Mobile genetic elements (e.g., transposable elements and viruses) display significant diversity with various life cycles, but how novel elements emerge remains obscure. Here, we report a giant (180-kb long) transposon, Teratorn, originally identified in the genome of medaka, Oryzias latipes. Teratorn belongs to the piggyBac superfamily and retains the transposition activity. Remarkably, Teratorn is largely derived from a herpesvirus of the Alloherpesviridae family that could infect fish and amphibians. Genomic survey of Teratorn-like elements reveals that some of them exist as a fused form between piggyBac transposon and herpesvirus genome in teleosts, implying the generality of transposon-herpesvirus fusion. We propose that Teratorn was created by a unique fusion of DNA transposon and herpesvirus, leading to life cycle shift. Our study supports the idea that recombination is the key event in generation of novel mobile genetic elements.
Mobile genetic elements are genetic entities that can move inside genomes of cellular organisms or between cells. These elements include transposable elements (TEs) and viruses as well as plasmids and self-splicing elements in bacteria1. All elements share the ability to parasitize cells and propagate by usurping the host machinery. In addition, all elements contain “hallmark genes” associated with replication (e.g., polymerase, helicase, transposase) and structural constitution (e.g., capsid protein), which are not shared by cellular organisms2. Despite those common characteristics, each class of elements has a different genome constitution (either DNA or RNA of double-stranded (ds) or single-stranded (ss) nucleotides), replication manner (e.g., DNA polymerization, RNA-dependent RNA transcription, reverse transcription) and life cycle (i.e., TEs for persistent stay and propagation inside host genomes and viruses for transient stay and massive transfer between cells). How does this diversity arise and what mechanism underlies this process? It has been proposed that they are evolutionarily linked with each other and that recombination between distantly related elements leads to the emergence of novel mobile genetic elements2. Indeed, in prokaryotic mobile genetic elements, massive gene exchange between phages, transposons and plasmids occurred, making the borders of different classes of elements ambiguous3. The existence of network-like evolutionary relationships has also been proposed for eukaryotic mobile genetic elements2, 4. One good example is the transition from LTR retrotransposons to retroviruses and vice versa5,6,7. In addition, phylogenetic analyses imply that, for other elements, the transition events between transposon and virus occurred in the distant past2, 8, 9. However, due to the low sequence similarity between diverged mobile elements and the incongruence of phylogeny for each gene10, 11, in most cases it remains ambiguous which element is actually a result of recombination events, leading to a transition in life cycle. Here, we report the analysis of a DNA transposon “Teratorn” in a small teleost fish medaka (Oryzias latipes), and provide direct evidence for genetic interaction between different classes of mobile elements, a fusion of a cut-and-paste DNA transposon and DNA virus.
This transposon was originally identified as an inserted DNA element in the medaka spontaneous mutant Double anal fin (Da)12. It was initially named Albatross, but recently renamed Teratorn (after the extinct group of very large birds of prey) to avoid confusion with a gene of the same name encoding a Fas-binding factor12. In our previous study, partial sequencing suggested that Teratorn is a DNA transposon that moves in a “cut-and-paste” manner, since it contains 18-bp terminal inverted repeats (TIR) at its termini and is flanked by target site duplications (TSDs). Interestingly, Teratorn was unusual in its relatively large size (at least 41-kb long12) for a transposon13, 14. We supposed that Teratorn is a giant DNA transposon with a unique evolutionary origin, life cycle and impact on the host genome. However, due to the presence of multiple copies as repetitive sequences, we failed to obtain the entire structure in the published draft genome constructed by shotgun sequencing15.
In this study, we perform bacterial artificial chromosome (BAC)-based full-length sequencing of several Teratorn copies and find that Teratorn is 180-kb long, which is significantly larger than any other transposon reported so far. We identify a transposase gene homologous to piggyBac superfamily DNA transposons, and detect its transposition activity both in vitro and in vivo. Surprisingly, Teratorn also contains the full genome of a herpesvirus, related to the family Alloherpesviridae, which infects fish and amphibians. In addition, genomic survey suggests that piggyBac-herpesvirus fusion also took place in yellow croaker (Larimichthys crocea) and nile tilapia (Oreochromis niloticus), implying the generality of transposon-herpesvirus fusion. We propose that Teratorn was created by a fusion of transposon and herpesvirus and as such, acquired a “bivalent” life form, behaving both as a transposon and a virus, assuring long-term survival in the host.
Teratorn is ~180-kb long
We screened a BAC library of the Hd-rR strain to obtain clones containing the entire Teratorn. Using PacBio long-read sequencing we determined the full-length sequences of six Teratorn individual copies (Supplementary Fig. 1a) and found that in five clones, Teratorn was ~ 180-kb and in one clone it was ~155-kb (Fig. 1a, b, Supplementary Fig. 1b, c. Supplementary Data 1). The 155 kb clone is a shorter version of the other five with a partial deletion. In addition, we identified another subtype of Teratorn, which exhibits ~88% sequence identity but little similarity in the repetitive region including terminal region, by blast search of the full-length Teratorn sequence against a new, higher quality, version of the medaka genome (Fig. 1c, d, Supplementary Fig. 2) (http://utgenome.org/medaka_v2/#!Top.md). Structurally, the two subtypes are very similar to each other. Except for an 80-kb long inversion in the middle, the overall structure, such as the size, gene order (see below) and position of long repeats, is conserved (Fig. 1b, c, Supplementary Fig. 2). Thus, we concluded that they were derived from a common ancestor, and referred to the initially identified copies as Teratorn ‘‘subtype 1’’ and the second one as Teratorn ‘‘subtype 2’’. Blast search and southern blot analysis of their terminal sequences suggested that there exist 30–40 copies of subtype 1 and ~5 of subtype 2 per haploid genome of the medaka Hd-rR inbred strain (Fig. 1e).
