Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae

Defne Arslan1, Matthieu Legendre1, Virginie Seltzer, Chantal Abergel2, and Jean-Michel Claverie2
Information Gnomique et Structurale, Centre National de la Recherche Scientique-Unit Propre de Recherche 2589, Aix-Marseille University, Institut de Microbiologie de la Mditerrane, Parc Scientique de Luminy, Case 934, FR-13288 Marseille, France Edited by James L. Van Etten, University of Nebraska, Lincoln, NE, and approved September 13, 2011 (received for review July 6, 2011)

Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the rst four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary signicance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i ) lineages specic gains or losses of genes, (ii ) lineage specic gene family expansion or deletion, and (iii ) the insertion/migration of mobile elements (intron, intein).
Mimiviridae

but divergent enough to provide a clear illustration of the evolutionary forces at work. Several Mimivirus-related megaviridae have been mentioned in recent literature: Mamavirus (15) (nearly identical to Mimivirus) and few others more briey described (Terra1, Terra2, Courdo, or Moumou) (16) and for which no genome sequence is available. Through a campaign of random aquatic environmental sampling, followed by culturing on a panel of acanthamoeba species, we isolated Megavirus from sea water sampled close to the shore off the ECIM marine station in Las Cruces, Chile (SI Materials and Methods). Megavirus is now routinely cultured on A. castellanii. Its complete genome sequence was determined by using a combination of 454-titanium and Illumina HiSeq approaches. Here, we present an electron microscopy study of the Megavirus replication cycle in A. castellanii and an analysis of its genome. Despite their substantial evolutionary divergence, all of the unique features noticed in Mimivirus are conserved in Megavirus, and delineate a core set of cellular-like functions that might be fundamentally linked to the origin and evolution of Megaviridae. Results and Discussion
Electron Microscopy Data. Megavirus and Mimivirus virion particles exhibit a very similar overall morphology, with a dense core nucleocapsid encased into an icosasedral-like capsid, itself covered by a layer of bers. However, details make them readily recognizable, even in mixed culture (Fig. 1A): The Megavirus bers are noticeably shorter (75 5 nm compared with 120 5 nm for Mimivirus) and cover a capsid slightly larger in diameter (440 10 nm, compared with 390 10 nm for Mimivirus). These dimensions refer to dehydrated virions as prepared for thin section transmission electronic microscopy (TEM), which induce an 20% shrinking of the capsids (17). Native Megavirus icosasedral capsids are thus 520 nm in diameter, corresponding to a total particle diameter of 680 nm. In addition, the hair of Megavirus virions often exhibits one or two patches of slightly longer and denser bers (nicknamed cowlicks) (Fig. 1A Inset). The Megavirus particles exhibit a clearly visible special vertex (Fig. 1B), corresponding to the Stargate already described for Mimivirus, a ve-pronged star structure the opening of which triggers the release of the nucleocapsid into the host cell cytoplasm (11, 14). Upon protease treatments, Megavirus par-

| DNA virus phylogeny | girus | viral translation | tree of life

he discovery of the viral nature of Acanthamoeba polyphaga Mimivirus (1), followed by the determination of its outstanding genome sequence (1,182 kb) (2) led to an irreversible change in the way microbiologists looked at viruses (36), even reviving the debate on their classication as living microorganisms (7, 8). After Mimivirus, no obvious limit could be set anymore on the expected size of a viral particle, or the complexity of its gene content, both of them now largely overlapping with that of the simplest cellular organisms, such as parasitic bacteria (9). Moreover, the Mimivirus genome was found to encode a number of functions thought to be trademarks of cellular organisms, such as aminoacyl tRNA synthetases (AARS), threatening to invalidate the absence of a translation system as the last inviolate criterion separating viruses from the cellular world (1, 10). Subsequent studies revealed additional Mimivirus idiosyncrasies such as its unique virion unloading/loading mechanisms (11), singular gene transcription signaling such as the hairpin rule (12), and its susceptibility to infection by a new type of satellite virus, called virophage (13, 14). Beyond the initial excitation of their discovery, the next step is now to assess to what extent these features are anecdotal or, in the contrary, deeply linked to the emergence and mode of evolution of giant DNA viruses with genome sizes >1 Mb (hereby referred to as Megaviridae). Such a task requires comparing Mimivirus with relatives situated at optimal evolutionary distances, close enough to allow the unambiguous assignment of homologous features,
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Author contributions: C.A. and J.-M.C. designed research; D.A., M.L., V.S., C.A., and J.-M.C. performed research; D.A., M.L., V.S., C.A., and J.-M.C. analyzed data; and M.L., C.A., and J.-M.C. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. JN258408). A Megavirus genome browser is available at www. giantvirus.org/megavirus/.
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D.A. and M.L. contributed equally to this work. To whom correspondence may be sent. E-mail: jean-michel.claverie@univmed.fr or chantal. abergel@igs.cnrs-mrs.fr

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1110889108/-/DCSupplemental.

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Fig. 1. Electron microscopy of Megavirus compared with Mimivirus. (A) Mimivirus (Upper) and Megavirus (Lower) particles in a same vacuole (coinfection). (A, Inset) Cowlicks (arrow) as often seen in the Megavirus ber outer layer. (B) Megavirus stargate. (B, Inset) Transversal section of a Megavirus particle below an open stargate. Megavirus (C ) and Mimivirus (D) seeds surrounded by a lipid membrane (arrows). Megavirus (E ) and Mimivirus (F ) early stages of the virion factories with the seeds at their centers. Megavirus (G) and Mimivirus (H) mature virion factories in full production. (Scale bars: AF, 200 nm; G and H, 1 m).

