Troyka represents a unique lineage of virus-like retroelements

Abstract

Eukaryotic reverse transcriptase genes are mostly incorporated into viruses or transposons. Among the six reported families of reverse-transcribing viruses, three families (Metaviridae/Gypsy, Belpaoviridae/BEL and Pseudoviridae/Copia) have proliferated mostly as transposons, collectively known as LTR retrotransposons. Troyka was reported as a unique lineage of the Metaviridae/Gypsy family. While most LTR retrotransposons generate 4 to 6 bp target site duplications (TSDs) upon integration and contain 5′-TG. CA-3′ termini, Troyka generates 3 bp TSDs and contains 5′-CG.CG-3′ termini. Here, the distribution and diversity of Troyka were extensively investigated from the available genome sequences. In addition to the six animal phyla reported previously, Troyka was characterized for the first time in Hemichordata, Priapulida, Annelida, Phoronida and Brachiopoda. The unique terminal nucleotides of Troyka are very well conserved. The phylogenetic analysis with various combinations of conserved domains supported the independent position of Troyka from any established retroelement groups, most likely as the sister lineage of the group including Metaviridae/Gypsy, Retroviridae, Caulimoviridae, Lokiretroviruses and Odin LTR retrotransposons. Troyka is here proposed as a new superfamily of LTR retrotransposons.

Impact Statement

This article introduces a new superfamily of LTR retrotransposons, showing the unseen diversity of virus-like retroelements. The reported information about the unique characteristics of Troyka LTR retrotransposons would encourage the improvement of currently available tools for repeat annotation, thus contributing to the better understanding of genome organization and evolution.

Data Availability

Supplementary material is available with the online version of this article, available through Figshare at https://doi.org/10.6084/m9.figshare.29341544 [1]

Introduction

Reverse transcriptase (RT) is an enzyme that catalyses DNA polymerization using an RNA template [2,3]. In eukaryotes, except for two cellular genes, telomerase reverse transcriptase (TERT) and reverse transcriptase (RVT), RT genes are seen in two forms of selfish DNA or RNA entities: transposons (retrotransposons) and viruses [4,5]. Phylogenetic analysis divided the RT into four major branches [4]: prokaryotic retroelements (P), non-LTR retrotransposons and RVT (L), Penelope-like elements and TERT (T), and other eukaryotic retroelements, including all viral forms of retroelements (V).

Reverse-transcribing viruses are classified into six families in the class Revtraviricetes by the International Committee on Taxonomy of Viruses (https://ictv.global/taxonomy). They are Retroviridae [6], Caulimoviridae [7], Hepadnaviridae [8], Pseudoviridae [9], Belpaoviridae [10] and Metaviridae [11,12]. Among them, the former three groups are full-fledged viruses, which can transmit between cells and whose virus particles are observed. However, it is considered that most elements in the latter three families are more likely to behave as a transposon, and thus, they are also called Copia (or Ty1/Copia), BEL (or BEL/Pao) and Gypsy (or Ty3/Gypsy) superfamilies of LTR retrotransposons, respectively [13,14]. It is notable that ‘family’ in the virus taxonomy almost corresponds to ‘superfamily’ in the transposon classification. These LTR retrotransposons generally encode one (gag-pol) or two proteins (gag and pol). Gag is a structural protein that shows similarity to the capsid and nucleocapsid proteins of vertebrate retroviruses (Retroviridae) [15]. Pol contains several enzymatic domains: retropepsin (RP or protease), RT, ribonuclease H (RH) and DDD/E transposase/integrase (IN). In Retroviridae, the original RH has lost the enzymatic function to become the ‘tether domain’ and a new RH has been acquired [16]. Some LTR retrotransposons encode additional proteins or protein domains. Chromovirus is a group of Gypsy LTR retrotransposons that acquired a chromodomain at the C-terminus of pol protein [14,17]. Several independent groups of LTR retrotransposons have acquired an envelope glycoprotein gene from viruses; Errantivirus has acquired an envelope gene likely from baculovirus [18], and Anakin has acquired an envelope gene from chuvirus [19]. Oppositely, not a few retrovirus lineages have lost their envelope gene secondarily and have become a ‘retrotransposon’; these retroviruses are called ‘endogenous retroviruses (ERVs)’ [20]. Odin LTR retrotransposons found in Cnidarians constitute a sister lineage of Lokiretroviruses and might be another example of endogenous retroviruses [21].