Teratorn belongs to the piggyBac superfamily
We first looked for a gene encoding transposase and found a gene homologous to piggyBac transposase inside Teratorn (magenta arrow in Fig. 1b for subtype 1, Supplementary Fig. 2a for subtype 2). piggyBac is one of the major “cut-and-paste” DNA transposon superfamilies and is widely distributed among eukaryotes, especially in metazoans13. The transposase gene of Teratorn exhibits high sequence similarity with other piggyBac superfamily transposase genes with essential amino-acid residues strictly conserved, e.g., the catalytic domain (DDD motif), especially the four aspartic acid residues required for the transposition reaction (Supplementary Fig. 3a)16. Furthermore, the sequence composition of TIRs and TSDs at its termini follows the rule of the piggyBac superfamily; piggyBac transposons contain TIRs with 12–19 bp, beginning with a “CCYT” motif, and preferentially target TTAA (Supplementary Fig. 3b)13, 14. These characteristics indicate that Teratorn belongs to piggyBac superfamily. Teratorn is thus the biggest among known transposons, as the largest transposons reported so far were a Polinton-like DNA transposon (up to 25-kb long)8, 17 and a Gypsy-like LTR retrotransposon (up to 25-kb long)18.
Teratorn contains the genome of a herpesvirus
In addition to the transposase gene, GENSCAN19 and GeneMarkS20 predicted ~90 putative genes, covering more than 60% of Teratorn (Fig. 1b, Supplementary Fig. 2a, Supplementary Tables 1 and 2, Supplementary Data 2). Remarkably, among the predicted genes, 17 genes show sequence similarity to those of herpesviruses (E-value < 0.01; blue arrows in Fig. 1b, Supplementary Fig. 2a, Supplementary Tables 1 and 2). Those include genes required for virus propagation, such as DNA replication (DNA polymerase, primase and UL21 homolog DNA helicase), virion maturation (capsid maturation protease), packaging of viral DNA (large subunit terminase) and virus structural proteins (major capsid protein, subunit 2 capsid triplex protein and envelope glycoprotein), and are known to be essential for the life cycle of herpesviruses.
Besides the essential genes, ~20 genes show no sequence similarity to those of other herpesviruses but are found in other organisms, which may have been secondarily obtained from infected host genomes or other sources such as bacteria and viruses (yellow arrows in Fig. 1b, Supplementary Fig. 2a, Supplementary Tables 1 and 2). Intriguingly, most of them appear to function in regulation of immune response or cell proliferation in hosts. The former includes cell surface proteins (CD276 antigen-like, CXCR-like, TNFR-like) and immune signal transduction factors (ZFP36-like, TARBP-like), while the latter includes cell cycle regulator (CDK-like), apoptosis inhibitor (Mcl1-like), and oncogene (pim-like). The existence of these gene repertoires is a common feature of herpesvirus species that infect the hematopoietic cell lineages (like beta herpesviruses and gamma herpesviruses of amniotes)21. The other ~50 genes (gray arrows in Fig. 1b, Supplementary Fig. 2a) show no significant blast hit against the current non-redundant protein database. Given that large DNA viruses tend to carry many unknown genes in addition to essential ones22, 23, it is likely that all predicted genes present in Teratorn (except for the tranposase gene) constitute a whole herpesvirus genome.
Herpesviruses are double-stranded DNA viruses that infect a wide variety of vertebrates and some invertebrates. Their genome is relatively large, ranging from 124-kb to 295-kb, and contains 70–200 genes24, 25. They share common characteristics, such as the way of DNA replication and packaging into the capsid, the three-dimensional structure of the capsid and the ability to establish a life-long persistent infection as a viral episome floating in the nucleus21. Herpesvirus species are classified into three families: Herpesviridae (amniotes), Alloherpesviridae (fish and amphibians), and Malacoherpesviridae (molluscs)24. Phylogenetic analysis based on amino-acid sequences of the terminase gene, the only gene confidently conserved among all herpesvirus species, suggests that Teratorn belongs to the family Alloherpesviridae (Fig. 2a). Within Alloherpesviridae, the same topology was obtained from the phylogenetic analysis using the concatenated amino-acid sequences of four genes (terminase, DNA polymerase, DNA helicase and major capsid protein, Fig. 2b). Importantly, Teratorn contains all 13 genes that are conserved among all alloherpesvirus species, and also shares other genomic features with alloherpesviruses such as the genome size, number of genes, GC content, and existence of long repeats (Fig. 2c, Supplementary Fig. 4)24, 25. All these data support the idea that Teratorn contains the complete genome of an alloherpesvirus.
Teratorn exists as a fusion of piggyBac and herpesvirus
The overall structure of the six Teratorn copies sequenced suggested that Teratorn is a fusion of the piggyBac-like DNA transposon and an alloherpesvirus. This raised the question of whether these two mobile genetic elements exist only in the fused form or also exist separately in the medaka genome. To address this question, we searched for genomic neighborhoods in all contigs from the Hd-rR genome assembly, which contain the transposase gene (Supplementary Fig. 5a). Among the 70 obtained contigs, all but one included the above herpesvirus genes at either 5′ or 3′ side to the transposase gene. For the remaining one contig, we were unable to examine the presence of viral genes because the assembly was interrupted by tandem repeats (Supplementary Fig. 5b). Thus, essentially no Teratorn copy exists as a typical configuration of “cut-and-paste” DNA transposons (the transposase gene and TIRs). Furthermore, no herpesvirus-like sequences exists alone in the medaka genome. Taken together, we conclude that all Teratorn copies exist as a fusion of the piggyBac-like transposon and herpesvirus genome (Fig. 1a, b).
Teratorn retains transposition activity
The above structural characteristics suggest that Teratorn has at least the transposon-like life cycle. To test this idea, we first examined whether the encoded transposase is active utilizing an in vitro assay that directly detects transposition activity (Fig. 3a–c, Supplementary Figs. 6–8)26,27,28. In this assay, using HEK293T cells, we co-transfected helper plasmid that ubiquitously expresses transposase, and indicator plasmid that includes the puromycin-resistant gene flanked by the Teratorn terminal sequences, and tested excision and chromosomal integration of the transposon cassette (Supplementary Fig. 6a, c). Excision of the transposon cassette from the indicator plasmid was examined by PCR using primers that flank the transposon cassette. We found that a PCR band was detected only when the helper plasmid was co-transfected with the indicator plasmid (Supplementary Fig. 6a, b). We then tested the integration activity of Teratorn transposase. We cultured transfected cells in puromycin-containing medium for about 2 weeks, to screen for cells that became puromycin-resistant after chromosomal integration of the cassette. However, few colonies were detected after puromycin selection (Supplementary Fig. 6c, d).