ticles appeared more fragile than the Mimivirus ones, showing a larger proportion of damaged particles in TEM preparation. The Megavirus isolate was initially tested for growth on A. grifni, A. polyphaga, and A.castellanii, and found to replicate in these three species. For comparison purpose, the replication of Megavirus was then studied in detail on A. castellanii (Neff American Type Culture Collection 30010). The infectious cycle of Megavirus lasts 17 h from the initial phagocytosis event to the time of maximal release of particles from fully mature virion factories, compared with 12 h for Mimivirus (MOI 10). The difference is mostly due to a slower progression from the seed stage (Fig. 1 C and D) to the fully bloomed virion factories (Fig. 1 G and H). Another macroscopic phenomenon specic of Megavirus is that 35% of the A. castellanii cells appear to die without undergoing productive infections. The reasons for this
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cytotoxicity is unknown but suggest that the laboratory A. castellanii strain behaves as a nonoptimal substitute for the unknown natural environmental host of Megavirus. We previously observed that Mimivirus induced a rounding of the infected A. castellanii cells, 6 h after infection. The same phenomenon, albeit slightly delayed, was observed with Megavirus. However, at variance with Mimivirus where rounded cells remained adherent, Megavirus infection caused most of them to come off their support, making the cultures more difcult to monitor by regular microscopy. A. castellanii cells infected by Megavirus exhibited three distinctive ultrastructural features in succession, as described for Mimivirus (11, 14, 18). First, the seed, corresponding to the Megavirus core nucleocapsid extracted from the external particle layers, appeared clearly separated from the cell cytoplasm by a well-delineated lipid membrane (Fig. 1 C and D).
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This membrane likely derives from the most internal of the two membranes visible inside the virus particles, as expected from the infection process (opening of the stargate, followed by the fusion of the rst virus membrane with the vacuole membrane). These seeds then progressively turn into early virion factories recognizable as electron-dense nucleic acid-rich regions (18) isolated from the surrounding cytoplasm by an exclusion zone constituted of a mesh of brils 16 nm in diameter (Fig. 1 E and F). The biochemical nature of these brils remains to be characterized. Finally, fully mature virion factories release a large number of particles from their periphery where three stages of maturation are seen: empty assembled particles budding from the factory edges, bald particles lled with the nucleocapsid, and bercovered mature particles ready to exit the host cell (Fig. 1 G and H). The burst size of Megavirus-infected A. castellanii cells is approximately one-half the thousand virions released by those infected by Mimivirus.
Megavirus Overall Genome Structure and Gene Content. The genome of Megavirus is a linear double-stranded DNA molecule with a size of 1,259,197 bp (74.76% A+T), making it the largest described viral genome. The whole sequence was assembled and corrected at once from a dataset of 278,663 454-titanium reads combined with 42,288,396 Illumina Hiseq (paired-end) reads (SI Materials and Methods). We annotated 1,120 putative proteincoding sequences (CDSs) and 3 tRNAs (1 Trp, and 2 Leu). Megavirus CDSs range from 29 aa to 2,908 aa in length, for an average of 338.4 aa (median = 281.5 aa). The distance separating consecutive CDSs was short (114.5 nt in average), resulting into a very high coding density (90.14%). Eight hundred sixty-two of the 1120 (77%) predicted Megavirus CDS have homologs in Mimivirus, whereas 793 of the 979 (81%) Mimivirus CDSs (19) have homologs in Megavirus. Using the best reciprocal match criterion (SI Materials and Methods), Megavirus and Mimivirus were found to share 594 orthologous proteins exhibiting a broad distribution of similarity centered on an average of 50% identical residues (Table S1A and Figs. S1 and S2). Most likely inherited from a Megavirus/Mimivirus common ancestor, the corresponding gene set provides a minimal estimate of the core genome of ancestral Megaviridae. Fig. 2 illustrates at one glance both the overall similarity and divergence of the Megavirus and Mimivirus genomes. They display a large central region of colinearity extending from mg210 to mg804 in Megavirus, and L192 to R730 in Mimivirus. This region is solely disrupted by the inversion of a central 338-kb genome segment and the translocation of a 76-kb distal segment. Interestingly, one of the boundary of the inversion coincides with the slope reversal of the A+C excess prole, a position associated to the origin of replication in bacteria (Fig. S3). However, this quasiperfect colinearity abruptly vanishes at the two extremities of the Megavirus chromosome, respectively corresponding to 193 kb and 327 kb. Although these regions still encompass several hundred of homologous genes, their location and orientation appear extensively shufed (Fig. 2). The process leading to the total loss of colinearity at the genome extremities remains unknown, because it is not correlated with a local enrichment in transposases, or to more divergent orthologous sequences. For instance, the highest conservation (93% identical residues) was found for a predicted cholinesterase (Mimivirus L906/Megavirus mg981) located at the extremity of the chromosomes. Interestingly, the same pattern is observed when comparing two poxviruses exhibiting a level of sequence divergence comparable to the one between Megavirus and Mimivirus (e.g., DNA polymerases exhibiting 65% identical residues) (Fig. S4). This observation suggests that Poxviruses and Megaviridae, despite their considerable differences, might share a genome replication strategy (e.g., coupling replication with recombination) (20, 21)
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A
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Megavirus genomic positions

233000 550000 871000 1259197

979

1181549

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750

913000

Mimivirus CDSs

500

612000

250

279000

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Fig. 2. Comparison of Mimivirus and Megavirus genomes. (A) Colinearity of Mimivirus and Megavirus CDSs. Each dot symbolizes the best BLAST match (e value 105) between the CDSs of the two viruses, in the same orientation (blue) or in reverse orientation (red). (B) The distribution of homologous CDSs. Megavirus 594 CDSs with Mimivirus orthologs are shown in red. The 268 additional Megavirus CDSs with a signicant (nonreciprocal) match in Mimivirus CDSs are shown in blue. CDSs specic to Megavirus are shown in yellow. Orthologs clearly cluster in the central region, whereas the two other categories of CDSs (e.g., duplicated in or specic to Megavirus) tend to cluster at the extremities.

