Evolution of gypsy Endogenous Retrovirus in the Drosophila obscura Species Group

Rafael P. V1,ázquez-Manrique*, Mariano Hernández{dagger}, M. José Martínez-Sebastián* and Rosa de Frutos3,*

*Departamento de Genética, Facultad de Ciencias Biológicas, Universitat de València, Burjassot, Spain; and
{dagger}Departamento de Genética, Universidad de La Laguna, La Laguna, Tenerife, Spain

Abstract

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.

Introduction

The Ty3/gypsy family of retroelements is widely distributed among eukaryotes. In addition to the yeast Ty3 and Drosophila melanogaster gypsy elements, a large number of families have been described in a wide range of organisms. Most of these elements have been found in Drosophila and plant species, but they are also present in protozoa, fungi, nematodes, vertebrates, and insects other than Drosophila. Retrotransposons constitute a variable fraction of their host genomes; they seem to account for a significant fraction of plant genomes, up to 50% in the case of Zea mays (SanMiguel et al. 1996Citation ). In D. melanogaster, a large number of different retrotransposon families have been found. In spite of this variety, the total amount of retrotransposons seems to be small, only 1.8% of the 2.9-Mb-long Adh region from D. melanogaster corresponded to identified transposable elements (Ashburner et al. 1999Citation ).

The Ty3/gypsy family is closely related to retroviruses. In the phylogenetic trees, based on the analyses of conserved domains from reverse transcriptase, integrase, and RNasa H, Ty3/gypsy elements cluster with retroviruses (Xiong and Eickbush 1988, 1990Citation ; Springer and Britten 1993Citation ; Malik and Eickbush 1999Citation ). Several features from the Ty3/gypsy elements are similar to those of retroviruses, for example, in both groups, reverse transcriptase always precedes integrase. This fact has been used as a systematic tool, and in this way, eukaryotic retrotransposon have been divided into Metaviridae (Ty3/gypsy elements) and Pseudoviridae (Ty1/copia elements) (Boeke et al. 1998a, 1998bCitation ). This resemblance is more remarkable in the Ty3/gypsy element skipper, where pro appears as a separate gene, as it does in retroviruses (Leng et al. 1998Citation ). Inside the Metaviridae family, a group of elements present a third open reading frame (ORF) resembling the retroviral gene env, which would place them even closer to retroviruses. Based on the absence or presence of env-like genes in the retroelements, the Metaviridae family has been divided into two genera, Metavirus and Errantivirus, respectively (Boeke et al. 1998aCitation ).

Kim et al. (1994)Citation provided the first evidence of horizontal transfer from a strain containing actively transposing gypsy elements to an "empty" strain, suggesting that the gypsy element from D. melanogaster is an infectious retrovirus. It was subsequently reported that the 2.1-kb mRNA produced by differential splicing generates a predicted protein with the characteristics of functional retroviral envelope protein. The gypsy Env protein is glycosylated and processed as in retroviruses and is found associated with viruslike particles (VLPs) from flies carrying the flam permissive mutation, which allows the mobilization of gypsy elements (Pélisson et al. 1994, 1997Citation ; Song et al. 1994, 1997Citation ; Bucheton 1995Citation ; Prud'homme et al. 1995Citation ; Chalvet et al. 1998, 1999Citation ). These facts suggest that viral infectivity of the D. melanogaster gypsy element is dependent on Env. It was recently established that viral particles formed by the MoMLV-based retroviral vector packaged with the gypsy Env protein are able to infect Drosophila cells, which strongly supports the role of Env as the infectious property of gypsy (Teysset et al. 1998Citation ).

The Errantivirus elements are present in fungui, plants, nematodes, and insects, and all of them possess env-like genes, which, in the case of 297, 17.6, TED, Tom, ZAM, Idefix, and Athila, display potentially functional structures such as those shown for gypsy (Song et al. 1994Citation ; Leblanc et al. 1997Citation ; Wright and Voytas 1998Citation ; Desset et al. 1999Citation ). However, ZAM and Idefix from D. melanogaster and TED from the lepidopteron Trichoplusia ni seem to be infective. The presence of env genes is not restricted to Errantivirus, and thus the copia/Ty1 SIRE-1 element encodes an envelope-like protein as well (Laten, Majumdar, and Gaucher 1998Citation ).