Since Teratorn possesses the additional TIRs at the boundary of a pair of long inverted repeats and a unique region, i.e. ‘internal TIRs’ (Fig. 1a–c, Supplementary Fig. 2a), we hypothesized that internal TIRs are also required for the integration reaction. Thus, we inserted it into the indicator plasmid so as to mimic the endogenous structure of Teratorn and performed the same assays again (Fig. 3a). We found that both excision and integration reaction took place this time (Fig. 3a–c). For the excision assay, sequencing of PCR products revealed that the transposon cassette was precisely excised at the boundary, which is the characteristics of piggyBac superfamily DNA transposons28 (Supplementary Fig. 7). For the integration assay, southern blot and sequencing analyses29 of genomic DNA of surviving colonies confirmed the chromosomal integration of the plasmid sequence via transposition, although all of the copies sequenced so far were integrated via internal TIRs (Supplementary Fig. 8). Since DNA cleavage reaction could occur both at external TIRs and internal TIRs in the artificial circular form of the indicator plasmid (Supplementary Fig. 8b), this data implies that, for integration reaction, external TIRs are less frequently used than internal TIRs for unknown reasons. In any case, these results indicate that the transposase of Teratorn is capable of mediating transposition in vitro.
To examine the transposition of Teratorn in vivo, we searched for integration site polymorphisms between the two groups of the Hd-rR medaka inbred strains, which had been kept separately for more than 20 years in our laboratory (University of Tokyo) and at the National Institute for Basic Biology. Southern blot analysis of Teratorn terminal sequences showed distinct band patterns between the two groups (Fig. 3d), suggesting that endogenous Teratorn transposition indeed occurred in vivo. Taken together, these results indicate that Teratorn still retains the activity of transposition in vivo and adopts the life cycle of the piggyBac family DNA transposon.
Teratorn includes intact herpesvirus genes
Teratorn is an active transposon, but could also retain some aspects of the herpesvirus. Indeed, Teratorn contains genes involved in virion morphogenesis (major capsid protein, capsid triplex protein, DNA packaging terminase, and capsid maturation protease, Figs. 1b and 2c, Supplementary Tables 1 and 2). Importantly, sequence comparison revealed that catalytic sites in the ATPase and nuclease domain of terminase30, 31, as well as the catalytic triad of capsid maturation protease32, are conserved for Teratorn (Fig. 4a, Supplementary Fig. 9). For major capsid protein and capsid triplex protein, moderate sequence similarity to other alloherpesvirus species was detected (Supplementary Fig. 10, blue). Although there is little sequence similarity to Herpesviridae family, similar pattern of secondary structure (α-helix and β-sheet) was detected, suggesting the conserved three-dimensional structure of major capsid protein (Supplementary Fig. 10, orange)33, 34. These data suggest that Teratorn equips virion morphogenesis machinery.
To know whether Teratorn is still active as a virus, we examined the extent of open reading frame (ORF) degradation in Teratorn copies. Since Illumina whole-genome shotgun-sequencing data are publically available for the Hd-rR strain35, which include sequences of all Teratron copies, we called SNPs and indels inside Teratorn by aligning Teratorn-derived reads to the Teratorn reference sequences. The reference sequences of subtype 1 and subtype 2 were constructed based on sequences from a single BAC clone (73I9) and on the consensus sequences deduced from all copies in the Hd-rR genome, respectively. We found that the number of nonsense mutations is much smaller than that of missense and synonymous mutations in coding regions (Tables 1 and 2, Supplementary Tables 3 and 4). Consistently, for the six Teratorn copies sequenced so far, all ORFs were found to be intact, suggesting that genes of Teratorn are currently functional.
Furthermore, we examined transcription of selected Teratorn genes including transposase and virus-related genes. Reverse transcription PCR (RT-PCR) analysis detected their ubiquitous and low levels of expression in nearly all tissues (e.g., brain, liver, muscles, gonads) and in developing embryos (5 dpf (days post fertilization)) (Fig. 4b, Supplementary Fig. 11). This pattern of expression could represent latency-associated conditions, and could change upon reactivation. To explore this possibility, we treated medaka embryonic-derived fibroblast cells with 5-azacytidine, N-butylate, and 12-O-Tetradecanoylphorbol 13-acetate (TPA), all of which are chemicals known to induce reactivation of latently infected herpesviruses in human cells21, 36, 37. We found that, although N-butyrate and TPA alone did not have an effect (Supplementary Fig. 12), some of the Teratorn genes were moderately derepressed after administration of 5-azacytidine; yet, the expression level of most genes was not sufficiently high (10–5–10–2 to ß-actin, Fig. 4c), as compared with herpesvirus genes in activated mammalian cells38.
Taken together, Teratorn encodes intact herpesvirus genes and thus likely utilizes the virus replication machinery for its propagation. However, conditions for full reactivation and virion formation remain elusive.
