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Home , Mavirus, Mimiviridae, Mobilome, Transpoviron, Virophage

Provirophages and as the diverse of giant

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 that infect giant addition, the encodes a retroviral-type integrase viruses of the family, has been recently described. Here and a -primed DNA polymerase B; these are ho- we report the simultaneous discovery of a giant of Acantha- mologous to the respective proteins of Maverick/ DNA moeba polyphaga (Lentille virus) that contains an integrated ge- transposons, which insert into of diverse , nome of a (Sputnik 2), and a member of a previously suggesting an evolutionary link between the Mavirus and the unknown class of , the transpovirons. The (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 , 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 replicates within the 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 protein particles, Sputnik 2 particles, and 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 of the igin, although each underwent multiple replacements. The

giant virus host and that, although less frequently, the transpoviron virophages are likely to be common parasites of nucleocytoplasmic 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 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 . 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 (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 to can be roughly grouped into four major classes: transposable prevent bacterial growth. Lentille virus particles were purified from elements (TEs), , viruses, and self-splicing elements such Sputnik 2 as previously described for 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 , , 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 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 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 ( 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, 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). Mimivirus and Lentille virus DNA single contig. The curated sequence of Sputnik 2 consists of 18,338 appeared as large fragments above the 339-kb low-range (LR) bp (Fig. 1D). marker (Fig. 1B, lanes 3 and 4, white arrows). In contrast, the A map of paired-end links joining contigs generated from the Sputnik 2 genome presented a multiband profile that apparently 454/Roche paired-end pyrosequencing run was produced (SI contains concatemeric genome units (Fig. 1B,lane5)produced Appendix, Fig. S3) to visualize the connections between the dif- from the circular genomic DNA. In vitro formation of concatemers ferent structures. One paired-read link was identified between by the end-to-end joining of the mature genomes is a well-known Sputnik 2 and Lentille virus. This common region corresponds to process for T7- (20) and, in the case of Sputnik 2, a near-identical sequence of 395 nt identified at the end of the digestion with EagI at the single site located between the V11 and Sputnik V6 gene and in the Lentille virus and Mamavirus (but not V12 genes (Fig. 1B, lane 13 and Fig. 1D) linearized the genome Mimivirus) gene that encodes a collagen-like repeat-containing units. Surprisingly, Southern blot hybridization using the Sputnik 2 protein (SI Appendix, Figs. S4 and S5 A and B). PCR products of genome as a probe identified a strong signal within the Lentille the sizes that were predicted under the assumption that the virus genome (Fig. 1C, black arrows), suggesting that the Sputnik 2 Sputnik genome integrated into the Lentille virus genome were genome is integrated into the genome of its viral host. obtained on purified Lentille virus particles from the right and left

Fig. 1. Lentille virus and Sputnik 2. (A)(Inset)In situ hybridization using Sputnik 2 probe (green), Lentille virus probe (red), and DAPI staining (blue) after 7 h p.i.; the merged image shows intense replication of both Lentille virus and the virophage inside the same viral factory of A. polyphaga cells; the arrow indicates colocalization of the virophage and Lentille virus signals. (B and C) PFGE (B) and Southern blot (C) using DIG-labeled Sputnik 2 probes showing the Sputnik genome in- tegrated into that of Lentille virus. Lane 1: λ marker; lane 2: low-range marker; lane 3: native Mimivirus genome; lane 4: native Lentille virus genome; lane 5: native Sputnik 2 genome; lane 6: ApaI-digested Mimivirus genome; lane 7: ApaI-digested Lentille virus genome; lane 8: EagI-digested Mimivirus ge- nome; lane 9: EagI-digested Lentille virus genome; lane 10: λ marker; lane 11: low-range marker; lane 12: ApaI-digested Sputnik 2 genome; lane 13: EagI- digested Sputnik 2 genome. (D) Map of the Sputnik 1 (outer circle) and Sputnik 2 genomes (inner circle). The ORF number is indicated above the genome along with orientations, clockwise in blue and counterclockwise in red. A line indentifies the EagI restriction site, which cuts once into the Sputnik 1 and 2 genomes. Inside: Sputnik 2 genome GC skew (orange/yellow) and GC content.