Sequences homologous to gypsy from D. melanogaster are distributed throughout Drosophila species. Southern hybridization signals have been detected in most of the analyzed species belonging to Sophophora and Drosophila subgenera (Stacey et al. 1986Citation ; de Frutos, Peterson, and Kidwell 1992Citation ; Loreto et al. 1998Citation ). The widespread presence of gypsy-like sequences suggests the existence of ancestral elements in the genomes of Drosophila species before early radiations. Full gypsy-like sequences in Drosophila subobscura and Drosophila virilis have already been analyzed (Mizrokhi and Mazo 1991Citation ; Alberola and de Frutos 1996Citation ), showing a structure similar to that of the gypsy element from D. melanogaster, which indicates that they are transcriptionally and transpositionally active. The three species posses well-conserved Env proteins, although the D. subobscura and D. virilis ones lack some essential domains needed to produce functional proteins (Alberola and de Frutos 1996Citation ). In this paper, we report a phylogenetic study based mainly on the analysis of the 5' region of the env genes from different species of the obscura group, including previously reported sequences from D. melanogaster, D. virilis, and Drosophila hydei. Our work shows that gypsy elements from the analyzed species of the obscura group appear as a monophyletic group, which is highly diverged from the D. melanogaster prototypic gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, supporting vertical transmission.

Materials and Methods

Drosophila Stocks
Ten species from the obscura group were analyzed. Drosophila obscura, Drosophila ambigua, Drosophila bifasciata, Drosophila pseudoobscura, Drosophila miranda, Drosophila persimilis, Drosophila azteca, and Drosophila affinis were obtained from The National Drosophila Species Resources Centre, Bowling Green State University. Drosophila madeirensis and Drosophila guanche, are laboratory strains from Tenerife (Canary Island).

PCR DNA Amplification
Genomic DNA from each species was prepared as in Junakovic, Caneva, and Ballard (1984)Citation , with some modifications. Degenerate primers were designed from alignments of gypsy elements from D. melanogaster, D. subobscura, D. virilis, and D. hydei. These primers amplify a fragment of approximately 450 bp corresponding to positions 5354–5801 of the D. subobscura gypsy element (fig. 1 ). Because the amplification was not successful in D. guanche and D. azteca, two new primers were used (positions 5229–5972). The sequences of these primers and their positions are as follows: DIR5354, 5'-CTG T(CT)C T(CT)C TTA AGG GGA GGG-3' REV5801, 5'-(AG)CC (AC)GC (AC)AC AAG CTT (CT)AA GGC-3' DIR5229, 5'-(CT)(GC)C (AT)AC CCG GCA AAA CCG CG-3' REV5972, 5'-(AT)GG AGT GTC GAC CAA (AG)TC GCC-3'.



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Fig. 1.—Structure of the Drosophila subobscura gypsy element. The analyzed region is indicated.

 
PCR reactions were generally carried out using 100 ng of genomic DNA, 80 pmol of each primer, 2 mM dNTPs, and 2.5 U of Taq polymerase. The amplifications were performed in a Perkin Elmer 2400 cycler under the following conditions: a single cycle for denaturing at 94 °C for 5 min, 30 cycles of 94°C for 45 s, 58°C for 1 min, and 72°C for 45 min, and the final extension at 72°C for 5 min.

Cloning and Sequence Analysis
Electroeluted fragments were cloned into pCRscript Amp (+) (Stratagene) at the EcoRV site. Vector restriction and ligation was carried out at the same time on the ligase buffer, at room temperature (20–25°C). Four to six clones from each species were sequenced in an automatic ABI-Prism sequencer. Multiple alignments were performed with the CLUSTAL X program (Thompson et al. 1997Citation ). Phylogenetic trees were inferred by the neighbor-joining method (Saitou and Nei 1987Citation ), based on the p-distance MEGA program, version 1.02 (Kumar, Tamura, and Nei 1993Citation ), and by the parsimony method with PAUP program, version 3.1.1.1 (Swofford 1993Citation ), using the heuristic option with random stepwise addition of sequences (10 replicates) and tree bisection-reconnection (TBR) branch swapping. Bootstrapping was performed using both neighbor-joining and parsimony methods (200 replicates).