Widespread colonization of Teratorn in the genus Oryzias
To examine whether Teratorn is widely present in medaka genomes, we performed a genomic search against the genus Oryzias. There are more than 20 medaka-related species inhabiting Southeast Asia, from India to Japan. They are subdivided into three groups, latipes species group, javanicus species group, and celebensis species group (Supplementary Fig. 13a, b)39. PCR analysis of five herpesvirus core genes (DNA polymerase, DNA helicase, ATPase subunit of terminase, major capsid protein, and envelope glycoprotein) and transposase gene of Teratorn in 13 medaka-related species revealed that Teratorn exists in the latipes and javanicus species groups, but not in the celebensis species group (Supplementary Fig. 13b). Phylogenetic analysis suggests that the phylogeny of Teratorn and transposase genes is almost the same as that of the host species (Supplementary Fig. 13c). BAC screening and sequencing of the large part of Teratorn in the species of javanicus species group revealed that the configuration of Teratorn, particularly the location of piggyBac transposase, is conserved among Oryzias genus (Supplementary Fig. 13d, e, sequences and gene annotations are included in Supplementary Table 5 and Supplementary Data 2). These data demonstrate that Teratorn has widely colonized in the genomes of Oryzias genus, presumably using the piggyBac transposition machinery. Similar topology of phylogenetic trees between host genes and Teratorn genes implies that Teratorn was vertically descended from the common ancestor of Oryzias genus. However, considering the high sequence similarity among copies inside one species as well as the absence of degraded copies for all species except for O. luzonensis, we still cannot rule out the possibility that recent invasion of Teratorn into each species took place.
Generality of piggyBac-herpesvirus fusion
To further understand how general the piggyBac-herpesvirus fusion event might be, we performed comprehensive genomic survey of Teratorn-like sequences against all publically available vertebrate genome data. We found that Teratorn-like sequences were detected in several teleost fish species (19 of the 67 species, tblastn E-value < 10−3, more than 9 of the 13 alloherpesvirus core genes of Teratorn). Intriguingly, in several species (yellow croaker (L. crocea), nile tilapia (O. niloticus), ocean sunfish (M. mola) and turquoise killifish (N. furzeri)), we identified piggyBac transposase genes next to the Teratorn-like elements (Supplementary Fig. 14, sequences and gene annotations are included in Supplementary Tables 6 and 7 and Supplementary Data 2), suggesting that the fusion of the two mobile elements similarly to the one in medaka. To further explore this possibility, we focused on yellow croaker (accession: GCA_000972845.1)40 and nile tilapia (accession: GCA_001858045.2) as these fish seem to have a high copy number of Teratorn-like elements, supported by multiple blast hits, compared with the other two fish (one remarkable blast hit for turquoise killifish and two hits for ocean sunfish, Supplementary Fig. 14). We searched for genomic neighborhoods in all scaffolds that harbor piggyBac transposase genes and compared to the reference sequence of the Teratorn-like element (Fig. 5a).
For yellow croaker, we obtained 40 scaffolds that include the piggyBac. Among the 17 obtained scaffolds that showed high sequence similarity with the reference piggyBac transposase (i.e., their p distance to the reference transposase sequence was < 0.041), five scaffolds contained the herpesvirus-like sequences next to the piggyBac transposase gene, in the same configuration as the reference shown in Fig. 5b. For 11 other scaffolds, we were unable to determine their neighborhoods because their length was too short. For one remaining scaffold, no herpesvirus-like sequence was found around the transposase gene, presumably caused by misassembly because the coverage of shotgun reads is peculiarly high at its flanking region (>100-fold to the transposase region). By contrast, for the remaining 23 contigs (their p distance was > 0.069), the transposase genes are not connected to any herpesvirus-like sequences. Indeed, phylogenetic analysis revealed that piggyBac copies adjacent to the herpesvirus-like sequence were clustered together (Fig. 5c). Thus, it is highly likely that the fusion of a particular piggyBac transposon and herpesvirus occurred in yellow croaker.
We next focused on nile tilapia (O. niloticus). Like in the analysis of yellow croaker, we searched for genomic neighborhoods for all copies of the same piggyBac and compared with the reference sequence of Teratorn-like element (MKQE0100015.1: 40163260–40398786), in which the herpesvirus-like sequence is flanked by the piggyBac sequences at both ends (Fig. 5d). This configuration was indeed observed in many of the identified elements (Fig. 5e). Importantly, in those elements, the regions flanked by the transposons are highly homologous, i.e., the region from the 5′ TIR of the 5′-side piggyBac to the 3′ TIR of the 3′-side piggyBac, while no sequence similarity was found outside (Supplementary Fig. 15), indicating the transposition of Teratorn-like element mediated by piggyBac. Therefore, we conclude that Teratorn-like element in nile tilapia is indeed the product of the fusion of piggyBac and herpesvirus, which allows for transposon-mediated propagation in the host genome. Taken together, these data suggest that the herpesvirus-piggyBac fusion event is not restricted to medaka, but frequently occurs in teleosts.
In the present study, we have characterized the mobile element Teratorn. Our study demonstrates that Teratorn has the gene encoding transposase of the piggyBac superfamily and retains transposition activity. One of the unique features of Teratorn is its size (~180-kb long), by far the biggest reported for a transposon. Thus, the transposition of Teratorn would be expected to have great impact on host genes and genomes. Indeed, among the previously published mutants, we found that at least four medaka spontaneous mutants are caused by insertion of Teratorn; rs-3 (ectodysplasin-A receptor)41, pc (glis3)42, abcdef (pkd1l1)43, and Da (zic1/zic4)12 (Supplementary Fig. 16). Because of its serious impact, the transposition activity of Teratorn is likely suppressed in vivo, possibly through an epigenetic mechanism. Indeed, our recent study demonstrated that Teratorn inserted in the zic1/zic4 locus is highly methylated44. Nonetheless, we detected insertion-site polymorphisms between the two long-separated groups of the medaka inbred line Hd-rR, suggesting that transposition of Teratorn occasionally occurs under natural conditions. Because of its size, Teratorn usually imposes deleterious effects on host gene function, but less frequently, can modulate the activity of long-range enhancers of a developmental gene, creating a novel trait in a host. One example is the medaka Da mutant (homozygous viable) in which Teratorn specifically disrupts the somite enhancers of the zic1/zic4 locus without affecting the neural enhancer, leading to drastic changes in body shape and fins from larva to adult12.