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208835109 Desnues et al. Downloaded by guest on September 23, 2021 flanks of the putative insertion site by using one primer in Sputnik Fig. S8C). The presence of the transpoviron in Lentille virus 2 and the other in the Lentille virus genome (SI Appendix, Fig. particles was further confirmed using PFGE (Fig. 2B)and S4, Table S6, and SI Materials and Methods). The PCR product Southern blot analysis (Fig. 2C) with a specific digoxigenin generated using the v5F-SpClR primer pairs was sufficiently pure (DIG)-labeled probe (SI Appendix,TableS6). The transpoviron (SI Appendix, Fig. S6) for direct sequencing. Multiple alignment was not detected in the Sputnik 2 genomic preparation by PFGE of the v5F-SpClR PCR sequence (SI Appendix, SI Results) with (Fig. 2B, lane 4) but was identified by Southern blotting (Fig. 2C, the Sputnik 2 and Lentille virus sequences (SI Appendix, Fig. S7) lane 4), in accordance with the low transpoviron sequence cov- showed a central common region of 402 nt flanked by a 3′ region erage observed in the Sputnik 2 sequence data (0.5×) and sug- similar to the Lentille virus sequence (identity of 55/58 nt) and gesting that only a small fraction of the Sputnik particles a5′ region similar to the Sputnik 2 sequence (identity of 121/122 encapsidates the transpoviron. The transpoviron was not detec- nt). Results from massive SOLiD genome sequencing, which gives ted in the uninfected amoeba genomes (either A. polyphaga or higher sequence coverage than pyrosequencing, confirmed the A. castellanii) (Fig. 2 B and C, lanes 5 and 6). Following infection common region as a hotspot of integration (SI Appendix, Fig. S8A, of A. polyphaga by Lentille virus, the signal of the transpoviron red circle). However, the presence of a further candidate in- was extremely intense (Fig. 2 B and C, lane 7), indicating active sertion site detected in the genome of Mamavirus or Lentille virus replication of the transpoviron in the infected amoebae. Sub- (SI Appendix, Fig. S5 A and C), along with the homogeneous sequent PCR (SI Appendix, Table S6) revealed early production distribution of mate pairs between Lentille virus and Sputnik 2 (SI of large amounts of transpoviron DNA starting at 4 h p.i. Appendix, Fig. S8A), suggests that Sputnik 2 may also insert in (SI Appendix, Table S7). An in situ hybridization analysis using multiple, possibly random, sites. a transpoviron-specific probe showed a diffuse signal spread all over the amoeba cytoplasm. At 7 h p.i. the signal was very bright Transpovirons, a Distinct Class of Mobile Genetic Elements. We also and appeared concentrated in rings surrounding the VFs (Fig. identified a contig of 7,420 bp (SI Appendix, Table S5). This 2A). No transpoviron was detected in A. polyphaga infected by abundant contig had a GC content of 24.7% and contained six Sputnik 2 alone (Fig. 2 B and C, lane 8). To investigate the ex- ORFs and long terminal inverted repeats of ∼530 bp in length. pression of the transpoviron during Lentille virus infection, RT- This contig is an extrachromosomal DNA molecule that is PCR was performed on RNA extracted from cultures of A. present in an extremely high copy number in Lentille virus par- polyphaga infected with Lentille virus or Sputnik 2. The mRNA ticles, as indicated by the sequencing coverage, which was 3- to for the predicted transpoviron helicase was detected in the 14-fold higher than the coverage of the Lentille virus for the Lentille virus-infected but not Sputnik 2-infected cultures, in- MICROBIOLOGY pyrosequencing and SOLiD runs (SI Appendix, Table S5). This dicating that expression of at least one transpoviron gene relies small linear resembles a TE and, because it is found on infection of A. polyphaga by Lentille virus (SI Appendix, Fig. inside a virus, we denoted it a “transpoviron.” The paired-end S9). The RT-PCR reactions with viral RNAs extracted from links inside the transpoviron, which uniformly cover the trans- Sputnik 2 particles failed to detect transpoviron-specific tran- poviron sequence without any break, along with the presence scripts. Thus, given that the transpoviron DNA was detected in of cohesive ends, imply the existence of circular intermediate purified Sputnik particles, we speculate that Sputnik is a tran- forms (SI Appendix,Fig.S3). Five pairs of paired-end reads sient host that could be a vehicle transferring transpoviron DNA having one end located in various parts of the Lentille virus to giant virus hosts. genome and the other end matching the transpoviron suggested nonspecific integration (SI Appendix,Fig.S3). Massive SOLiD Distinct Transpovirons Are Associated with Different Groups of sequencing revealed that the transpoviron can insert randomly Mimiviridae. Our current collection of Mimiviridae includes 19 into the Lentille virus genome (SI Appendix,Fig.S8B) and also, virus isolates, namely Mimivirus (7)/Mamavirus (11), 16 isolates although less frequently, into the Sputnik genome (SI Appendix, partially characterized previously (21), and 2 additional, recently

Fig. 2. Identification of the transpoviron. (A)(In- set) In situ hybridization using a Lentille virus probe (green), transpoviron probe (red), and DAPI nucleic acid staining (blue) at 7 h p.i. in A. polyphaga cells. (B) PFGE showing the Lentille virus genome (aster- isk) and the transpoviron as a second band between the 6.55- and 9.42-kb bands of the LR marker (lane 3) identified with a white arrow. (C) Southern blot using DIG-labeled transpoviron probes. Lane 1: λ marker; lane 2: low-range marker; lane 3: Lentille virus genome; lane 4: Sputnik 2 genome; lane 5: A. polyphaga; lane 6: A. castellanii; lane 7: A. poly- phaga infected with Lentille virus and Sputnik 2; lane 8: A. polyphaga infected with Sputnik 2; lane 9: A. polyphaga infected with Mimivirus.