Results

Phylogenetic Analyses of gypsy Sequences
DNA amplification was obtained from all of the analyzed species with the exception of D. guanche, indicating that they are widely distributed among the obscura group. The presence of deleted gypsy elements in D. guanche, which would explain the absence of amplification in this species, had already been described by de Frutos, Peterson, and Kidwell (1992)Citation . The genomic DNA of all analyzed species was probed against the amplified PCR fragment from D. subobscura obtained in our study. Hybridization was observed in all the species except D. guanche (data not shown), indicating that in this species the deletion of gypsy elements completely includes the analyzed region.

Figure 2 shows the phylogenetic relationships between gypsy elements from Drosophila species. The tree is based on the comparison of the 450-bp PCR sequences obtained in our study and the gypsy sequences of D. melanogaster (Z31368, the proptotypic element, and AC006215), D. virilis (M38438), and D. hydei (X74538, X74539, and X74543). The amplified fragment includes the 3' end of the pol gene, the 5' region of the env gene, and the intergenic region between them (fig. 1 ). We selected the intergenic region because of scarce variability described for the coding regions of Drosophila gypsy elements (Alberola and de Frutos 1996Citation ). In order to determine the potential functionality of Env, we also included the N-terminal env region. The phylogenetic tree was constructed by the neighbor-joining method (Saitou and Nei 1987Citation ) using the yoyo element as the outgroup (Zhou and Haymer 1998Citation ). An identical topology was obtained with the parsimony method. The branching of the tree was supported by high bootstrap values. The most remarkable facts that could be inferred from the analysis of the phylogenetic tree are as follows:



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  Fig. 2.—Neighbor-joining tree based on total nucleotide divergence from the analyzed gypsy sequences. The bootstrap values on the nodes are percentages for 200 replicates. The tree was rooted by using yoyo as an outgroup. Sequences of Drosophila melanogaster, Drosophila hydei, and Drosophila virilis are named according to accession numbers. In sequences from species of the obscura group, the number of each clone is indicated

 
  1. The gypsy elements from species of the obscura group cluster as a monophyletic group which splits into two subgroups, one corresponding to the Nearctic species and the other corresponding to the Palearctic ones. The Nearctic species separate into pseudoobscura and affinis subgroups, and the Palearctic species separate into obscura and subobscura subgroups. These phylogenetic relationships among gypsy sequences are almost consistent with the ones established for the corresponding species (see Powell [1997]Citation for a revision). Within the obscura group, D. bifasciata has an uncertain phylogenetic position, different studies assign it to species of either obscura, subobscura, or pseudoobscura subgroups, as well as a separate clade, depending on which phylogenetic marker is considered (Loukas, Krimbas, and Vergini 1984Citation ; González et al. 1990Citation ; Barrio et al. 1992Citation ; Beckenbach, Wei, and Liu 1993Citation ; Barrio, Latorre, and Moya 1994Citation ; Acosta et al. 1995Citation ; Barrio and Ayala 1997Citation ; Gleason et al. 1997Citation ). It is remarkable that in our work, gypsy sequences from D. bifasciata cluster with the obscura subgroup species. The two sister clades in which the Nearctic species are divided are almost coincident with the phylogeny of the species. Although the two D. affinis sequences cluster with D. azteca, and with D. hydei and D. virilis, respectively, the differences are not significative. The five clones from D. azteca are the most diverged sequences among the analyzed obscura-like elements.
  2. The gypsy sequences D. hydei (X74538), D. hydei (X74539), and D. virilis (M38438) cluster with species from the affinis subgroup. The close relationships between D. subobscura and D. virilis gypsy elements with respect to that of D. melanogaster has previously been reported (Alberola and de Frutos 1993, 1996Citation ). The obscura group belongs to Sophophora subgenera, and both D. hydei and D. virilis belong to Drosophila subgenera. It has been proposed that divergence between both subgenera occurred 30–60 MYA (Throckmorton 1975Citation ; Beverley and Wilson 1984Citation ; Powell and DeSalle 1995Citation ; Powell 1997Citation ). Our present data agree with early results and support the existence of horizontal transfer events in the evolutionary history of gypsy elements among Drosophila species.
  3. The obscura-like gypsy elements have strongly diverged from the gypsy sequences of D. melanogaster and D hydei (X74543), where highly diverged copies coexist. The presence of different gypsy subfamilies in D. melanogaster had previously been described (Lambertsson, Andersson, and Jhoansson 1989Citation ; Lyubomirskaya 1990, 1993Citation ; Chalvet et al. 1998Citation ). It is interesting to note that from all analyzed gypsy sequences, the prototypic active D. melanogaster (Z31368) is the most diverged. Two obscura-like sequences (X74538 and X74539) and a diverged copy of the element (X74543) coexist in D. hydei. These three elements are truncated and very degraded copies and are located on chromosome Y (Hochstenbach et al. 1994, 1996Citation ).