that favor the rearrangement, gain, or loss of genes at the extremities of viral chromosomes. Again, the dramatic 190-kb genome reduction exhibited by a recently described spontaneous Mimivirus mutant is mainly due to large deletions occurring at both ends of the genome (22).
Unique Transcriptional Features in Megaviridae. Previous analyses of the Mimivirus genome uncovered two distinctive features within its noncoding moiety. The rst one was the perfect conservation of the octameric motif AAAATTGA in front of 45% of Mimivirus CDSs (23). The second was the presence of unrelated palindromic sequences (capable of generating hairpins with a minimal stem length of 15 bp) at the 3 end of 72% of all Mimivirus mapped transcripts (12). The AAAATTGA motif was later shown to be strongly correlated to early expressed transcripts (24), and the predicted hairpins were demonstrated to serve as polyadenylation signals (12, 24). We examined the 100nt region upstream of the predicted start codon of the 1,120 Megavirus CDSs. Overall 446 (40%) were found to contain the exact AAAATTGA motif. This proportion was 33.8% (201/594) among Megavirus genes with orthologs in Mimivirus. For 170 of these (85%), the AAAATTGA sequence was also found upstream of Mimivirus orthologs. This ratio shows that (i ) Megavirus and Mimivirus are using the same motif to specify early gene expression, and (ii ) that the expression pattern of orthologous genes is globally well conserved. Detailed statistics on the distribution of early and late promoter elements are presented in Table S1B. Similarly, we searched the Megavirus genome 3 intergenic regions for palindromic sequences obeying the same constraints used to identify them in Mimivirus. Nine hundred fty-four Megavirus CDSs (85%) genes were found to be followed by
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a suitable predicted hairpin. The proportion was also 85% for Megavirus genes orthologous to Mimivirus genes exhibiting a hairpin. This correlation again suggests that these hairpins are the termination signal of Megavirus transcripts and that the transcript structures are well conserved between the two viruses. This prediction was experimentally veried by sequencing the 3 end of the cDNA of Megavirus mg464, orthologous to Mimivirus major capsid (MCP) gene, L425. These two CDSs, 78% identical at the nucleotide level, encode two proteins sharing 79% of identical residues. As shown in Fig. S5, the polyadenylation of the Megavirus MCP mRNA occurs within the predicted hairpin, albeit 14 nucleotides upstream of the site used in Mimivirus. Given the low level of sequence similarity between these 3 UTRs (<49% identical), this result provides the demonstration that the polyadenylation of Megaviridae transcripts is uniquely guided by secondary structure information, rather than a sequence signal. In keeping with the cytoplasmic localization of its replication cycle, Megavirus possesses orthologs of all of the genes previously predicted to encode components of Mimivirus transcription machinery: the two largest RNA polymerase subunits (mg373, mg339); 11 additional transcription factors: mg307, mg332, mg344, mg577, mg519, mg563, mg552, mg544, mg462, mg438, mg414, and one TATA-box binding like protein (mg435); and one polyA polymerase (mg561). All those genes are located in the middle of the colinear Megavirus/Mimivirus genome segments and exhibit a higher-than-average sequence similarity (64 10% of identical residues). A recurrent feature of viral genomes is the small proportion of genes for which a function can be predicted (e.g., Megavirus possesses 610 anonymous genes, 54.5% of its genome). It is then tempting to use the intensity of expression of a given gene as an indicator of its importance (i.e., essentiality), eventually to prioritize functional studies. Unexpectedly, this rationale appears to be false, because the expression level of Mimivirus genes exhibited no correlation with their conservation in Megavirus (Fig. S6).
Mimivirus Genes Unique Among dsDNA Viruses Are Conserved in Megavirus. Mimivirus was found to possess many genes never

Fig 3. Phylogenetic reconstruction of IleRS sequences. The midpoint-rooted neighbor-joining tree was generated from a 381-aa alignment of conserved positions. The tree topology and bootstrap values were very similar when using different alignment programs, reconstruction methods, and substitution models) (SI Materials and Methods and Materials and Methods). The Megavirus (star) and CroV IleRS sequences are branching off the eukaryote domain before the radiation of the cytoplasmic IleRSs.