Desnues et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 isolated viruses, Ochan and Marais (SI Appendix, SI Materials and Methods). Phylogenetic analysis of these isolates based on the multiple alignment of partial sequences of the DNA polymerase gene revealed three lineages, which were denoted A (Mimivirus group), B (Moumouvirus group), and C (Courdo11 virus group) (22). We extended this phylogenetic analysis by including the highly conserved genes for the DNA polymerase, D5-like helicase, and a viral factor (Fig. 3; the corresponding acces- sion numbers are in SI Appendix,TableS8). The recently de- scribed Megavirus chilensis (23) falls into group C, and group B Fig. 4. Organization of genes in transpoviron genomes. The approximate now contains the Moumou, Monve, and Ochan viruses. position and strand orientation of each ORF (>150 nt) is indicated by an In the sequencing data for Courdo7, Monve, and Mamavirus, arrow. The ORFs with homologs outside the transpovirons are color-coded contigs with 2- to 10-fold excess coverage were detected. These and their predicted biochemical activities are indicated (TM, protein with a predicted transmembrane helix; C2H2, Zn-finger protein; Super Family I sequences of 6,594, 6,717, and 7,438 bp, respectively, were identi- fi (SFI) Helicase). An ORF that is homologous between different transpovirons is ed as transpovirons by similarity comparison with the originally rendered in blue, and completely uncharacterized ORFs (ORFans) are ren- isolated, Lentille virus-associated transpoviron. Thus, transpovirons dered in white. were detected in Mimiviruses from all three groups (Fig. 3). The available sequences of Megavirus chilensis do not harbor trans- povirons, but we cannot rule out that reads corresponding to the The transpovirons encompass six to eight predicted protein- transpoviron have been rejected. Thus, the current genomic survey coding genes; two genes are conserved in all four sequenced reveals a broad even if sporadic occurrence of transpovirons among genomes, whereas the rest of the genes are missing in one or more the Mimiviruses. In contrast, the virophages so far show a more of the transpovirons (Fig. 4 and SI Appendix, Table S9). The narrow spread, with Sputnik detected only in association with the largest and most highly conserved protein encoded in all trans- Mimiviruses (group A) and the distantly related Mavirus virophage povirons consists of a C-terminal superfamily I helicase domain, associated with CroV that represents the outgroup to the A, B, and which contains the seven signature helicase motifs and is pre- C groups (Fig. 3). dicted to be an active DNA helicase (SI Appendix, Fig. S12), and an uncharacterized N-terminal region. Helicase domains are also Comparative Genomics of the Transpovirons and the Links Between found in other MGEs such as eukaryotic helitrons, diverse bac- Components of the Giant Virus Mobilome. Comparative genomic terial and archaeal plasmids, as well as diverse small viruses (25– analysis of the transpovirons showed general conservation of 28). Typically, these MGEs possess small circular genomes (or gene organization, with some variations (Fig. 4 and SI Appendix, form circular replicative intermediates in the case of the heli- Figs. S10 and S11). Long terminal inverted repeats (TIRs) were trons) and replicate via the rolling-circle (RC) mechanism (27, detected only in the Lentille virus and Mamavirus transpovirons, 29). The indications of a circular intermediate in transpoviron in which they were nearly identical in sequence. The Monve and replication (see above) imply that the transpovirons might use Courdo7 transpovirons lacked the TIRs, possibly because their a similar replication strategy. The RC replication mechanism genome sequences were incomplete at the ends. The TIRs are relies on key replication proteins that consist of a helicase domain extremely AT-rich (>80% AT), and upon closer inspection were and an RC replication initiator endonuclease domain that typi- found to consist of six imperfect, direct tandem repeats (SI Ap- cally comprises the N-terminal part of the respective two-domain pendix, Fig. S11). The presence of long TIRs in the transpovirons protein (28). However, extensive sequence-similarity searches resembles the genome structure of the mavericks (polintons), the including direct comparisons to Rep proteins failed to detect large eukaryotic DNA TEs (24) that notably are related to significant similarity or any of the diagnostic motifs (28) in the virophages (15); other DNA TEs have shorter TIRs (if any). N-terminal region of the large transpoviron protein. The second conserved transpoviron gene encodes a C2H2 Zn- finger protein that is a homolog of the V4 and V14 gene products of Sputnik; a more distant homolog of this protein is also encoded by the Mavirus (SI Appendix, Fig. S13). The transpovirons of Lentille virus, Mamavirus and Monve (but not Courdo7), encode another protein with a homolog in Sputnik, a DNA-binding subunit (SI Appendix, Fig. S14). The remaining pre- dicted proteins of the transpovirons do not have detectable homologs. The mechanism of transpoviron replication remains unknown. Nevertheless, the results of the transpoviron gene analysis are compatible with the expected features of a mobile element that partly relies for replication on its own encoded proteins (in particular the predicted helicase of the transpoviron) and partly on proteins of the host (apparently the Mimiviruses that would supply the DNA polymerase and other components of the replication machinery). The presence, in the transpovirons, of two genes with homologs in Sputnik implies that recombination between the two components of the giant virus mobilome con- tributes to their evolution. Phylogenetic analysis of the trans- posase subunits clearly supports the common origin of this gene in Fig. 3. The spread of transpovirons and virophages in giant viruses. The the transpovirons and Sputnik (SI Appendix, Fig. S14). In con- figure shows the pattern of presence–absence of transpovirons and viroph- ages among the three groups of giant viruses isolated in our laboratory trast, phylogenetic analysis of the helicases places the predicted fi transpoviron helicase into a branch with a distinct group of bac- (black) and by others (blue). The identi ed transpovirons and virophages are fi mapped onto the phylogenetic tree obtained from concatenated alignments terial homologs (SI Appendix, Fig. S15). These ndings indicate of DNA polymerase B, D5-like ATPase/helicase, and a transcription factor of that, like Sputnik and the Mavirus, the transpovirons have a chi- giant viruses. Accession numbers are indicated in SI Appendix, Table S8. meric origin, with different genes derived from distinct sources

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208835109 Desnues et al. Downloaded by guest on September 23, 2021 including genes from other components of the giant mobilome as Virus Cloning. A supernatant containing Sputnik and Lentille virus was sub- well as genes of apparent cellular derivation. mitted to heat inactivation at 68 °C for 1 h or to desiccation for 48 h. The viral −1 −10 In an attempt to identify potential recombination events between suspension was then diluted in PAS by 10-fold dilution from 10 to 10 . transpovirons and Mimiviruses and/or virophages, we searched all Each dilution was inoculated into four wells of a suspension of fresh of the available Mimivirus sequences for regions homologous to amoebae and observed daily for lysis under an inverted microscope. The supernatant of the last dilution producing lysis in 1/4 wells was subcultured the transpovirons. A short sequence segment homologous to the fi fi onto fresh amoebae to produce Lentille virus. Puri ed Lentille virus particles Monve transpoviron was identi ed in the Sputnik genome (SI were examined with a transmission electron microscope (Morgagni 268D; Appendix,Fig.S5D). Although this observation does not reveal Philips) as previously described (34). evidence of transpoviron recombination with Mimiviruses, taken together with the findings on Sputnik integration into the Mimivirus PFGE and Southern Blotting. Plug preparation and treatment and PFGE of viral genome, it suggests that all components of the triple system Mim- DNA were performed as previously described (7). Restriction enzyme (ApaI ivirus–virophage–transpoviron are linked through recombination. and EagI; New England BioLabs) digestion of agarose-embedded DNA was performed using 20 U of the restriction enzyme of interest in the appro- Discussion priate buffer at 37 °C for 4 h. The solution was