Surprisingly, Teratorn contains all essential genes shared by alloherpesvirus species with a similar genome configuration (Fig. 2c, Supplementary Fig. 4). We also found genes involved in the promotion of cell proliferation, inhibition of apoptosis, and immunomodulation, which is typical of lymphotropic herpesviruses. Importantly, in the majority of Teratorn copies, the above key genes are maintained intact and transcribed at low levels in medaka hosts. Based on these findings, we propose that Teratorn was descended from a herpesvirus that has propagated in the medaka genome by utilizing the piggyBac transposon system (Fig. 6).
Besides accidental chromosomal integration, there are only two endogenous herpesviruses, human herpesvirus 6 (HHV-6) and tarsier endogenous herpesvirus45, 46, reported so far, out of > 100 herpesvirus species. They belong to the Betaherpesviridae and have an array of telomeric repeats (TMRs) at their termini, which are identical to the telomere sequences (TTAGGG)45. These herpesviruses are specifically integrated into the telomere region via homologous recombination through TMRs, presumably catalyzed by U94, an endonuclease-like gene homologous to parvovirus Rep45, 47. However, it remains unclear whether these endogenous viruses are genuine genomic parasites, because the tarsier endogenous herpesvirus has accumulated deleterious mutations in ORFs46 and HHV-6 has not yet reached a fixation state among human populations (approximately 1% of the human population)47. In contrast, Teratorn has acquired the ability of transposition using its own transposase and colonized in the genomes of medaka species keeping the viral genes intact. Although the mechanism remains a mystery, one possible scenario is that an ancestral Teratorn was accidentally created by fusion of the two elements in other organisms, and the virus form of Teratorn then invaded into the medaka lineage. Since one of the hallmarks of herpesviruses is to establish a life-long persistent infection inside hosts as viral episomes without integration21, we reason that chromosomal integration is an adaptive consequence to escape from host immunity and ensure a stable transmission of their progeny across host generations. The absence of genes involved in nucleotide metabolism in Teratorn (e.g., thymidine kinase (TK), deoxyuridine triphosphatase (dUTPase), and uracil DNA glycosylase), which are utilized for massive virus propagation by modulating the nucleotide pool21, is suggestive, because those genes might no longer be needed after endogenization. Taken together, Teratorn represents the first example of herpesvirus adaptation to intragenomic life cycle (Fig. 6).
The entire life cycle of Teratorn is still largely unknown, although it behaves at least as an active transposon in vivo. However, given that most herpesvirus-related genes also appear functional, those genes might be coordinately utilized for its propagation together with the transposase gene. Indeed, there is circumstantial evidence that chromosomally integrated HHV-6 (ciHHV-6) can be reactivated, e.g., in ciHHV-6-positive cultured cells48, healthy ciHHV-6-positive individuals and X-linked severe combined immunodeficiency (X-SCID) patients47. Reactivation of these HHV-6 genes appeared to be accompanied by the formation of circular viral DNA molecules via excision of the telomeric t-loop47, 49. Teratorn could also undergo similar processes during reactivation; excision mediated by the piggyBac-like transposase followed by genome replication and virion formation (Fig. 6b). However, we have not succeeded in full activation of Teratorn. 5-azacytidine treatment of medaka fibroblasts only caused moderate reactivation of viral genes, and under this condition, we failed to detect virus proteins derived from Teratorn by western blot (major capsid protein, capsid triplex protein, and membrane glycoprotein, Supplementary Fig. 17). Given that all essential viral genes, especially genes involved in virion morphogenesis such as capsid proteins, DNA packaging terminase and capsid maturation protease, appear to be functional, it is still likely that, under as-yet-unknown conditions, Teratorn actually produces virus particles that infect new individuals. Similarly, virions have not yet been detected experimentally from Polinton/Maverick superfamily transposons, replicative large DNA transposons widespread among eukaryotic genomes8, 17, although they contain a set of intact virus-like genes required for virion formation, including A32-like DNA packaging ATPase, Ulp1-like cysteine protease and two capsid proteins9, 50.
Despite the potentially hazardous consequences of viral genes, there might be some benefits to the host having this large mobile element. One possible scenario is that Teratorn serves as a shield against an otherwise lethal virus, by inhibition of virus entry by receptor block, inhibition of functional virion assembly or establishment of immunotolerance. On the other hand, Teratorn shows characteristics of selfish genetic elements, since all herpesvirus essential genes and the transposase gene remain intact. Thus, we propose that Teratorn might serve two purposes: one is as a guardian against exogenous virus infection, and the other is as a selfish intragenomic parasite. Recently, it was shown that endogenous virophages in some protozoan genomes were reactivated by superinfection of giant viruses (preys of the virophages), which facilitates host survival and their own propagation51, 52. Teratorn could undergo a similar response during infection with exogenous viruses. In any case, further experimental efforts to detect virions will be necessary to understand the life cycle of Teratorn and the biological significance of the existence of Teratorn in the medaka genome.
As mentioned above, Teratorn is a mobile element that experienced the fusion between virus and cut-and-paste DNA transposon in eukaryotes. Importantly, our genomic survey implied that the herpesvirus-transposon fusion event is not restricted to medaka but is rather frequent among teleost lineages (Fig. 5, Supplementary Fig. 14). Teratorn, however, cannot be the only example in the network of eukaryotic mobile elements. Indeed, all viruses have the potential to shift into the intragenomic life cycle if they acquire an integration system from other sources. In this context, Polintons are of particular interest, since recent phylogenetic studies suggested that Polintons have evolutionary links with other mobile genetic elements (adenovirus, virophages, linear plasmids, and bacteriophages) and serve as a hotbed for recombination leading to a change in their life cycle8, 9, 11. Furthermore, recombination events between distantly related viruses have been reported, such as chimeric viruses (chimera of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA(+)) virus) and bidnavirus (chimera of parvovirus, Polinton, reovirus, and baculovirus)2, 9, 53, 54. Thus, recombination and fusion between mobile elements of distinct classes occur more frequently than previously thought, leading to the diversification of mobile genetic elements. Further identification of novel and peculiar mobile genetic elements will provide new insights into mechanisms underlying their diversification and evolution.