before identied outside of cellular genomes (Table S1C). The most unexpected were those related to protein translation, a trademark of cellular organisms. Determining whether these oddities were anecdotal (e.g., due to random horizontal gene transfers) or fundamentally linked to the origin and evolution of all Megaviridae required the identication of a Mimivirus relative at a suitable evolutionary distance. The Mimivirus genome exhibits eight components central to protein translation, including the rst four AARS (ArgRS, CysRS, MetRS, and TyrRS) ever found in a virus (2). Megavirus orthologs were found for all of them (respectively encoded by mg804, mg807, mg771, mg907), strongly suggesting that they were present in the Megavirus/Mimivirus common ancestor. More unexpectedly, three additional AARS are found in the Megavirus genome. The AsnRS (mg743) is a member of the class-II AARS, whereas both the TrpRS (mg844) and the IleRS (mg358) are class-I AARS, like all of the ones found in Mimivirus. The signicance of this nding is twofold: rst, it demonstrates that viral AARS are not limited to class-I enzymes, and second, it makes the scenario of independent acquisition of these genes by HGT increasingly unlikely. Interestingly, the 730-kb genome of the Cafeteria roenbergensis virus, a very distant relative of Mimivirus, also encodes an IleRS (25). Both of these viral IleRS sequences are from the cytoplasmic archaeal/eukaryotic type, but do not exhibit phylogenetic afnity with a known eukaryotic clade (Fig. 3). The Megavirus AsnRS is branching at the origin of the mitochondrial enzyme type, before the radiation of the eukaryotes (Fig. S7A). The Megavirus TrpRS is of the archeal/eukaryotic type, again
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branching out before the radiation of the eukaryotes (Fig. S7B). In our opinion, these three additional Megavirus AARS were part of an ancestral Megaviridae genome and were subsequently lost in the Mimivirus lineage. These seven AARS were most likely the remnant of a complete set of 20 AARS inherited from an ancestral cellular genome. The Mimivirus genome was also found unusually packed with DNA repair enzymes, capable of correcting damages caused by UV light, ionizing radiation, or chemical mutagens (2). Megavirus exhibits orthologs to all these previously identied genes, including a specic type of mismatch repair enzyme MutS (mg543) common to large DNA viruses and specically abundant in the marine environment (26). In addition, Megavirus exhibits a DNA photolyase (mg779), an enzyme using the energy of light to repair thymidine dimers. A remnant of this gene, interrupted by a transposase, can be found in Mimivirus (R853R855). The presence of this functional photolyase in Megavirus, among its many other DNA repair enzymes, might participate to its increased resistance to UV irradiation: At a level sufcient to totally inactivate Mimivirus (30 min, 20 cm under a 30 W UV lamp, 253 nm), close to 100% of Megavirus particles remained infectious. One hour of irradiation at the same intensity was required to cause a 90% decrease in Megavirus infectivity. The three types of topoisomerases found in Mimivirus: one of type II (R480), a bacterial-like type I (L221), and a pox-like type I (R194) are also found in Megavirus (mg403, mg323, mg859). However, Megavirus mg859 is a much closer homolog (50% identical) to the topoisomerase 1b found in CroV (crov152), suggesting that the Mimivirus R194 gene has a distinct origin. Mimivirus exhibited a number of enzymes related to protein folding, and various sugar and amino acid manipulating enzymes not usually found in viruses. With the exception of two enzymes of the cholesterol biosynthetic pathways, all these genes have well conserved homologs in Megavirus (Table S1C), showing that they are part of the Megaviridae core gene set.
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Finally, Megavirus mg431 encodes a uridine monophosphate kinase, an enzyme previously undescribed in a DNA virus. This enzyme is 44% identical to its closest homologs in bacteria where it is the rate-limiting enzyme of the pyrimidine salvage pathway (Table S1C).
Lineage-Specic Genome Features. The progressive accumulation of point mutations (random drift) appears to be the main cause of the divergence between the Megavirus and Mimivirus genomes. The orthologous CDSs, as well as the orthologous intergenic regions, exhibit an average similarity of 65% identical nucleotides, higher than the 50% identical residues shared by orthologous proteins (Fig. S1). Two hundred fty-eight Megavirus CDSs exhibit no obvious homolog in Mimivirus and, reciprocally, 186 Mimivirus CDSs have no homolog in Megavirus. More than 85% of these lineagespecic CDSs correspond to proteins without functional predictions. They appear to cluster at the ends of the Megavirus chromosome (Fig. 2B). These CDSs might also correspond to fast evolving proteins pushed below our similarity threshold (e < 105). More likely, they correspond to lineage specic losses along the Mimivirus or Megaviruses branches. Genome reduction is a universal, fast, and irreversible process among intracellular parasitic microorganisms (27, 28) that might also apply to the evolution of Megaviridae from their more complex ancestors (3). An alternate scenario would attribute the 258 Megavirus private genes to horizontal gene transfers (HGT) that occurred after the divergence of the Megavirus/Mimivirus branches. However, when screened against the NR protein sequence database, these Megavirus private genes exhibited a much lower percentage of matches (17% vs. 52%) than the genes with orthologs in Mimivirus. Moreover, the few matching genes (44/258) exhibited no peculiar afnity with potential gene donors. This result argues against recent HGTs (at least from known viruses or cellular organisms) as the major cause in the difference in gene content between Megavirus and Mimivirus. The difference in the gene content of the two Megaviridae is also due to the differential expansion or reduction of large paralogous families. Six hundred fty-two of the Megavirus predicted proteins are in one copy (not matching elsewhere in the genome at e < 105), representing 58.2% of the gene content. The corresponding number is 585 (59.8%) for Mimivirus. The genome of Megavirus is thus truly more complex than the one of Mimivirus and not simply repeat-rich or more redundant. Although the distributions of single- vs. multiple-copy genes are globally similar for the two viruses, specic protein families experienced lineage specic expansions. For instance, the third largest Mimivirus gene cluster (referred to as the N172 L cluster; ref. 29) corresponding to 14 paralogues is represented by a single copy in Megavirus. Conversely, the set of 10 FNIP repeat-containing proteins constituting the N165 cluster in Mimivirus (29) is inated to 55 members (mg34 paralogues) in Megavirus. Finally, only a few differences between the Megavirus and Mimivirus genomes resulted from lineage-specic movements of mobile elements. Megavirus exhibits ve segmented CDSs. The major capsid protein (mg464, with two introns), the largest RNA polymerase subunit (mg373, with one intron), and the DNA polymerase (mg582, with one intein) exhibit the same exact topology than their Mimivirus orthologs. Orf mg500, encoding a HSP70 chaperonin-like protein, contains a type I intron, whereas its Mimivirus ortholog (L393) does not. The second largest RNA polymerase subunit (mg339 orthologous to Mimivirus L244) exhibited the most intricate rearrangements with changes in the numbers and locations of introns, and the insertion of an intein (30) (Fig. S8). The duplication and movement of transposases (ve in Mimivirus; only two in Megavirus)