Phylogenetic analyses revealed the monophyly of the six families of reverse-transcribing viruses along with several non-viral and viral groups, but distinct from the two other major groups of eukaryotic retrotransposons: non-LTR retrotransposons and Penelope-like elements [4]. The retroelement groups which show close affinity to the reverse-transcribing viruses are HEART, Nackednavirus and DIRS. HEART is an enigmatic group of retroelements whose mobilization mechanism is not yet resolved [22]. Nackednavirus represents a sister lineage of Hepadnaviridae but lacks an envelope protein [23]. DIRS, which may be further classified into PAT, Ngaro, VIPER and TATE, are a unique group of retrotransposons whose RT is closely related to that of LTR retrotransposons but which encodes a tyrosine recombinase instead of DDD/E transposase for its genome integration [24,26]. Although the phylogenetic relationships among these retroelements have not yet been confirmed, the monophyly of Retroviridae, Caulimoviridae and Metaviridae/Gypsy was indicated based on the RT phylogeny [16,21, 27]. It is consistent with the conserved domain order in the Pol proteins of retroviruses and Metaviridae/Gypsy, as well as of Belpaoviridae/BEL [14].

Troyka is a small group of LTR retrotransposons that are assigned to the Gypsy superfamily in the current Repbase taxonomy. Troyka was first reported from the cnidarian Nematostella vectensis and the amphioxus Branchiostoma floridae in 2008 [28]. To date, Troyka has been reported from six animal phyla, Cnidaria, Chordata, Echinodermata, Arthropoda, Nemertea and Mollusca [13]. Troyka is unique in its terminal signatures: 5′-CG.CG-3′, while most LTR retrotransposons terminate with 5′-TG and CA-3′. Troyka generates 3 bp target site duplications (TSDs), while other LTR retrotransposons generate 4 to 6 bp TSDs. These unique sequence features led me to reinvestigate the position of Troyka in the RT phylogeny.

In this study, the wide distribution, terminal signature and the 3 bp TSDs of Troyka LTR retrotransposons are confirmed. The phylogenetic analysis of virus-like retroelements revealed that Troyka constitutes an independent branch from any reported virus-like retroelement groups.

Methods

Characterization of Troyka LTR retrotransposons

All Troyka LTR retrotransposon sequences were downloaded from Repbase (https://www.girinst.org/repbase/) [13] as of 24 July 2024.

blast search was performed with reported Troyka proteins in Repbase as queries against protein datasets or genome DNA sequence datasets at the National Center for Biotechnology Information (NCBI) blast website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Genomes with significant blast hits were downloaded from the NCBI Assembly (https://www.ncbi.nlm.nih.gov/datasets/genome/). As many significant blast hits resulted in the characterization of non-Troyka retroelements, such as Metaviridae/Gypsy, only the genome assemblies from which Troyka elements were characterized in this study are listed in Table S1, available in the online Supplementary Material.

Censor [29] searches were performed with the protein of Troyka-1_TeGr from the blood clam Tegillarca granosa in Repbase as queries against each genome assembly. Censor hits were extracted and clustered with BLASTCLUST 2.2.25 in the NCBI blast package with the thresholds at 75% length coverage and 75% sequence identity. The consensus sequence for each cluster was generated with the 50% majority rule applied with the help of homemade scripts. Censor searches were again performed with the consensus sequence of each cluster against the respective genome assembly. Up to 10 Censor hits were extracted with 10,000 bp flanking sequences on both sides. Consensus sequences were regenerated to be elongated to find LTRs. The full-length consensus sequences were determined based on the presence of LTRs at both ends and flanking 3 to 6 bp TSDs. All characterized consensus sequences are available as supplementary materials (Data S1) and are also submitted to Repbase (https://www.girinst.org/repbase/).

Terminal signatures and TSDs

To confirm the termini and TSDs, four groups of organisms whose genomes have been sequenced are chosen for the analysis: four Polites species (Polites rus, Polites lutea, Polites cylindrica and Polites divaricata) in Cnidaria, five Mytilus species (Mytilus galloprovincialis, Mytilus californianus, Mytilus coruscus, Mytilus edulis and Mytilus trossulus) in Mollusca, three Heliocidaris species (Heliocidaris crassispina, Heliocidaris erythrogramma and Heliocidaris tuberculata) in Echinodermata and five species in Leuciscinae (Rutilus rutilus, Abramis brama, Squalius cephalus, Scardinius erythrophthalmus and Vimba vimba) in Chordata. Censor searches were performed with the genomes of P. rus, M. trossulus, H. erythrogramma and R. rutilus against the Troyka consensus sequences from these respective genomes. The entire Troyka sequence and flanking sequences were extracted. The junctions and TSD were determined manually. The entire Troyka and one TSD sequence are removed to prepare the pre-inserted sequence. Censor searches were performed with the genomes of sibling species against the pre-inserted sequence to detect the orthologous loci.