Hd-rR, d-rR, and HNI inbred strain of Oryzias latipes were maintained in our laboratory. Fish strains of medaka-related species were obtained from the laboratory stocks maintained at Niigata University and National Institute for Basic Biology. Lists of each species and their original collection sites are described in Supplementary Table 8. All experimental procedures and animal care were performed according to the animal ethics committee of the University of Tokyo.
Screening and sequencing of BAC clones
Teratorn insertion sites were identified by screening of the 5′ and 3′ flanking regions by blastn of Teratorn terminal sequences against the public genome assembly of medaka Hd-rR inbred strain15, followed by mapping of those obtained sequences to the genome of another medaka inbred strain, HNI, to ascertain which of the 5′ and 3′ flanking sequences are derived from the same loci. For the 12 Teratorn copies with identified integration sites, BAC clones that were PCR-positive for both Teratorn ends were screened from the medaka Hd-rR BAC library55. Sequences of the primers for screening are in Supplementary Table 9. For the screened six individual Teratorn copies, BAC DNA was purified by QIAGEN Large-Construct Kit (QIAGEN). Sequencing of BAC clones was implemented using PacBio RS-II. Assembly was carried out using HGAP pipeline.
For medaka related species (library name: IMBX for O. dancena; OHB1 for O. hubbsi; OJV1 for O. javanicus; LMB1 for O. luzonensis), BAC clones were screened by PCR of the four Teratorn genes (piggyBac-like transposase, membrane glycoprotein, DNA polymerase and DNA packaging terminase). Sequences of the primers for screening are in Supplementary Table 9. BAC DNA was then purified using NucleoBond® Xtra BAC (MACHEREY-NAGEL). The sequencing library was prepared by sonicating to a size ranging from 800 to 1200 bp, followed by adaptor ligation and amplification using KAPA Hyper Prep Kit (KAPA BIOSYSTEMS). Paired-end sequencing of the prepared libraries was executed on the Illumina Miseq platform. After filtering out low-quality reads by trimmomatic v0.3356, de novo assembly was carried out by CLC Genomic Workbench (https://www.qiagenbioinformatics.com/).
Gene annotation was initially carried out by the GeneMarkS web server20. For genes that start with codons other than “ATG”, prediction by ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used instead. If adjacent multiple ORFs seemed to be derived from a single gene (i.e., different portions of the same gene were obtained as blastp output), gene annotation by GENSCAN web server19 was used to generate a more plausible gene model including introns.
Identification of subtype 2 Teratorn
Subtype 2 Teratorn was identified by blastn search of subtype 1 Teratorn sequence against the updated version of Hd-rR genome assembly (http://utgenome.org/medaka_v2/#!Top.md). The putative full-length subtype 2 Teratorn sequence was constructed by conjugating partial sequences of two contigs (ctg7180000008209: 1–30344, ctg7180000008207: 2815545–2982619), followed by calling consensus sequence by Bcftools57, based on the Illumina whole-genome shotgun short reads (see below).
Multiple alignment of active piggyBac transposase genes
Amino acid sequences of piggyBac superfamily transposase genes (piggyBac in T. ni, Uribo2 in X. tropicalis, yabusame-W in B. mori, piggyBat in M. lucifugus, AgoPLE in A. gossypii, and PLE-wu in S. frugiperda) were downloaded from GenBank. Those transposases were aligned using Clustal W with default parameters58. The multiple alignment was visualized using TrimAl59.
For the phylogenetic analysis of Teratorn among Herpesvirales, amino-acid sequences of DNA polymerase, DNA helicase and major capsid protein of Alloherpesviridae species, as well as DNA packaging terminase of Herpesvirales species and T4 phage, downloaded from GenBank, were used. Multiple sequence alignment was built up using MUSCLE in MEGA6 package60. Poorly aligned regions were removed using trimAl with -strictplus option59. Maximum-likelihood analysis was carried out using MEGA6. Le_Gascuel_2008 model, considering evolutionary rate difference among sites by categorizing into five values to fit Gamma distribution, with a proportion of sites being invariable, was used as substitution model with 1000 bootstrap replicates.
For the analysis of Teratorn transposase copies in the Hd-rR genome, nucleotide sequences of all Teratorn transposase copies, screened from the genome assembly data by blastn, were used. Multiple sequence alignment was built up using MUSCLE. Neighbor-joining analysis was carried out using MEGA6. Jukes-Cantor model, considering uniform evolutionary rate among sites, was used as substitution model with 1000 bootstrap replicates.
For the analysis in the genus Oryzias, partial sequences of several Teratorn genes were screened from the genome of each species as described below. Multiple alignment of Teratorn genes and concatenated sequences of host genes were constructed by MUSCLE. Maximum-likelihood analysis was carried out using Tamura-Nei method as substitution model with 1000 bootstrap replicates, without considering evolutionary rate difference among sites.
Search of neighboring region to the piggyBac transposase
Contigs or scaffolds including the transposase gene were screened by blastn search of the transposase gene, followed by extraction of flanking region by BEDtools (upstream 25 kb and downstream 25 kb region for medaka, 60 kb flanking region for yellow croaker, and 20 kb flanking region for nile tilapia). Gene order around the transposase genes were then assessed by displaying dot plot matrix between the reference Teratorn-like element and the obtained contigs, using mafft on line server61. Neighbor-joining analysis were carried out based on the partial region of the transposase gene using Tamura-Nei method as substitution model with 1000 bootstrap replicates, without considering evolutionary rate difference among sites.
Plasmid construction for the transposition assay
Indicator plasmid was constructed as follows. First, the puromycin-resistant gene cassette was extracted from the pMXs-puro retroviral vector by digesting with BamHI and XhoI. In parallel, the AcGFP expression cassette was amplified by PCR. These two DNA fragments were then conjugated using in-fusion HD cloning kit (TaKaRa). This puroR-AcGFP fragment was further conjugated with Teratorn TIRs at both sides by joint-PCR, and was subcloned into pCR-BluntII-TOPO (Invitrogen). Finally, Teratorn internal TIR was inserted into the boundary of the 5′ TIR and the puroR-AcGFP cassette so as to mimic the endogenous Teratorn structure. Helper plasmid was constructed by RT-PCR of the Teratorn transposase followed by subcloning into pCSf107mT vector62. Sequences of the primers for the construction of the plasmids are in Supplementary Table 9.