also caused the disruption of a Mimivirus photolyase (R853R855) and a second photolyase paralogue in Megavirus (mg400). Conclusion We present the analysis of a Mimivirus relative isolated from a marine environment. With a genome 77 kb (6.5%) larger than Mimivirus, Megavirus shows that the limit is not yet reached in the complexity of giant DNA viruses infecting acanthamoeba or other phagotrophs from yet-uncharted protozoan clades. The potential origin of giant mimivirus-like genomes has been hotly debated, basically opposing two views. One is depicting Mimivirus as an extremely efcient gene pickpocket, explaining its large genome as the result of considerable HGTs from its host, bacteria, or other viruses (7, 20). This scenario has been criticized in detail elsewhere (4, 8, 14). The opposite view claims that the level of HGT remained marginal (10%) and that most of the Mimivirus genes originated from an even more complex viral ancestor, itself eventually derived from an ancestral cellular genome (4, 8). The origin of the many cell-specic functions uniquely encoded by Mimivirus is central to this debate. Thanks to their optimal evolutionary distance, the comparison between the Mimivirus and Megavirus genomes allowed us to delineate a common gene set most likely derived from their common ancestor. This ancestral gene set was found to include most of the Mimivirus cell-like key functions, in particular the ones associated to protein translation. Moreover, three additional AARS were identied in Megavirus, to our opinion ruling out HGT (i.e., seven independent gene acquisitions) as the origin of these genes. In contrast, our analyses corroborate the scenario whereby the last Megaviridae common ancestor originated from a cellular organism (thus endowed of a translation apparatus), from which todays Megaviridae (Fig. S7C) mostly derived by a number of lineage specic genome reduction events. The analysis of other distant Megaviridae genomes will provide an increasingly clearer picture of this evolutionary process. Materials and Methods
Megavirus chilensis was produced in A. castellanii and puried as described for Mimivirus (12) where the discontinuous gradient of CsCl was replaced by a sucrose gradient (10/20/30/40%), the viral pellet resuspended in PBS was layered on, and was centrifuged at 5,000 g for 30 min. DNA Extraction. The puried Megavirus pellet was resuspended in 50 mM TrisHCl at pH 7.5 and incubated for 30 min at 37 C in the presence of 1.25 mg/mL lysozyme. Lysis was performed by adding 0.1 mg/mL proteinase K incubated 30 min at 55 C, 1% laurylsulfate 30 min at room temperature, and 10 mM DTT overnight at room temperature. After phenol-chloroforme extraction and alcohol precipitation, the recovered DNA was resuspended in RNase-Dnase free water. Electron Microscopy. The A. castellanii-infected cells were washed in PBS and resuspended in 2% paraformaldehyde 0.5% glutaraldehyde in PBS for 1 h at room temperature. After three washes in PBS, cell pellet was xed in 2% osmium tetroxyde, washed once in PBS, dehydrated in 70, 95, and 100% ethanol, and embedded in Epon-812. Ultrathin sections were poststained with 4% uranyl acetate and lead citrate and were observed by using FEI Tecnai TEM operating at 120 kV. SI Materials and Methods contains the details of the procedures used for the virus isolation and its genome sequencing, assembly, and annotation. ACKNOWLEDGMENTS. We thank A. Bernadac and P. Bergam for help with the electron microscopy, D. Byrne and A. Lartigue for sequence validations, A. Chambouvet for early culture attempts, and Dr. Moriah Szpara for reading the manuscript. DNA sequencing was performed by the University of Oklahomas Advanced Center for Genome Technology (Prof. B. Roe) and GATC Biotech. This work was supported by the Centre National de la Recherche Scientique, Agence Nationale de la Recherche Grant ANRBLAN08-0089, and a fellowship from the Direction Gnrale de lArmement (to D.A.). The sampling expedition was sponsored by the ASSEMBLE initiative (European Commissions seventh framework program).

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1. La Scola B, et al. (2003) A giant virus in amoebae. Science 299:2033. 2. Raoult D, et al. (2004) The 1.2-megabase genome sequence of Mimivirus. Science 306: 13441350. 3. Claverie JM (2006) Viruses take center stage in cellular evolution. Genome Biol 7:110. 4. Claverie JM, Abergel C (2010) Mimivirus: The emerging paradox of quasi-autonomous viruses. Trends Genet 26:431437. 5. Raoult D, Forterre P (2008) Redening viruses: Lessons from Mimivirus. Nat Rev Microbiol 6:315319. 6. Villarreal LP, Witzany G (2010) Viruses are essential agents within the roots and stem of the tree of life. J Theor Biol 262:698710. 7. Moreira D, Lpez-Garca P (2009) Ten reasons to exclude viruses from the tree of life. Nat Rev Microbiol 7:306311. 8. Claverie JM, Ogata H (2009) Ten good reasons not to exclude giruses from the evolutionary picture. Nat Rev Microbiol, 7:615, author reply 615. 9. Claverie JM, et al. (2006) Mimivirus and the emerging concept of giant virus. Virus Res 117:133144. 10. Abergel C, Rudinger-Thirion J, Gieg R, Claverie JM (2007) Virus-encoded aminoacyltRNA synthetases: Structural and functional characterization of mimivirus TyrRS and MetRS. J Virol 81:1240612417. 11. Zauberman N, et al. (2008) Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. PLoS Biol 6:e114. 12. Byrne D, et al. (2009) The polyadenylation site of Mimivirus transcripts obeys a stringent hairpin rule. Genome Res 19:12331242. 13. La Scola B, et al. (2008) The virophage as a unique parasite of the giant mimivirus. Nature 455:100104. 14. Claverie JM, Abergel C (2009) Mimivirus and its virophage. Annu Rev Genet 43:4966. 15. Colson P, et al. (2011) Viruses with more than 1000 genes: Mamavirus, a new Acanthamoeba castellanii mimivirus strain, and reannotation of mimivirus genes. Genome Biol Evol 3:737742. 16. La Scola B, et al. (2010) Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry. Intervirology 53:344353.

