Complete fusion of a transposon and herpesvirus created the Teratorn mobile element in medaka fish

Abstract

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. Teratorn is a large mobile genetic element originally identified in the small teleost fish medaka. Here, the authors show that Teratorn is derived from the fusion of a piggyBac superfamily DNA transposon and an alloherpesvirus and that it is widely found across teleost fish.

Fig. 1

Sequence characteristics of Teratorn. a The overall structure of Teratorn. b Gene map of the subtype 1 Teratorn copy (73I9; named from the BAC clone ID). Predicted genes are classified into four categories depicted by colored arrowheads; magenta, piggyBac-like transposase gene; blue, herpesvirus-like genes; yellow, cellular homologues; gray, unannotated genes. Terminal inverted repeats (TIRs) of piggyBac-like transposon are depicted by boxed triangles. c Comparison of the whole structure of subtype 1 and subtype 2 Teratorn. d Neighbor-joining tree of all Teratorn transposase copies in the genome of medaka Hd-rR inbred strain. Note that they are separated into two clusters. The scale bar represents the number of substitutions per site. e Southern blot displaying individual Teratorn insertions in the genome of medaka Hd-rR inbred strain. The ~300-bp 5′ terminal region of Teratorn of each subtype was used as hybridization probes

Fig. 2

Teratorn contains a full herpesvirus genome. a Maximum-likelihood tree based on the amino-acid sequence of the DNA packaging terminase gene, the only gene confidently conserved among all herpesvirus species. Bootstrap values of branching are indicated at the nodes. Note that, among the three families (Herpesviridae, Alloherpesviridae, and Malacoherpesviridae) in the Herpesvirales order, Teratorn belongs to the family Alloherpesviridae (infecting fish and amphibians). b Maximum-likelihood tree based on the concatenated amino-acid sequences of major capsid protein, DNA helicase, DNA polymerase, and DNA packaging terminase from all alloherpesvirus species with sequenced genome. Within Alloherpesviridae, Teratorn is only distantly related to any other species. c Comparison of genomic structure of subtype 1 Teratorn and several alloherpesvirus species, as representatives of genera in the family Alloherpesviridae. The colored squares indicate each herpesvirus core gene, and black boxes depict repeat sequences. Note that Teratorn contains all 13 core genes conserved among all alloherpesvirus species, as well as long repeats. The scale bars in b and c represent the number of substitutions per site

Fig. 3

Teratorn retains transposition activity. a A schematic of the transposition assay. In the helper plasmid, Teratorn transposase gene was expressed under the CMV promoter. In the indicator plasmid, a GFP reporter and a puromycin-resistant gene were flanked by the 5′ and 3′ TIR. Note that additional TIR was inserted at the boundary of 5′ TIR and GFP cassette so as to mimic the endogenous Teratorn structure (internal TIRs). Transposition activity was examined by co-transfection of those two plasmids into HEK293T cells, followed by either PCR-based detection of transposon cassette excision from the indicator plasmid (excision assay, left) or chemical selection of transgenic cell lines (integration assay). Thick arrows indicate primer pairs used for the excision assay. b Excision assay. “+” and “−” indicate the presence and absence of helper plasmid, respectively. The number indicates the target region of PCR depicted in a (1, flanking the transposon cassette, amplifying only when excision reaction takes place; 2, targeting a terminus of transposon cassette, positive control). Note that PCR product flanking the transposon cassette was detected only when the helper plasmid was co-transfected (subtype 1, 202 bp; subtype 2, 250 bp; arrowheads). c Integration assay. Thirteen days after 7.5 μg/ml of puromycin selection, colonies were stained with methylene blue. Note that multiple colonies were observed only when the helper plasmid was co-transfected, indicating that the transposon cassette was integrated into chromosomes of HEK293T cells via transposition. d Southern blotting detecting individual Teratorn insertions in the Hd-rR individuals kept in the University of Tokyo (U-Tokyo) and at the National Institute for Basic Biology (NIBB), using sequences of Teratorn 5′ and 3′ ends as hybridization probes. Note that band patterns are different between the two individuals (arrowheads)