Sequence alignment and phylogenetic analysis

The protein sequences longer than 700 residues encoded by Troyka LTR retrotransposons were aligned with MAFFT [30] with default parameters. The alignment was manually investigated to remove weakly aligned sequences, caused by frameshift or deletion. The final alignment contains 457 sequences of 1,409 sites. A maximum likelihood tree was generated at the PhyML 3.0 server (http://www.atgc-montpellier.fr/phyml/) [31] with an approximate likelihood-ratio test. The substitution model Q.insect+R+F was used based on the Bayesian information criterion (BIC). The phylogenetic tree was rooted at the midpoint and visualized with FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

To examine the relationship of Troyka to other groups of virus-like retroelements, the alignments of protein domains from Metaviridae/Gypsy, Belpaoviridae/BEL, Pseudoviridae/Copia, Retroviridae and Caulimoviridae were downloaded from the Gypsy Database (https://gydb.org/) [14] on 24 July 2024. DIRS, ERV4 [32] and endogenous foamy virus [33] sequences were extracted from Repbase (https://www.girinst.org/repbase/) [13] and selected to represent the diversity of these groups. Nackednaviruses [23], HEART [22], Pliego virus [34], Lokiretroviruses [35] and Odin LTR retrotransposons [21] were obtained from the sequences reported in publications.

The multiple alignment of each domain (RP, RT, RH and IN) was generated using MAFFT [30] and muscle [36] with default parameters. As well as the single domain alignments, the concatenated sequences of multiple domains were also used for the phylogenetic analysis. The alignments used for the phylogenetic analysis were (1) the RT domain only, composed of 370 sequences of 573 sites; (2) the concatenated sequences of RT and RH domains, composed of 298 sequences of 875 sites; (3) the concatenated sequences of RP, RT and IN domains, composed of 243 sequences of 1,238 sites; and (4) the concatenated sequences of RP, RT, RH and IN domains, composed of 173 sequences of 1,336 sites.

Maximum likelihood trees were generated at the PhyML 3.0 server (http://www.atgc-montpellier.fr/phyml/) [37] with 100 bootstrap supports. The substitution model Q.pfam+R+F was selected based on the BIC. The phylogenetic tree was unrooted and visualized with FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Results and discussion

Phylogenetic distribution

Repbase keeps LTR retrotransposons and retroviruses split into two parts: LTR and internal portion. As of 24 July 2024, Repbase [13] contained 34 Troyka entries (17 LTRs, 16 complete internal portions and 1 incomplete internal portion) from 11 species of 6 animal phyla (Cnidaria, Arthropoda, Mollusca, Nemertea, Echinodermata and Chordata). Although the analysis was not systematic, this study characterized 586 LTRs and 589 internal portions from 256 species (Tables 1 and S1). Among them, 581 pairs of LTRs and internal portions were annotated. The internal portions for the five LTRs were not determined. The LTRs of the three internal portions were completely identical to the LTRs of sibling species. The LTR for Troyka-1_MagHon-I from Magallana (Crassostrea) hongkongensis is identical to Troyka-2_MagAng-LTR from Magallana angulata, the LTR for Troyka-1_OxySte-I from Oxygymnocypris stewartii is identical to Troyka-1_GymEck-LTR from Gymnocypris eckloni and the LTR for Troyka-3_EptAta-I from Eptatretus atami is identical to Troyka-3_EptOki-LTR from Eptatretus okinoseanus. They represent the activity of Troyka in the common ancestors of sibling species. The remaining five internal portions correspond to internally deleted derivatives.

Table 1. Distribution of Troyka LTR retrotransposons. The numbers of species whose genomes contain Troyka sequences are shown in parentheses.