17. Xiao C, et al. (2009) Structural studies of the giant mimivirus. PLoS Biol 7:e92. 18. Mutsa Y, Zauberman N, Sabanay I, Minsky A (2010) Vaccinia-like cytoplasmic replication of the giant Mimivirus. Proc Natl Acad Sci USA 107:59785982. 19. Legendre M, Santini S, Rico A, Abergel C, Claverie JM (2011) Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing. Virol J 8:99. 20. File J, Siguier P, Chandler M (2007) I am what I eat and I eat what I am: Acquisition of bacterial genes by giant viruses. Trends Genet 23:1015. 21. Esteban DJ, Hutchinson AP (2011) Genes in the terminal regions of orthopoxvirus genomes experience adaptive molecular evolution. BMC Genomics 12:261. 22. Boyer M, et al. (2011) Mimivirus shows dramatic genome reduction after intraamoebal culture. Proc Natl Acad Sci USA 108:1029610301. 23. Suhre K, Audic S, Claverie JM (2005) Mimivirus gene promoters exhibit an unprecedented conservation among all eukaryotes. Proc Natl Acad Sci USA 102: 1468914693. 24. Legendre M, et al. (2010) mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus. Genome Res 20:664674. 25. Fischer MG, Allen MJ, Wilson WH, Suttle CA (2010) Giant virus with a remarkable complement of genes infects marine zooplankton. Proc Natl Acad Sci USA 107: 1950819513. 26. Ogata H, et al. (2011) Two new subfamilies of DNA mismatch repair proteins (MutS) specically abundant in the marine environment. ISME J 5:11431151. 27. Merhej V, Royer-Carenzi M, Pontarotti P, Raoult D (2009) Massive comparative genomic analysis reveals convergent evolution of specialized bacteria. Biol Direct 4:13. 28. Blanc G, et al. (2007) Reductive genome evolution from the mother of Rickettsia. PLoS Genet 3:e14. 29. Suhre K (2005) Gene and genome duplication in Acanthamoeba polyphaga Mimivirus. J Virol 79:1409514101. 30. Perler FB (2002) InBase: The intein database. Nucleic Acids Res 30:383384.

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Supporting Information
Arslan et al. 10.1073/pnas.1110889108
SI Materials and Methods Virus Isolation. Megavirus chilensis was isolated from coastal waters in front of the ECIM marine station from Las Cruces, Chile. One liter of seawater was supplemented with 4% of rice media (supernatant obtained after autoclaving 1 L of seawater with 40 grains of rice) and let to incubate for 1 mo in the dark at room temperature. The rationale of such procedure is to get rid of the phototrophic microorganisms while allowing the heterotrophic bacteria to grow for a while, when they then feed the phagocytic/heterotrophic protozoans that nally expand to a population allowing eventual viruses to multiply (1). Seawater with rice medium was then ltered rst through a polycarbonate Isopore membrane lter of 1.2-m pore size and then through 0.2-m pore size membrane lter (RTTP04700, GTTP04700; Millipore). The 0.2-m pore size membrane was then treated with gentamicin at 1 mg/mL nal concentration, 10% penicillin/ streptomycin and 5% fungizone for 3 d. Supernatant was inoculated to several acanthamoeba species cultured in microplates and monitored for cell lysis.
Giant Virus Naming. We believe it is useful and desirable that the name of a newly isolated microorganism convey some of its most distinctive properties. After the initial naming of Mimivirus (for microbe mimicking), already not a very good name because the prex mimi does not convey a helpful scientic notion, newly isolated related viruses are receiving increasingly random/funny names such as Mamavirus, Moumouvirus, Courdovirus, and Terra (2). Although it is traditionally the privilege of the rst authors describing a new microbe to give it whatever name of their choosing, we believe the current trend is counterproductive and should give way to more informative names. With the few examples now at hand, it is clear that a distinctive feature of the above giant viruses (or of their close ancestors) is to possess genome in excess of a megabase. Hence, the term Megavirus, and the proposed family/genus Megaviridae that will be proposed to the International Committee on Taxonomy of Viruses. Chilensis then refers to the location where this virus was rst isolated. Finally, we broke with tradition not incorporating the hosts species to the virus name. This decision is justied by the fact that Megavirus and other Mimivirus relatives are capable of replicating in a variety of acanthamoeba species, whereas the phagocytic protozoan that is the natural host of Megavirus chilensis is not known, as will be the situation for most viruses isolated from the environment using the acanthamoeba coculture protocol. Genome Assembly. The Megavirus genome was assembled by using a combination of 454-titanium and Illumina Hiseq paired-end reads. We rst assembled the 42,288,396 Hiseq paired-end reads by using the Velvet assembler (3) with the following parameters:
1. Massana R, del Campo J, Dinter C, Sommaruga R (2007) Crash of a population of the marine heterotrophic agellate Cafeteria roenbergensis by viral infection. Environ Microbiol 9:2660e2669. 2. La Scola B, et al. (2010) Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry. Intervirology 53:344e353. 3. Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821e829. 4. Chevreux B, Wetter T, Suhai S (1999) Genome sequence assembly using trace signals and additional sequence information. Computer science and biology. Proceedings of the German Conference on Bioinformatics 99:45e56. 5. Boneld JK, Whitwham A (2010) Gap5editing the billion fragment sequence assembly. Bioinformatics 26:1699e1703. 6. Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: A self-training method for prediction of gene starts in microbial genomes. Implications for nding sequence motifs in regulatory regions. Nucleic Acids Res 29:2607e2618.

