisted of 10 con- ther performed pulsed-field gel electrophoresis (PFGE; Fig. 1B)on tigs organized in one scaffold with an average coverage of 29× (SI native, ApaI- (which does not cut the Sputnik 2 genome), and EagI- Appendix, Table S5). A contig of 18,332 bp with a higher coverage (cuts once inside the Sputnik 2 genome) digested Lentille virus (86×) represented the Sputnik-related sequence. The DNA from genome (Fig. 1B, lanes 4, 7, and 9, respectively), Mimivirus genome purified Sputnik 2 particles was also sequenced separately on a (lanes 3, 6, and 8, respectively), and Sputnik 2 genome (Fig. 1B, GS FLX titration plate, generating 1,700,533 bp assembled in a lanes 5, 12, and 13, respectively).
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