Fig. 4

Teratorn encodes intact herpesvirus genes. a Multiple alignment of amino-acid sequences around catalytic centers of DNA packaging terminase and capsid maturation protease gene in Teratorn (magenta), herpesvirus species of Alloherpesviridae (blue), Herpesviridae (orange), Malacoherpesviridae (black) and bacteriophages (green). Note the conservation of catalytic residues of terminase (walker A, walker B, C-motif and adenine-binding motif in the ATPase domain (magenta), catalytic triads Asp-Glu-Asp in the nuclease domain (blue)), as well as the catalytic triad of protease (His-Ser-His/Glu; magenta) in Teratorn. b RT-PCR of Teratorn genes in 5 dpf (days post fertilization) medaka embryos. “+” and “–” indicate that the reverse-transcription reaction was carried out or not, respectively. Cycle number of RT-PCR was 40. The colors indicate the categories of genes shown as in Fig. 1 (red, piggyBac transposase; blue, herpesvirus genes with known funtion; yellow, cellular homologues that seem to be involved in evasion of host immunity (ZFP36-like, CXCR-like and DNA methyltransferase-like) and cell proliferation (CDK-like, pim-like and ZnSCAN-like). c qPCR analysis of Teratorn genes in medaka fibroblast cells administered with or without 2 μM of 5-azacytidine, 3 mM of N-butyrate and 500 ng/ml of 12-O-Tetradecanoylphorbol 13-acetate (TPA). “+” and “–” indicate that each chemical was administrated or not. The value indicates the ratio of molar concentration relative to β-actin. Note that expression levels of most genes were moderately increased by chemical administration, although the expression level was still low. Statistical significance was tested by one-sided Welch Two Sample t-test. Each data point indicates the raw value of each experiment, and bars represent the mean ± SEM of replicates. Number of biological replicates are as follows; n = 3 for no chemical treatment, n = 3 for 5-azacytidine treatment, n = 4 for 5-azacytidine, TPA and N-butyrate treatment

Fig. 5

piggyBac-herpesvirus fusion in other species. a The procedure of screening all scaffolds that include the piggyBac transposase gene inside Teratorn-like element in yellow croaker (Larimichthys crocea) and nile tilapia (Oreochromis niloticus). First, contigs that contain the transposase gene were screened from the genome by blastn. For all contigs obtained, genomic neighborhoods around the transposase genes were tested by displaying alignment with the reference sequence of Teratorn-like element. In parallel, the phylogenetic relationship of the transposase copies was analyzed. b Reference sequence of Teratorn-like elements of yellow croaker. Predicted ORFs (exons) are depicted by colored arrows according to the categories; magenta, piggyBac transposase; blue, herpesvirus genes; yellow, cellular homologues; gray, unannotated genes. c Neighbor-joining tree based on the sequences of piggyBac transposase copies obtained by blast search is displayed. One region (1369–1598) of the reference transposase sequence was utilized for the phylogenetic tree construction. Each sequence is named by the contig name (e.g., KQ042839.1). piggyBac copies are categorized into four groups, according to the linkage to the herpesvirus genes. Note that piggyBac copies adjacent to the herpesvirus-like sequence (magenta) are clustered together, suggesting that a particluar type of piggyBac element is fused with the herpesvirus-like sequence. d Reference sequence of the Teratorn-like element of nile tilapia. e Neighbor-joining tree based on the sequences of piggyBac transposase copies obtained by blast search is displayed. piggyBac copies are categorized into six groups as described in the inset, according to the linkage to the herpesvirus-like sequence. Note that there are multiple piggyBac copies linked to the herpesvirus-like sequence in the same configuration as the reference sequence (either 5′ and 3′ end of Teratorn like element, yellow and magenta), although phylogenetically polyphyletic. The scale bars in c and e represent the number of substitutions per site

Fig. 6

Model of Teratorn derivation from a herpesvirus that shifted to an intragenomic life cycle by gaining the piggyBac transposon system. a The entire structure of Teratorn. Teratorn is a fusion of a piggyBac-like transposon and a herpesvirus. b Comparison of life cycles of normal herpesviruses and Teratorn. Normal herpesviruses do not integrate their genome into chromosomes of host cells during their life cycles; instead, they form episomal DNA molecules and persist inside cells, accompanied by recursive reactivation (left). Teratorn might be derived from a herpesvirus that had shifted its life cycle to an intragenomic transposon-like parasite, by gaining the piggyBac transposon system. The transposition mechanism might be either via (1) DNA replication and virus particle formation or (2) conventional cut-and-paste transposition. Virus particles might be either (1–1) infectious and transmissible to other cells/individuals or (1–2) non-infectious and remain inside cells (right)