Transfection and excision and integration assay
HEK293T cell line was a kind gift from Prof. Yoshinori Watanabe (University of Tokyo). Cells were maintained in Dulbecco's Modified Eagle’s Medium (DMEM) with 10% FBS and 100U/ml penicillin and 100 µg/ml streptomycin (Gibco) at 37 °C with 5% CO2. The day before transfection, cells were seeded in 6-well plates to achieve 70–90% confluent on the next day. Lipofectamine 3000 reagent (Invitrogen) was used to co-transfect cells with 1500 ng of helper plasmid and 2500 ng of indicator plasmid per well. For the excision assay, 40% of cells were passaged to a new dish, cultured for about 2 days to become fully confluent and the plasmid was extracted by the Hirt method63. PCR was carried out using primers that flank the transposon cassette of the indicator plasmid. Sequences of the primers for the excision assay are in Supplementary Table 9. In the integration assay, 40% of transfected cells were passaged to a new dish and cultured in DMEM with 5.0, 7.5, or 10.0 µg/ml of puromycin for about 2 weeks. Medium was changed every 2 or 3 days. Surviving colonies were isolated by peeling off and sucking up with a pipette and transferred to a new dish. When cell clones grew in sufficient number, genomic DNA was isolated using Wizard Genomic DNA extraction kit (Promega) for Southern blotting and PCR-based integration site determination.
In all, 5–10 µg of genomic DNA extracted from medaka adult whole body or HEK293T cells were digested with appropriate restriction enzymes for at least one hour and precipitated with ethanol. Digested DNA was resolved by gel electrophoresis with 25–30 V overnight, and transferred to a Hybond-N+ nylon membrane (GE Healthcare). AlkPhos Direct Labeling and Detection System with CDP-Star (GE Healthcare) was used for hybridization and signal detection. Purified PCR products of Teratorn terminal sequences were used as hybridization probes to visualize individual Teratorn insertions. Sequences of the primers for the preparation of template DNA for probes are in Supplementary Table 9.
Extension primer tag selection linker-mediated PCR
Extension primer tag selection linker-mediated PCR ((EPTS)LM-PCR) was carried out as previously described29. First, 1.5 µg of genomic DNA from HEK293T cells was digested with HaeIII or ApoI overnight. After precipitation with ethanol, a biotinylated primer specific to Teratorn terminal sequence was used for tagging the transposon insertion sites. In this step, Phusion High-Fidelity DNA Polymerase (Thermo) was used for DNA extension (98 °C for 3 min, 68 °C for 30 min, and 72 °C for 30 min). After purifying the PCR products using the Wizard® SV Gel and PCR Clean-Up System (Promega), biotin-tagged DNA was isolated by incubating with Dynabeads® M280 Streptavidin (Thermo) for one hour, followed by twice washing out of non-target DNA. Finally, double-stranded oligonucleotide phosphorylated linkers were ligated at 16 °C overnight. PCR with primer pairs specific to Teratorn terminal sequence and linker oligonucleotide were carried out to amplify Teratorn insertion sites. Sequences of the primers for this analysis are in Supplementary Table 9.
Multiple alignment of herpesvirus genes
Multiple alignment of amino acid sequences of DNA packaging terminase, capsid maturation protease, major capsid protein and subunit 2 capsid triplex protein were constructed by PROMALS3D64. Sequences of each species were downloaded from Genbank. Catalytic centers of terminase and protease gene was characterized based on Rao V.B. and Feiss M.30, Selvarajan Sigamani S. et al.31, and Cheng H. et al.32. Domains of major capsid protein was characterized based on Huet A. et al.33.
Variant calling and annotation
Reference medaka genome data was reconstructed as follows, in an attempt to map all Teratorn-derived reads to the reference Teratorn sequences. Teratorn was masked from the public medaka draft genome15 by blastn of both Teratorn subtypes, followed by maskFastaFromBed command of BEDtools. Then, the genome was conjugated with the sequences of Teratorn. Illumina whole-genome shotgun read data was downloaded from DDBJ Sequence Read Archives (accession: DRR002213). After filtering out low-quality bases and adapter sequences from short reads by trimmomatic v0.3356, reads were aligned to the reconstructed reference genome using BWA (Burrows-Wheeler Aligner)-MEM65, with the default parameter settings. Removal of PCR duplicates was carried out by Picard (http://picard.sourceforge.net). Local realignment was executed using RealignTargetCreator and IndelRealigner tools in GATK, with the default parameters66. Variant calling was performed by UnifiedGenotyper tool in GATK, with the following parameters (ploidy, 30 for Teratorn subtype 1 and 5 for subtype 2; stand-call-conf, 30; stand-emit-conf, 20; glm, BOTH). Variant annotation was implemented using SnpEff, with the default parameters67.
Expression analysis of Teratorn genes in medaka
Total RNA was isolated from 5 dpf (days post fertilization) embryos and adult fish tissues (brain, liver, fin, muscle, gut, ovary, testis) of medaka Hd-rR strain, using ISOGEN (Nippon Gene), according to the manufacturer’s protocol. After removal of genomic DNA by DNaseI digestion (Invitrogen), Complementary DNA (cDNA) was synthesized using SuperScript III (Invitrogen). RT-PCR was carried out using Phusion DNA Polymerase (Thermo Fisher) for 40 cycles. Sequence of the primers for RT-PCR are in Supplementary Table 9.