removed, refreshed, and The discovery of the Mimivirus and subsequent identification of incubated overnight. The digested agarose blocks and molecular weight other giant viruses revealed unexpected complexity of viral markers (Low Range PFG Marker and Lambda Ladder PFG Marker; New England BioLabs) were equilibrated in 0.5× TBE (50 mM Tris, 50 mM boric genomes that, with over 1,000 protein-coding genes, are more acid, 1 mM EDTA). Each agarose block was placed in the well of a 1% PFGE complex than many parasitic and symbiotic bacteria and are agarose gel (Sigma) in 0.5× TBE. The pulsed-field gel separation was made comparable to the most compact genomes of free-living bacteria on a CHEF-DR II apparatus (Bio-Rad), with pulses ranging from 5 to 25 s at and archaea (7). The present work shows that giant viruses are a voltage of 5 V/cm for 22 h at 14 °C. Gels were either stained with ethidium associated with a commensurately complex mobilome that bromide and analyzed using a Gel-Doc 2000 system (Bio-Rad) or used to encompasses three of the four major classes of mobile elements, prepare Southern blots. Resolved uncut or digested genomic DNA by pulsed- namely self-splicing elements, transposable elements or linear field gel electrophoresis was treated and transferred onto Hybond N+ (GE plasmids (transpovirons), and viruses (virophages that can form Healthcare) with a vacuum blotter (model 785; Bio-Rad) and UV–cross-linked provirophages after integration into the host giant virus genome). for 2 min. The blots were then hybridized against the digoxigenin-labeled Different components of the giant virus mobilome share homol- probe as recommended by the manufacturer (DIG-System; Roche Diag- ogous genes, and genomic comparisons point to DNA transfer nostics), except detection of the hybridized probe was performed using a horseradish peroxidase-conjugated monoclonal mouse anti-digoxigenin between the mobilome components and the host virus but also antibody (Jackson Immunoresearch; 1:20,000). After washings, blots were MICROBIOLOGY within the mobilome itself. Thus, the giant virus mobilome is revealed by chemiluminescence assays (ECL; GE Healthcare). The resulting a network that potentially could provide routes and vehicles for signal was detected on Hyperfilm ECL (GE Healthcare) by using an auto- gene exchange and might make substantial contributions to the mated film processor (Hyperprocessor; GE Healthcare). shaping of mosaic viral genomes. The giant viruses and their mobilomes together are part of even more expansive, dynamic Probe Labeling and Fluorescence in Situ Hybridization. Slides for in situ hy- genetic networks: the amoebae with their diverse bacterial para- bridization were prepared from a culture of A. polyphaga infected with sites and symbionts and their own viruses (30). Lentille virus. At 7 h postinfection, a culture sample (200 μL) was placed in Of special note is the transpoviron, a distinct plasmid that a cytospin chamber, centrifuged at 800 rpm for 10 min in a Shandon Cyto- depends on giant viruses for its replication and spread. Sub- spin 4 (Thermo Electron), and fixed for 10 min with a drop of methanol. A stantial analogies can be found between the transpovirons and negative control consisting of uninfected A. polyphaga culture was also virus-associated plasmids present in bacteria and archaea. In performed simultaneously. Slides were treated for 15 min at 37 °C with 0.1 mg/mL RNase A (Boehringer) in 2× SSC. After two washes in PBS, prepara- particular, the well-studied bacteriophage P4 (also known as fi “ ” tions were dehydrated in an ethanol series and air-dried. Speci c DNA a phasmid ) is a plasmid that replicates episomally in the ab- probes were generated by PCR (SI Appendix, Table S6 and SI Materials and sence of the helper bacteriophage P2 but is encapsidated into Methods) and labeled using either the DIG- or Biotin-High Prime DNA La- virions and thus can infect new bacterial cells in the presence of beling Kit (Roche Applied Sciences) following the manufacturer’s recom- the helper (31, 32). A similar replication strategy has been de- mendations. Labeled probes were ethanol-precipitated and resuspended scribed for the archaeal virus plasmid pSSVx that depends on the in a solution containing 50% diformamide, 2× SSC, 10% (wt/vol) dextran fuselloviruses SSV1 