k = 95, ins_length = 280, cov_cutoff = 200 and exp_cov = 382. We next mapped the 278,663 454-titanium reads onto the assembled contigs by using Mira (4) to extend them. Gap5 (5) software was used to join the resulting overlapping contigs into a single one. We nally remapped the Hiseq reads at high stringency to correct sequencing errors. The 454 technology generated a large number of local errors due to the miscalling of homopolymeric sequences in the Megavirus A+T rich genome. Steep drops in the Illumina read coverage were used to guide the visual inspection of the sequence and its manual correction (usually a single A or T nucleotide insertion or deletion). The total Illumina data used for this nishing step corresponds to 1/ 10th of a ow cell channel used in a multiplexed fashion with nine other unrelated sequencing projects. A few positions were conrmed by PCR followed by Sanger sequencing. The nal Megavirus genome sequence corresponds to a single 1,259,197nt-long contig.
Gene Annotation. The Megavirus protein coding regions (CDSs) were identied by using the GeneMarkS algorithm (6). Transfer RNAs were searched by using tRNAscan-SE (7) with the general tRNA model. The functional assignment of these predicted Megavirus genes was performed by using a combination of BlastP searches against public databases using an e value threshold of 105 and protein motif identication using Interproscan (8). Megavirus/Mimivirus orthologous gene pairs were dened based on the best-reciprocal blast hit criterion between the two proteomes, again using BlastP at an e value threshold of 105. Megavirus (respectively Mimivirus) paralogues correspond to predicted proteins exhibiting BlastP similarity within the Mimivirus (respectively Megavirus) proteome at the same threshold but failing the reciprocal best match criterion. These correspond to Megavirus/Mimivirus specic gene duplications. The last category of CDSs, specic to each virus, corresponds to those not exhibiting a BlastP hit at the conservative e value threshold of 105. Phylogenetic Analysis. The most similar homologs of Megavirus aminoacyl tRNA synthetases were rst identied by using the Blast-Explorer tool (9) on the Phylogeny.fr (10) server. A subset of sequences was selected based on the alignment quality (preserving enough informative positions) and their phylogenetic distribution among the main domains (Archaea, Eukarya, Eubacteria). An optimal multiple alignment was then computed by using MAFFT version 6 (11) on the CBRC-AIST server (mafft. cbrc.jp/alignment/server/). Several trees were then reconstructed from this alignment by using a simple neighbor-joining algorithm (with the JTT model) or PhyML (with the WAG model) (10). The topology of the reconstructed trees and the condence values were very similar for both methods.
7. Schattner P, Brooks AN, Lowe TM (2005) The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33(Web Server issue):W686W689. 8. Hunter S, et al. (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37(Database issue):D211eD215. 9. Dereeper A, Audic S, Claverie JM, Blanc G (2010) BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol 10:8. 10. Dereeper A, et al. (2008) Phylogeny.fr: Robust phylogenetic analysis for the nonspecialist. Nucleic Acids Res 36(Web Server issue):W465W469. 11. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286e298.

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A
40 50

Conservation (nt.)

Genic
30

Counts

Intergenic

0 0

10

20

20

40

60

80

100

Conservation (%)

B
Counts
5 10 15

Conservation (aa.)

20

40

60

80

100

Conservation (%)
Fig. S1. Sequence divergence between Megavirus and Mimivirus. (A) Orthologous genes and intergenic nucleotide sequences. (B) Orthologous protein sequences. Notice that the nucleotide sequences exhibit more similarity than the amino acid sequences, in part due to the large nucleotide composition bias (75% A+T).

Megavirus 1120

Mimivirus 979

Megavirus Megavirus match only Mimivirus 258 268

Orthologs

Mimivirus Mimivirus match only Megavirus 199 186

594

Fig. S2.

Comparison of Mimivirus and Megavirus gene contents. The various subsets in this Venn diagram are not represented to scale.

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A+C cumulative excess

A+C excess

10000 0

8000

6000

4000

2000

200000

400000

600000

800000

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Genomic position

Fig. S3. A+C excess prole of the Megavirus genome. The slope reversal (red arrow) approximately coincides with one of the boundaries of the large inverted segment disrupting the colinearity between the Megavirus and Mimivirus genomes (Fig. 2).

Rabbitpox vs. Variola

165 124 82 41 1 1 49 98 147 196 170 128 85 42 1 1

Deerpox vs. Variola

49

98

147

196

Fig. S4. Diagonal similarity plots (dot plots) of pairs of poxvirus genomes. These pairs of viruses exhibit global sequence similarity levels comparable to the one exhibited by the Mimivirus/Megavirus pair (DNA polymerase sharing 65% identical residues). The colinearity is conserved in the central region of the genomes and abruptly vanishes at both ends of the chromosomes.

Stop

polyA

B
MCP_Megavirus Hairpin

polyA

MCP_Mimivirus Hairpin

Fig. S5. (A) Optimal alignment of the 3 UTR regions of the major capsid protein transcripts in Mimivirus and Megavirus. The two sequences only share 49% identical nucleotides. (B) Predicted hairpin structures and experimentally validated polyadenylation sites (red arrows).

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A
Gene expression
8 6
R2 0.003

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Gene expression
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Solid data 1
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60 80 100

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Conservation Conserved genes (%) Conserved genes (%)

Conservation Conserved genes (%)

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Gene expression

Gene expression

20 40 60 80

20 40 60 80

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Gene expression

Fig. S6. Absence of correlation between the level of expression of Mimivirus genes and their conservation in Megavirus. (A) Shown is gene expression level (log scale) vs. % of identical residues between orthologs in three independent transcriptome datasets. Mimivirus gene expression (red; Left) was measured based on 454 mRNA sequence reads (1) and a total RNA dataset by using Solid sequencing technology (green; Right) (2). A third Solid dataset (ner grid through the infection cycle) with 196 million reads from total RNA is also shown (blue; Center). (B) Percentage of genes with a Megavirus ortholog vs. their expression level in Mimivirus distributed in 20 bins from the lowest (blue) to the highest (red).
1. Legendre M, et al. (2010) mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus. Genome Res 20:664e674. 2. Legendre M, Santini S, Rico A, Abergel C, Claverie JM (2011) Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing. Virol J 8:99.

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56 100

P.aerophilum.Archaea.Crenarchaeota.Thermoprotei T.volcanium.Archaea.Euryarchaeota.Thermoplasmata 56 100 A.saccharovorans.Archaea.Crenarchaeota.Thermoprotei T.gammatolerans.Archaea.Euryarchaeota.Thermococci P.horikoshii.Archaea.Euryarchaeota.Thermococci M.paludicola.Archaea.Euryarchaeota.Methanomicrobi Megavirus.AsnRS.mg743 O.lucimarinus.Eukaryota.Viridiplantae P.patens.Eukaryota.Viridiplantae P.tricornutum.Eukaryota.stramenopiles V.fischeri.Eubacteria.Gammaproteobacteria.Vibrionales N.punctiforme.Eubacteria.Cyanobacteria