Phylum	Class	Order (no. of species with Troyka)
Cnidaria	Hexacorallia	Actiniaria (6), Scleractinia (17), Antipatharia (1)
	Octocorallia	Alcyonacea (1), Scleralcyonacea (2), Malacalcyonacea (1)
	Hydrozoa	Leptothecata (1), Anthoathecata (2), Siphonophorae (1)
	Scyphozoa	Semaostomeae (2), Rhizostomeae (2)
	Cubozoa	Carybdeida (1)
Echinodermata	Crinoidea	Comatulida (2)
	Asteroidea	Forcipulatida (5), Valvatida (2), Paxillosida (2)
	Ophiuroidea	Amphilepidida (3)
	Echinoidea	Temnopleuroida (2), Camarodonta (5), Clypeasteroida (1), Diadematoida (2)
	Holothuroidea	Aspidochirotida (1), Apodida (1)
Hemichordata	Enteropneusta	Harrimaniidae* (1), Ptychoderidae* (1)
Chordata	Leptocardii	Amphioxiformes (3)
	Ascidiacea	Stolidobranchia (2)
	Myxini	Myxiniformes (4)
	Chondrichthyes	Heterodontiformes (1)
	Actinopterygii	Notacanthiformes (1), Anguilliformes (2), Clupeiformes (10), Cypriniformes (38), Characiformes (1), Stomiiformes (1), Myctophiformes (4), Lampriformes (2), Gadiformes (2), Kurtiformes (1), Blenniiformes (1), Carangiformes (2), Perciformes (5), Spariformes (2)
Arthropoda	Thecostraca	Balanomorpha (2), Scalpellomorpha (1), Sacculinidae* (1)
	Malacostraca	Decapoda (2)
Nemertea	Pilidiophora	Heteronemertea (2)
Priapulida	Priapulimorpha	Priapulimorphida (1)
Mollusca	Bivalvia	Venerida (12), Adapedonta (2), Cardiida (9), Lucinida (7), Myida (4), Pterioida (4), Mytilida (11), Ostreida (13), Arcoida (3), Pectinida (5)
	Gastrapoda	Patellidae* (4), Lottiidae* (2), Trochida (3), Lepetellida (4), Neomphalida (1)
	Cephalopoda	Seppida (2), Myopsida (2)
	Scaphopoda	Gadilida (1), Dentaliida (1)
	Polyplacophora	Chitonida (2)
	Caudofoveata	Chaetodermatida (1)
Annelida	Sipuncula	Golfingiida (1)
	Polychaeta	Xenopneusta (1), Phyllodocida (2), Eunicida (1), Sabellida (5), Scolecida (1), Spionida (1), Terebellida (2), Magelonidae* (1)
Phoronida	–	Phoronidae* (1)
Brachiopoda	Lingulata	Lingulida (1)

*No order is assigned.

In addition to the six animal phyla mentioned above, Hemichordata, Priapulida, Annelida, Phoronida and Branchiopoda also include species whose genomes have Troyka insertions. No Troyka was found outside of Metazoa.

Troyka is particularly widely distributed in Cnidaria and Mollusca. In Cnidaria, Troyka is distributed in five classes (Hexacorallia, Octacorallia, Hydrozoa, Scyphozoa and Cubozoa). Among the 30 Hexacorallia genomes analysed, 24 genomes contain at least 1 Troyka element. In Mollusca, Troyka is distributed in seven classes (Bivalvia, Gastropoda, Cephalopoda, Scaphopoda, Polyplacophora and Caudofoveata) and is especially rich in Bivalvia; among the 86 genomes analysed, 70 contain at least one Troyka element. Although the distribution is patchy in Chordata, out of 66 genomes from Cypriniformes, 38 contain Troyka insertions. In Arthropoda, Troyka was found only in two groups of crustaceans, Thecostraca (barnacles and crabhook barnacles) and Decapoda (shrimps). Although no genomic sequence of Troyka was characterized, the blast search against transcriptome shotgun assembly (TSA) in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) suggested the presence of Troyka in the family Gammaridae in Amphipoda too (Eulimnogrammarus verrucosus, Marinogammarus marinus, Gammarus lacustris and Hirondellea gigas).

There is no instance of Troyka in terrestrial animals. Troyka was not observed from terrestrial Chordata (Amphibia, Reptilia, Aves and Mammalia), Arthropoda (Hexapoda, Myriapoda and Arachnida), Annelida (Clitellata) or Mollusca (Caenogastropoda and Heterobranchia in Gastropoda), despite the vast genome sequence information of mammals, birds and insects (Table 1). It suggests that Troyka has not successfully inhabited the land.