or SSV2 and appears to have acquired genes sulfate, 50 mM sodium phosphate, pH 7 (final concentration of 1 ng/μL). from a fusellovirus (33). The discovery of the transpoviron shows Probe solution (10 μL) and preparation were denatured simultaneously that virus-associated plasmids exist in all three domains of for 4 min at 80 °C and hybridized in a moist chamber at 37 °C overnight. × cellular . Slides were washed three times for 5 min at 45 °C with 2 SSC containing 50% formamide, five times for 2 min with 2× SSC at room temperature (RT), It is unlikely that the present study exhausts the diversity of the and once for 5 min with TNT buffer [100 mM Tris·HCl (pH 7.5), 150 mM NaCl, giant virus mobilome; additional virophages and transpovirons, and 0.05% Tween 20]. The preparation was then incubated for 30 min at 37 °C perhaps distinct classes of mobile elements, are likely to be dis- with TNB buffer [100 mM Tris·HCl (pH 7.5), 150 mM NaCl, 0.5% blocking covered. Indeed, the transpoviron had not been detected until the reagent] and 30 min at 37 °C with 2 µg/mL rhodamine anti-DIG antibody isolation of Lentille virus from a human sample described here. (Roche Applied Sciences) and 2 μg/mL of Alexa Fluor 488 streptavidin (Invi- Furthermore, we failed to detect closely related homologs of trogen) diluted in TNB buffer. Slides were finally washed three times for 5 min transpoviron genes in the available databases of environmental with TNT buffer, dehydrated in an ethanol series, air-dried, and mounted with sequences, although close homologs of many Mimivirus and 10 μL of Prolong antifade. Slides were observed under epifluorescence mi- Sputnik genes were readily detectable (11). Thus, specific con- croscopy with a Leica microscope equipped with a DS1-QM (Nikon) black and ditions and/or habitats could be required for accumulation of white camera. Native images were colored and merged using ImageJ free transpovirons and probably other elements comprising the giant software (35). virus mobilome. Characterization of such conditions will likely lead Combined Fluorescence in Situ Hybridization and Immunofluorescence. FISH to the discovery of additional genetic elements associated with giant using a DIG-labeled Sputnik 2 probe was performed from a culture of viruses and facilitate elucidation of their replication mechanisms A. polyphaga infected with Lentille virus at time 0, hour 2, hour 4, hour 6, and the relationships between different mobilome components. and hour 23 postinfection. Slides were prepared as described above for FISH. For immunofluorescence, preparations were incubated for 30 min with Materials and Methods rabbit anti-Mimivirus serum previously adsorbed on A. polyphaga and Isolation, Culture, and Purification of Viruses. Lentille virus and Sputnik 2 were washed with PBS-Tween as previously described (11). Lentille virus particles isolated from contact lens fluid of a patient with keratitis (19) as previously were detected with goat anti-rabbit Ig-Alexa 546, and DIG-labeled Sputnik 2 described (11). probe was detected with a FITC anti-DIG antibody.

Desnues et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 Genome Sequencing, Bioinformatic Analysis, and Sequence Accession Numbers. CLC Genomics workbench from CLC Bio (SI Appendix, Table S5). Broken (i.e., DNA extracted from a clonal preparation of Lentille virus particles was first pairs broken with respective orientation or with a distance above 65 nt) pyrosequenced with a 454/Roche GS FLX system using shotgun technology mate pairs matching different reference genomes were extracted and vi- and then pyrosequenced with a 454/Roche GS FLX Titanium system using sualized using an in-house script written in R (www.r-project.org)(SI Ap- paired-end technology. Raw data for the two sequencing runs are presented pendix, SI Materials and Methods). in SI Appendix, Table S1. Pyrosequencing reads from shotgun and paired- The ORFs in contigs were identified using GeneMark (36) and Prodigal 2.5 end technologies were then assembled with Newbler version 2.5.3 (Roche (37) and annotated using BLASTN, BLASTX, and BLASTP (38), PFam (39), and Applied Science) using default parameters either separately (SI Appendix, tRNAscan (40). For in-depth annotation, the nonredundant protein se- Tables S2 and S3 for shotgun and paired-end