Test of reactivation of Teratorn genes by chemicals
Medaka embryonic fibroblast cell line was previously established in our laboratory. Cells were maintained in L-15 medium (Gibco), supplemented with 15% FBS (biosera), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco) at 30 °C. For reactivation, cells were administered with 2 μM of 5-azacytidine (Sigma) for 5 days. For a subset of samples, 500 ng/ml of 12-O-Tetradecanoylphorbol 13-acetate (Sigma) and 3 mM of sodium butyrate (Sigma) were also administered for the last 2 days. This experiment was repeated for three (no chemical administration, 5-azacytidine only) or four (TPA + N-butyrate, TPA + N-butyrate + 5-azacytidine) times. Total RNA extraction and cDNA synthesis were carried out as described above. Quantitative reverse transcription PCR (qRT-PCR) was carried out with the Stratagene mx3000p system (Agilent), using the THUNDERBIRD SYBR qPCR Mix (ToYoBo). β-actin was used as an internal control. Sequence primers for qRT-PCR are in Supplementary Table 9. Ratio of molar concentration of Teratorn genes relative to β-actin was quantified based on standard curves generated from purified PCR products, using Qubit® 2.0 Fluorometer (Thermo Fisher) to measure mass concentration.
In the qRT-PCR experiment described above, no statistical method was used to predetermine sample size. Sample sizes were based on previously reported experiments that are similar to the present study. No data were excluded from the analysis. For genes that met the assumption of normal distribution (all genes except for major capsid protein, pim, and terminase, Shapiro-Wilk test, p < 0.05), statistical significance was tested by one-sided Welch Two Sample t-test. All statistical analyses were performed using R software (version 3.2.4).
Partial sequences of major capsid protein (amino acids 934–1074), capsid triplex protein (amino acids 51–170) and membrane glycoprotein (amino acids 711–830) were subcloned into pGEX4T-1 vector (GE Healthcare). After culture of transformed bacteria, GST-tagged protein fragment was purified using Glutathione Sepharose 4B (GE Healthcare). Polyclonal antibodies were raised by immunization of mouse with the GST-tagged truncated proteins (100 μg) for about 2 months, followed by extraction of serum. Sequences of the primers for antigen production are in Supplementary Table 9.
For reactivation of Teratorn genes, medaka fibroblast cells were administered with 5-azacytidine (0.0 μM, 1.0 μM, 1.5 μM, 2.0 μM) for 5 days. For a subset of samples, 500 ng/ml of 12-O-Tetradecanoylphorbol 13-acetate (Sigma) and 2 mM of sodium butyrate (Sigma) were also administered for the last 2 days. For positive control, HEK293T cells transfected with the plasmids that express the GFP-tagged antigen protein fragment were used. Cells were lysed with 1× SDS buffer, separated by polyacrylamide gels (5.5% for major capsid proteins and membrane glycoprotein, and 10.0% for capsid triplex protein, respectively) and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies (anti-major capsid protein, anti-capsid triplex protein, and anti-membrane glycoprotein, 1:2000 dilution) over night at 4 °C. After several washes, membranes were incubated with secondary antibody (Anti-Mouse IgG HRP-Linked Whole Ab Sheep (GE Healthcare, NA931V), 1:2500 dilution) for 1 h at room temperature. Protein bands were visualized using the ECL Select Western Blotting Detection Reagent (GE Healthcare) and detected by ImageQuant (GE Healthcare).
Screening of Teratorn genes in the genus Oryzias
Partial sequences of five herpesvirus genes (DNA polymerase, DNA helicase, major capsid protein, membrane glycoprotein, and ATPase subunit of terminase) were screened from genomic DNA of each medaka related species by PCR, using degenerate primers constructed from the multiple alignment of Teratorn-like sequences in several teleost fish (S. salar, O. mykiss, E. lucius, O. niloticus, P. nyererei, C. semilaevis). For genes in which degenerate primers didnot work, primers designed from sequences of subtype 1 and subtype 2 Teratorn of O. latipes were used instead. piggyBac-like transposase gene was screened using primers that were designed from O. latipes genome. Sequences of the primers for the screening are in Supplementary Table 9.
Search of Teratorn-like elements in other species
Tblastn search of 13 herpesvirus core genes of Teratorn was carried out against all available teleost genomes with default parameters. In addition, tblastn of the four genes (DNA polymerase, DNA helicase, DNA packaging terminase, and major capsid protein) was performed against amphibians, chondrichthyes or sarcopterygi in the web browser. Contigs or scaffolds that include a series of herpesvirus-like sequences were screened as follows. First, location of the 13 herpesvirus core genes was identified by tblastn. After merging the genomic loci which are within 40 kb of one another, sequences of the defined region and the flanking 40 kb region were extracted from the draft genome by BEDtools.
Consensus sequence calling of Teratorn-like elements
In yellow croaker, preliminary consensus sequence was constructed using Unipro UGENE68, based on multiple alignment of seven contigs that include the relatively long range region of Teratorn-like sequence (> 40 kb). Then the consensus sequence was created by mapping of the Illumina whole-genome shotgun reads (Accession: SRP041375), followed by consensus sequence calling by vcftools57. In nile tilapia, all gained Teratorn-like sequences were aligned to the longest one (MKQE01000015: 40163260-40398786) using BWA (Burrows-Wheeler Aligner)-MEM65, followed by extraction of consensus sequence using Unipro UGENE.
Nucleotide sequence data and gene annotation of Teratorn and Teratorn-like elements are provided and described within the Supplementary Data of this manuscript. Sequence of subtype 1 Teratorn (73I9) has been submitted to the DDBJ/EMBL/GenBank databases (Accession No: LC199500). All other data supporting the findings of this study are available from the corresponding author upon request.
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We thank Dr W. Qu for the help of identification of Teratorn integration sites, Drs. T. Kimura and Y. Takehana for the help of BAC screening, Drs. A. Kato and J. Arii for valuable discussion and experiments of reactivation of Teratorn, Prof. Deshou Wang and Mr. Xianbo Zhang for the help of fosmid screening to determine the sequence of Teratorn-like elements in nile tilapia, Dr A. Terashima for critical reading of the manuscript, and Mrs. I. Fukuda for fish care. The five Oryzias species (O. sarasinorum, O. nigrimas, O. marmoratus, O. matanensis, O. profundicola) used in the present study were provided by Niigata University through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This work was supported by CREST, JST (Grant No: JPMJCR13W3).
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