Protein domain structure

The LTR length ranges between 100 and 400 bp. The internal portion is between 1,553 and 7,691 bp in length. The majority of Troyka LTR retrotransposons encode a single Gag-Pol protein which includes CCHC zinc finger, RP, RT, RH and IN, in the identical order of Metaviridae/Gypsy and Belpaoviridae/BEL. Only a few Troyka elements encode Gag and Pol as separate proteins. No Troyka element encodes an envelope protein. The HHpred [38] analysis with Troyka Gag proteins showed their similarity to the Gag proteins from Copia, Ty3, HIV-1 and the Gag-derived protein dArc [39] in both capsid and nucleocapsid domains. The zinc finger at the C-terminus of Troyka Gag protein can be shown as CX₂CX_8-10HX₄C, which is distinct from CX₂CX₄HX₄C seen in other virus-like retroelements (Retroviridae, Metaviridae/Gypsy, Pseudoviridae/Copia, Belpaoviridae/BEL and Caulimoviridae) [15]. No tether domain is present between RT and RH domains, unlike Retroviridae, Lokiretroviruses and Odin LTR retrotransposons. The GPF/Y motif, which is a hallmark of IN of Metaviridae/Gypsy, Retroviridae, Odin and Ginger1 DNA transposons [40], was not found in Troyka, either.

Some short Troyka elements appear not to encode a functional Gag-Pol or Pol protein. Such non-autonomous elements are especially abundant in sea urchins (Echinoidea); 62 of 70 non-autonomous Troyka elements found in this study were from sea urchins.

Transmission patterns

The patchy distribution of Troyka in animals implies the horizontal transfer during evolution. The phylogenetic analysis using the entire Troyka Pol protein sequences revealed that Troyka elements from related organisms tend to cluster together, indicating their long-term vertical transmission (Fig. 1). At the same time, multiple events of horizontal transfer of Troyka were also indicated.

Fig. 1. Phylogenetic tree of Troyka LTR retrotransposons based on the entire Pol protein. Branches are coloured based on the host phylogeny of Troyka. Three lineages from ray-finned fishes are indicated with arcs V1–V3. The original phylogenetic trees and protein alignments are available as Data S2 and S3. The species abbreviations in Troyka names are shown in Table S1.

Troyka from ray-finned fishes constitutes three lineages. The lineage V1 consists of three Troyka elements from Anguilliformes (Troyka-1_SynKau, Troyka-2_GymJav) and Kurtiformes (Troyka-1_ApoImb). The lineage V2 consists of nine Troyka elements from Clupeiformes (Troyka-1_ClH, Troyka-1_SprSpr, Troyka-1_SarPil, Troyka-2_EngEnc, Troyka-2_SarPil, Troyka-2_SalLon and Troyka-3_SarLon), Characiformes (Troyka-1_AsMe) and Gadiformes (Troyka-1_PolPol). All other 38 Troyka elements from ray-finned fishes belong to the lineage V3, which includes Troyka from Cypriniformes, Clupeiformes, Carangiformes, Lampriformes, Notacanthiformes and Myctophiformes. The two fish genomes from Clupeiformes, European anchovy Engraulis encrasicolus and Indian oil sardine Sardinella longiceps, contain both V2 and V3 lineages of Troyka.

Troyka elements from the same family in the order Cypriniformes clustered together (Figs 1 and S1), suggesting their vertical inheritance. However, the relationships between host families and the Troyka phylogeny are not consistent (Fig. S1). The family Cobitidae belongs to the suborder Cobitoidei, while Xenocyprinidae, Gobionidae, Leuciscidae and Cyprinidae belong to the suborder Cyprinoidei [41]. Troyka elements from Cyprinidae are more closely related to those from Cobitidae than Lauciscidae, Gobionidae or Xenocyprididae. This discrepancy could be explained by the horizontal transfer of Troyka between related organisms. Horizontal transfer of transposons is much more frequent in ray-finned fishes than in mammals and birds [42]. It is very likely that horizontal transfer between closely related organisms occurs more often than between distantly related organisms. The maintenance of multiple lineages of Troyka in Cypriniformes is less likely, considering that no genome from Cypriniformes contains multiple active Troyka elements.