assemblies) or combined (SI quence database and the database of environmental sequences at the Na- Appendix, Table S4). The paired-end links between reads were visualized tional Center for Biotechnology Information (National Institutes of Health) with the Gephi open graph visualization platform (freely accessible at http:// were searched using PSI-BLAST (41). Additional searches of protein family gephi.org) from the parsing of a New454PairEndStatus Newbler log file and profile databases were performed using the HHpred program (42). Multiple using the Force Atlas 2 layout. To gain higher coverage and better finishing alignments of protein sequence were constructed using the MUSCLE pro- in closing the Lentille virus genome, a third sequencing round was per- gram (43). Maximum-likelihood phylogenetic trees were constructed using formed, using SOLiD V4 chemistry on one full slide associated with 49 other the TREEFINDER program (44) [WAG matrix, fixed rate model (45) G[Opti- projects on an Applied Biosystems SOLiD 4 machine. The paired-end library mum]:4, 1,000 replicates, Search Depth 20]; the bootstrap support was cal- was constructed from 1 μg of purified genomic DNA of Lentille virus. All of culated as expected-likelihood weight. DNA repeats were identified using – these 50 genomic DNA were barcoded, with the module 1 96 barcodes Inverted Repeats Finder (46). provided by Life Technologies. Libraries were pooled in equimolar ratios and fi emulsion PCR was performed according to Life Technologies speci cation. ACKNOWLEDGMENTS. This work was funded by the Centre National de la Raw data from the Lentille virus project are presented in SI Appendix, Table Recherche Scientifique (crédits recurrent). N.Y. and E.V.K. are supported by S1. Reads were mapped on the contigs previously obtained after assembly of the intramural funds of the US Department of Health and Human Services the two pyrosequencing runs with 99% identity and 99% coverage using the (National Library of Medicine, National Institutes of Health).

1. Frost LS, Leplae R, Summers AO, Toussaint A (2005) Mobile genetic elements: The 24. Pritham EJ, Putliwala T, Feschotte C (2007) Mavericks, a novel class of giant trans- agents of open source evolution. Nat Rev Microbiol 3(9):722–732. posable elements widespread in eukaryotes and related to DNA viruses. Gene 390(1- 2. Feschotte C, Pritham EJ (2007) DNA transposons and the evolution of eukaryotic 2):3–17. genomes. Annu Rev Genet 41:331–368. 25. Kapitonov VV, Jurka J (2001) Rolling-circle transposons in eukaryotes. Proc Natl Acad 3. Kazazian HH, Jr. (2004) Mobile elements: Drivers of genome evolution. Science 303 Sci USA 98(15):8714–8719. (5664):1626–1632. 26. Kapitonov VV, Jurka J (2007) Helitrons on a roll: Eukaryotic rolling-circle transposons. 4. Siefert JL (2009) Defining the mobilome. Methods Mol Biol 532:13–27. Trends Genet 23(10):521–529. 5. Sun AJ, et al. (2010) Functional evaluation of the role of reticuloendotheliosis virus 27. Khan SA (1997) Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev long terminal repeat (LTR) integrated into the genome of a field strain of Marek’s 61(4):442–455. disease virus. 397(2):270–276. 28. Ilyina TV, Koonin EV (1992) Conserved sequence motifs in the initiator proteins for 6. Hertig C, Coupar BEH, Gould AR, Boyle DB (1997) Field and vaccine strains of fowlpox rolling circle DNA replication encoded by diverse replicons from eubacteria, eucar- virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus. yotes and archaebacteria. Nucleic Acids Res 20(13):3279–3285. Virology 235(2):367–376. 29. Vadivukarasi T, Girish KR, Usha R (2007) Sequence and recombination analyses of the 7. Raoult D, et al. (2004) The 1.2-megabase genome sequence of Mimivirus. Science 306 geminivirus replication initiator protein. J Biosci 32(1):17–29. (5700):1344–1350. 30. Raoult D, Boyer M (2010) Amoebae as genitors and reservoirs of giant viruses. In- 8. Colson P, et al. (2011) Viruses with more than 1,000 genes: Mamavirus, a new tervirology 53(5):321–329. Acanthamoeba polyphaga mimivirus strain, and reannotation of Mimivirus genes. 31. Briani F, Dehò G, Forti F, Ghisotti D (2001) The plasmid status of satellite bacterio- Genome Biol Evol 3:737–742. phage P4. Plasmid 45(1):1–17. 