56

71 84

100 88

S.elongatus.Eubacteria.Cyanobacteria P.acanthamoebae.Eubacteria.Chlamydiae P.gingivalis.Eubacteria.Bacteroidetes L.buccalis.Eubacteria.Fusobacteria C.perfringens.Eubacteria.Firmicutes.Clostridia C.saccharolyticum.Eubacteria.Firmicutes.Clostridia 100 D.purpureum.Eukaryota.Amoebozoa D.discoideum.Eukaryota.Amoebozoa N.gruberi.Eukaryota.Heterolobosea T.adhaerens.Eukaryota.Metazoa.Placozoa

97 90 68

65 93 100 100

D.rerio.Eukaryota.mitochondria G.gallus.Eukaryota.mitochondria T.guttata.Eukaryota.mitochondria

0.05

Phyco.OtV-1

Phyco.O sV-5

Phy co.O tV-2 Ph yco .Ol V -1 Ph yc o.M pV -1

C
x.F Po

x apo .Tan Pox ma

99

10 100 0

Po x. O Un rf c.O LV -1 Un c.O LV 10 -2 0
Unc.C eV

99

97

100

67

yxo x.M Po

y Ph

-1 pV .B o c
yc Ph o.P

x lpo ow

83 R4 -F V C

76

0 10

.AT yco Ph

-1 CV

0 10

Unc.PpV
Unc.CroV
Unc.P oV

100

98
10 0
100 100

CV-1 o.PB Phyc

V-AR Phyco.PBC 158

90

Phyco.PBCV-NY2A

Phyco .EhV-8
100
sV Ph yco .Es V-1 Ph yc o. Ha VD NA -1 Phy co.F

60

s viru ega

As fa r.A He SF rpe V s. H um an Her -6 pes .Hu ma 10 n-7 0

a-2 err c.T s Un ru ivi im M

0 10 0 0 1
0 10
76

100

100

100

us vi r ne an us us La Vir c. ille Un rse a c.M Un V

96
ru

I o.W Irid

IV Irido.I

Irido.LDV-Chin

Irido.ISKNV

Herpe s.Rod ent.P e

-3

Fig. S7. Phylogenetic analysis of selected megavirus protein sequences. All alignments were computed by using the default option of the MAFFT server (1). (A) AsnRS (mg743): This neighbor-joining tree was computed from the 300 conserved positions. The tree is rooted on the Archaea branch. The nodes are labeled with their bootstrap values when >50. A very similar tree was computed by using the default option of the Phylogeny.fr server (alignment with MUSCLE and tree reconstruction by PhyML) (2). Despite being one of the least canonical of the AARS (sensu Woese et al.; see ref. 3), the Megavirus AsnRS nicely separates the archeal enzymes from all of the eukaryotic (including mitochondrial) types known to intermix with bacterial enzymes. (B) TrpRSs (mg844): This PhyML tree (rooted on the Archaea branch) was computed on the Phylogeny.fr server (2) from 327 conserved positions. The nodes are labeled with their bootstrap values when >50. A similar tree was computed by using the default option of the MAFFT server (tree reconstruction by neighbor joining). The Megavirus TrpRS is branching off the eukaryotic domain before the radiation of all clades. (C ) DNA polymerase (mg582). This neighbor-joining tree was computed from the 523 ungapped positions of an alignment of 38 DNA polymerases sequences from the main large DNA virus families: Poxviridae, Iridoviridae, Herpesviridae, Asfarviridae, Marseilleviridae, and Phycodnaviridae. Megavirus, Mimivirus, and Terra-2 form a tight cluster (in red) within a larger well supported group of unclassied (unc) aquatic viruses including those with the largest known genome sizes. This group (red and green) shares a number of distinctive features and is proposed to constitute a new family: the Megaviridae. PoV, Pyramimonas orientalis virus (560 kb); CroV, Cafeteria roenbergensis virus (730 kb); PpV, Phaeocystis pouchetii virus (485 kb); CeV, Chrysochromulina ericina virus (510 kb); OLV, Organic Lake virus (1 and 2); HaVDNA, Heterosigma akashiwo DNA virus; PBCV, Paramecium bursaria Chlorella virus; ATCV, Acanthocystis turfacea Chlorella virus; BpV, Bathycoccus sp. RCC1105 virus; OsV, Ostreococcus virus; Legend continued on following page

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OtV, Ostreococcus tauri virus; MpV, Micromonas sp. RCC1109 virus; OlV, Ostreococcus lucimarinus virus; EhV, Emiliania huxleyi virus; FsV, Feldmannia species virus; EsV, Ectocarpus siliculosus virus; WIV, Wiseana iridescent virus; IIV, Invertebrate iridescent virus; LDV, Lymphocystis disease virus; ISKNV, Infectious spleen and kidney necrosis virus; ASFV, African swine fever virus.
1. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286e298. 2. Dereeper A, et al. (2008) Phylogeny.fr: Robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36(Web Server issue):W465W469. 3. Woese CR, Olsen GJ, Ibba M, Sll D (2000) Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiol Mol Biol Rev 64:202e236.

Mimivirus L244
1548
175

1197

460

866

125

681

320

787

78

125

993

325

288

L247

L246

L245

mg341
1548
146

mg340
340

909

1661

1084

34

135

1071

394

332

Megavirus mg339
Fig. S8. Complex reorganization of the RPB2 gene in Megavirus. Exons are shown in blue, introns in brown, and the intein in green. Numbers correspond to DNA segment sizes in base pairs. The rst exon is the only gene segment for which there is a one-to-one correspondence between Mimivirus and Megavirus. The second Megavirus exon incorporates most of the coding sequence of Mimivirus second and third exons, in addition to a 1,084-bp intein (1). The purple and orange boxes correspond to the last (H) and the rst (S) amino acid of the N-terminal and C-terminal exteins, respectively. The third Megavirus exon corresponds to the end of the Mimivirus third exon and its entire fourth exon.

1. Perler FB (2002) InBase: The intein database. Nucleic Acids Res 30:383e384.

Other Supporting Information Files

Table S1 (DOC)

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