The lineages V1 and V2 are nested in a Troyka lineage from corals (Hexacorallia, Cnidaria) and the lineage V3 is in a Troyka lineage from scallops (Pectinidae, Bivalvia, Mollusca). These phylogenetic relationships suggest the occurrence of multiple ancient horizontal transfer events between major animal groups. The sister lineage of V3 is composed of two Troyka elements from Mimachlamys varia and Ylistrum balloti. Their sister lineage is constituted by Troyka elements from Mizuhopecten yessoensis, Argopecten irradians and Pecten maximus. The phylogeny of Troyka is not consistent with the phylogeny of host scallops; Mimachlamys and Mizuhopecten belong to the subfamily Pedinae, while Ylistrum, Argopecten and Pecten belong to the subfamily Pectininae. It is suggested that Troyka has been maintained in scallops with the combination of vertical transmission and infrequent horizontal transfer events among scallops. The lineage V3 likely originated by the horizontal transfer from scallops to fish.

The phylogeny of Troyka indicates that they are maintained mainly through vertical inheritance but relatively frequent horizontal transfer among related organisms and rare cross-phylum horizontal transfer cannot be negligible. The exclusive presence of Troyka in the aquatic environment might contribute to this pattern of maintenance. The relative stability of extracellular DNA in aquatic than terrestrial environments could be a positive factor for horizontal transfer.

Terminal signatures and TSDs

The 5′-CG.CG-3′ termini of LTRs of Troyka are the distinguished feature of Troyka in LTR retrotransposons. To confirm the termini and the alteration upon integration, the genome comparison of closely related species was performed (Fig. 2). Four groups of organisms whose genomes were sequenced were chosen for the analysis: four Polites species (P. rus, P. lutea, P. cylindrica and P. divaricata) in Cnidaria, five Mytilus species (M. galloprovincialis, M. californianus, M. coruscus, M. edulis and M. trossulus) in Mollusca, three Heliocidaris species (H. crassispina, H. erythrogramma and H. tuberculata) in Echinodermata and five species in Leuciscinae (R. rutilus, A. brama, S. cephalus, S. erythrophthalmus and V. vimba) in Chordata. The 5′-CG.CG-3′ termini and 3 bp TSDs were confirmed in all insertions whose empty loci were identified, except for one case of Troyka-3_MyTr in the genome of M. trossulus, which generated 4 bp TSDs (CATG) (Fig. 2).

Fig. 2. Sequence comparison between the occupied and the unoccupied sites of Troyka LTR retrotransposons. The consensus sequences of Troyka LTR retrotransposons are shown in red and bold. The Troyka insertions are in red. TSDs are highlighted in yellow. (a) Troyka insertions in P. rus and their orthologous loci in P. lutea. (b) Troyka insertions in M. trossulus and their orthologous loci in M. edulis or M. galloprovincialis. (c) Troyka insertions in H. erythrogramma and their orthologous loci in H. tuberculata or H. crassispina. (d) Troyka insertions in R. rutilus and their orthologous loci in A. brama or S. cephalus.

Among the 603 Troyka LTRs characterized, 573 contain CGCCA at the 5′ terminus. The other 26 LTRs show a single transition (9 CACCA, 5 CGTCA, 4 CGCCG and 3 CGCTA) or a single transversion (2 CGCCC, 1 CGCCT, 1 CGCAA and 1 CGGCA) from CGCCA. Only four LTRs show more than two substitutions (1 CATCA, 1 CGCTG, 1 CGGCC and 1 CGGTG).

The 3′ termini of Troyka LTRs show more variations. Among the 603 Troyka LTR sequences, 522 contain TGGCG at the 3′ terminus. The 57 LTRs show a transition (18 TGACG, 4 TGGTG and 2 TGGCA) or a transversion (23 TGTCG, 8 TTGCG, 1 TGCCG and 1 TGGCT) from TGGCG. The positions −4 and −3 of TGGCG seem not to be restricted strictly, as the substitutions are found mostly (69 out of 81) only at these two positions.

These substitutions are not just mutations accumulated after transposition or errors introduced during consensus reconstruction. One Troyka lineage, composed of five entries (Troyka-2_PatPel, Troyka-2_PatUly, Troyka-8_PatVul, Troyka-10_PatCae and Troyka-10_PatVul) from the genus Patella, shares the TGTCG 3′ terminus. Another lineage, composed of three entries (Troyka-3_PelNoc, Troyka-1_MasPap and Troyka-04_ClyHem) from the two cnidarian classes (Hydrozoa and Scyphozoa) shares the TAACG 3′ terminus. One Troyka lineage from sea urchins includes multiple variants of 3′ terminus: 6 TTGCG, 3 TTACG, 2 TTTCG, 1 TGTCG and 1 TGGTG (Fig. S2A). One basal Troyka lineage from Mollusca and Annelida also shows multiple variants of the 3′ terminus: 5 TGACG and 3 TGTCG (Fig. S2B). In this lineage, two elements also show the variants at the 5′ terminus: CGTCA instead of CGCCA. These data indicate that the substitutions have been maintained during a certain evolutionary time and that the restriction of LTR termini is relaxed in these lineages.