9. Van Etten JL (2003) Unusual life style of giant chlorella viruses. Annu Rev Genet 37: 32. Lindqvist BH, Dehò G, Calendar R (1993) Mechanisms of genome propagation and 153–195. helper exploitation by satellite phage P4. Microbiol Rev 57(3):683–702. 10. Simon AE, Roossinck MJ, Havelda Z (2004) virus satellite and defective in- 33. Arnold HP, et al. (1999) The genetic element pSSVx of the extremely thermophilic terfering RNAs: New paradigms for a new century. Annu Rev Phytopathol 42: crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus. Mol Microbiol 34 415–437. (2):217–226. 11. La Scola B, et al. (2008) The virophage as a unique parasite of the giant mimivirus. 34. Boyer M, et al. (2011) Mimivirus shows dramatic genome reduction after intra- Nature 455(7209):100–104. amoebal culture. Proc Natl Acad Sci USA 108(25):10296–10301. 12. Sun S, et al. (2010) Structural studies of the Sputnik virophage. J Virol 84(2):894–897. 35. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image 13. Desnues C, Raoult D (2010) Inside the lifestyle of the virophage. Intervirology 53(5): analysis. Nat Methods 9(7):671–675. 293–303. 36. Lukashin AV, Borodovsky M (1998) GeneMark.hmm: New solutions for gene finding. 14. Iyer LM, Abhiman S, Aravind L (2008) A new family of polymerases related to su- Nucleic Acids Res 26(4):1107–1115. perfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases. Biol 37. Hyatt D, et al. (2010) Prodigal: Prokaryotic gene recognition and initiation Direct 3:39. site identification. BMC Bioinformatics 11:119. 15. Fischer MG, Suttle CA (2011) A virophage at the origin of large DNA transposons. 38. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment Science 332(6026):231–234. search tool. J Mol Biol 215(3):403–410. 16. Fischer MG, Allen MJ, Wilson WH, Suttle CA (2010) Giant virus with a remarkable 39. Finn RD, et al. (2008) The Pfam protein families database. Nucleic Acids Res 36(Da- complement of genes infects marine zooplankton. Proc Natl Acad Sci USA 107(45): tabase issue):D281–D288. 19508–19513. 40. Lowe TM, Eddy SR (1997) tRNAscan-SE: A program for improved detection of transfer 17. Yau S, et al. (2011) Virophage control of Antarctic algal host-virus dynamics. Proc Natl RNA genes in genomic sequence. Nucleic Acids Res 25(5):955–964. Acad Sci USA 108(15):6163–6168. 41. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein 18. Koonin EV, Yutin N (2010) Origin and evolution of eukaryotic large nucleo-cyto- database search programs. Nucleic Acids Res 25(17):3389–3402. plasmic DNA viruses. Intervirology 53(5):284–292. 42. Söding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein ho- 19. Cohen G, Hoffart L, La Scola B, Raoult D, Drancourt M (2011) Ameba-associated mology detection and structure prediction. Nucleic Acids Res 33(Web Server issue): keratitis, France. Emerg Infect Dis 17(7):1306–1308. W244–W248. 20. Son M, Hayes SJ, Serwer P (1988) Concatemerization and packaging of bacteriophage 43. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high T7 DNA in vitro: Determination of the concatemers’ length and appearance kinetics throughput. Nucleic Acids Res 32(5):1792–1797. by use of rotating gel electrophoresis. Virology 162(1):38–46. 44. Jobb G, von Haeseler A, Strimmer K (2004) TREEFINDER: A powerful graphical analysis 21. La Scola B, et al. (2010) Tentative characterization of new environmental giant viruses environment for molecular phylogenetics. BMC Evol Biol 4:18. by MALDI-TOF mass spectrometry. Intervirology 53(5):344–353. 45. Whelan S, Goldman N (2001) A general empirical model of protein evolution derived 22. Colson P, de Lamballerie X, Fournous G, Raoult D (2012) Reclassification of giant vi- from multiple protein families using a maximum-likelihood approach. Mol Biol Evol ruses composing a fourth domain of life in the new order Megavirales. Intervirology 18(5):691–699. 55(5):321–332. 46. Warburton PE, Giordano J, Cheung F, Gelfand Y, Benson G (2004) Inverted repeat 23. Arslan D, Legendre M, Seltzer V, Abergel C, Claverie JM (2011) Distant Mimivirus structure of the human genome: The X-chromosome contains a preponderance of relative with a larger genome highlights the fundamental features of Megaviridae. large, highly homologous inverted repeats that contain testes genes. Genome Res 14 Proc Natl Acad Sci USA 108(42):17486–17491. (10A):1861–1869.

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