The 5′-CG.CG-3′ termini of LTRs, along with 3 bp TSDs, are a clear indicator for the classification, even if they are not conclusive.

Transcription

The transcription of several Troyka elements was confirmed by the blast search against the TSA database at NCBI (Table S2). Long transcripts that cover substantial portions of Troyka insertions were detected for Troyka-1_NV (N. vectensis, sea anemone), Troyka-1_MyGa (M. galloprovincialis, mussel), Troyka-1_MyEd (M. edulis, mussel), Troyka-1_PeMa and Troyka-2_PeMa (P. maximus, scallop) and Troyka-2_HaRuf (Haliotis rufescens, abalone) (Fig. S3). Most of these transcripts contain non-Troyka sequences, indicating they are read-through transcripts and are not involved in the function or transposition of Troyka elements. The transcript of Troyka-2_HaRuf (GJDU01131704.1) may represent a transcript that is used for reverse transcription, as it starts in the 5′ LTR and ends in the 3′ LTR.

Recently active Troyka elements

Several Troyka elements seem to be active or have been active very recently. The autonomous Troyka elements, with an average sequence identity of greater than 99.9%, were chosen to investigate their recent activities in the respective genomes (Table S3). For each Troyka element, from 2 to 69 insertions were characterized with 3 bp TSDs. Recently active Troyka elements include Troyka-1_AcEq (24 insertions) from Actinia equina, Troyka-1_DenCri (25 insertions) from Dendrophyllia cribrosa, Troyka-2_HelCra (18 insertions) from H. crassispina, Troyka-1_MimVar (26 insertions) from Mimachlamys varia and Troyka-1_ParEch (43 insertions) and Troyka-4_ParEch (69 insertions) from Paraescarpia echinospica. Unfortunately, no transcription data are currently available for these Troyka elements, but these Troyka elements would be good candidates for functional characterization of this distinct lineage of retroelements.

Phylogenetic position in virus-like retroelements

The unique characteristics of termini and target alteration let me reanalyse the phylogenetic position of Troyka in virus-like retroelements. Troyka is a type of LTR retrotransposon and was originally reported to belong to Metaviridae/Gypsy [28]. Well-studied groups of virus-like retroelements are Metaviridae/Gypsy, Belpaoviridae/Bel, Pseudoviridae/Copia, Retroviridae, Caulimoviridae and Hepadnaviridae [11,14]. Here, Lokiretroviruses are excluded from Retroviridae as they cluster with Odin LTR retrotransposons [21]. As TATE, VIPER and HEART have only a few sample sequences and they often contain mutations at conserved residues in the RT domain, these three groups were excluded from the analysis. The preliminary phylogenetic analyses indicated that TATE and VIPER are closely related to other members of DIRS retrotransposons and HEART is related to Hepadnaviridae and Nackednaviruses.

The RH domain of Retroviridae, Lokiretroviruses and Odin LTR retrotransposons is not orthologous to those from Troyka [18,21, 35]. The RP domain is not found in Hepadnaviridae, Nackednaviruses or DIRS. IN domains are not found in Caulimoviridae, Hepadnaviridae, Nackednaviruses or DIRS. The phylogenetic trees based on the solo RT domain (RT tree) and the concatenated sequences of RT and RH domains (RT-RH tree), of RP, RT and IN (RP-RT-IN tree) and of RP, RT, RH and IN (RP-RT-RH-IN tree) were generated (Fig. 3). The monophylies of Pseudoviridae/Copia, Belpaoviridae/BEL, Troyka, Caulimoviridae and Blubervirales (Hepadnaviridae and Nackednaviruses) were well-supported. The monophyly of Lokiretroviruses and Odin LTR retrotransposons (Loki-Odin) was also supported, but the monophyly of Retroviridae and Loki-Odin was only weakly supported (35% in RT and 45% in RP-RT-IN). The monophyly of DIRS retrotransposons was not confirmed, even excluding TATE and VIPER.

Fig. 3. Phylogenetic trees of LTR retrotransposons and related retroelements based on the alignments of (a) the RT domain only; (b) the concatenated RT and RH domains; (c) the concatenated RP, RT and IN domains; and (d) the concatenated RP, RT, RH and IN domains. All trees are unrooted. Bootstrap values of 100 replicates for retroelement groups are shown in parentheses if available. In (a) and (c), Metaviridae/Gypsy was not indicated as a monophyletic lineage. The original phylogenetic trees and protein alignments are available as Data S4–S11.

Metaviridae/Gypsy did not constitute a well-supported distinct branch in the virus-like retroelements. The lineage of Metaviridae/Gypsy often includes the Caulimoviridae, Retroviridae and Loki-Odin branches. The inclusion of Caulimoviridae in the Metaviridae/Gypsy was also observed in previous studies [4,22], indicating that Caulimoviridae originated from Metaviridae/Gypsy. The two established internal groups of Metaviridae/Gypsy, chromoviruses and errantiviruses, were supported by over 50% bootstrap values in all trees.

When focusing on retroelements having LTRs, five retrotransposon groups (Pseudoviridae/Copia, Belpaoviridae/BEL, Troyka, Metaviridae/Gypsy and Odin) and two virus groups (Retroviridae and Lokiretroviruses) have been recognized (Fig. 4). The relationships among Pseudoviridae/Copia, Belpaoviridae/BEL, Metaviridae/Gypsy and Retroviridae have been investigated in detail [22,35, 43]. Lokiretroviruses and Odin LTR retrotransposons were reported as a sister lineage of Retroviridae [21], and the phylogenetic analysis here is consistent with the previous studies (Fig. 3).

Fig. 4. Schematic diagram of the evolution of LTR retrotransposons. Bootstrap values of 100 replicates in the phylogenetic trees of the RT only (Fig. 3a), RT-RH (Fig. 3b), RP-RT-IN (Fig. 3c) and RP-RT-RH-IN (Fig. 3d) are shown in parentheses. Minus indicates the corresponding cluster was not indicated. ‘nd’ indicates the sequences corresponding to the lineage were not included in the analysis. Canonical protein domain structures are shown at right. Vertical lines indicate the presence of the GPF/Y motif in the IN domain. CA, capsid; NC, nucleocapsid; RP, retropepsin; RT, reverse transcriptase; RH, ribonuclease H; IN, integrase.

Troyka is clearly distinguished from Retroviridae, Lokiretroviruses or Odin LTR retrotransposons as Troyka lacks the tether domain and their RH is orthologous to those from Metaviridae/Gypsy (Fig. 4). The phylogeny clearly distinguishes Troyka from Pseudoviridae/Copia or Belpaoviridae/BEL (Fig. 4). The remaining question is the relationship of Troyka with Metaviridae/Gypsy. The phylogeny of various sets of protein domain alignments generated fundamentally the same phylogenetic relationships: Troyka as a sister lineage of the cluster of Metaviridae/Gypsy, Caulimoviridae, Retroviridae and Loki-Odin (Fig. 3). There is no support for Troyka to be included in Metaviridae/Gypsy. The paraphyly of Metaviridae/Gypsy against Caulimoviridae, Retroviridae and Loki-Odin should be reinvestigated with an expanded dataset, and it would lead to the division of Metaviridae/Gypsy into several families in the virus taxonomy and superfamilies in the transposon taxonomy. The GPF/Y motif in the IN domain observed in Metaviridae/Gypsy, Retroviridae, Odin and Ginger1 DNA transposons suggests their common ancestry [40]. The absence of the GPF/Y motif in the IN domain is another support for the position of Troyka outside of Metaviridae/Gypsy, although some Metaviridae/Gypsy lineages, such as chromoviruses and errantiviruses, do not have the GPF/Y motif, and thus, the lack of the GPF/Y domain could be a derived feature. Finally, the unique characteristics of termini and TSDs of Troyka justify their distinction from Metaviridae/Gypsy. Here, Troyka is proposed as a new superfamily of LTR retrotransposons in the transposon taxonomy. It could also be considered to propose a new family Trojicaviridae inside the order Ortervirales in the virus taxonomy in the future.

Abbreviations

BIC

Bayesian information criterion

ERVs

endogenous retroviruses

IN

integrase

RH

ribonuclease H

RP

retropepsin

RT

reverse transcriptase

TERT

telomerase reverse transcriptase

TSA

transcriptome shotgun assembly

TSDs

target site duplications