Env Region
In addition to the pol 3' end and the intergenic region, the 450-bp analyzed fragment encompasses the 5' region of the env gene. The last region extends over the signal peptide (SP) and a stretch of approximately 100 amino acids of the Env protein surface domain (SU), including the gypsy splicing sites. As in retroviruses, the gypsy Env protein from D. melanogaster is generated from a subgenomic RNA produced by splicing. Remarkably, this splicing event generates a new start codon (Pélisson et al. 1994Citation ) with an AT pair provided by the 5' site and a G from the 3' site. We performed multiple alignments of the putative 3' splice sites from all of the analyzed sequences and the prototypic gypsy sequence of D. melanogaster (Z31368). All of them display a conserved motif, with the exception of the six D. azteca clones and D. hydei (X74543), where some deletions are present. This 3' splice motif includes the above-mentioned G in all sequences except D. hydei (X74539) and D. virilis (M38438). This chimerical structure for the ATG start codon of the Env protein is frequently found in retroviruses. Taking this ATG triplet as a start codon, the predicted polypeptides obtained are highly similar to those found in the functional Env product of D. melanogaster (fig. 3A ). The N-terminal region of the retroviral Env protein invariably contains a short hydrophobic signal peptide (Swanstrom and Wills 1997Citation ); we checked for the presence of this signal in all of the analyzed sequences with the PSORT program and found the N-terminal signal peptide extending through the first 15 amino acids, while in D. melanogaster (Z31368) the length was 13 residues, which is in agreement with the data from Pélisson et al. (1994)Citation . In D. melanogaster (AC006215) and D. hydei (X74538) sequences, a stop codon is present, originating truncated proteins. In the obscura species, the first 100 amino acids of the Env could produce functional proteins, as in D. melanogaster, although point mutations or single indels downstream of the analyzed region could originate frameshift or truncated proteins. A full gypsy element from D. subosbcura (X72390) had already been analyzed by Alberola and de Frutos (1996)Citation . Several Env regions from different D. subobscura strains were analyzed later by Alberola, Bori, and de Frutos (1997)Citation . In these works, an ATG codon located downstream of a putative one generated by splicing was considered as a functional start codon, and all of the analyzed sequences presented frameshift mutations induced by single indels, originating truncated proteins. We reanalyzed these sequences, taking the ATG generated by splicing as the start codon, but in all the cases the results where the same as before, with single indels causing truncated proteins (fig. 3B ).



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Fig. 3.—Structure of the Env proteins. A, Drosophila melanogaster (Z31368, AC006215), Drosophila hydei (X74538). The obscura species refer to Drosophila madeirensis, Drosophila obscura, Drosophila ambigua, Drosophila bifasciata, Drosophila pseudoobscura, Drosophila persimilis, Drosophila miranda, and Drosophila affinis. B, Env regions from different strains of Drosophila subobscura. White boxes indicate signal peptides. Shaded boxes indicate sequences that are not in frame. Upper vertical white bars indicate N-glicosylation sites, and lower bars indicate conserved cysteine residues

 
Discussion

The close phylogenetic relationships between Ty3/gypsy family of retrotransposons and retroviruses have been firmly established (Xiong and Eickbush 1988, 1990Citation ; Springer and Britten 1993Citation ; Wright and Voytas 1998Citation ; Malik and Eickbush 1999Citation ; Lerat and Capy 1999Citation ). However, the phylogenetic relationships between members of this family are not conclusive. This family constitutes a heterogenous group which is broadly extended among eukaryotes, from fungi to vertebrates. In this work, we analyzed the evolutionary relationships among gypsy elements belonging to Drosophila species. In the gypsy group of retroelements, gypsy presents characteristics of endogenous retroviruses (Kim et al. 1994Citation ; Pélisson et al. 1994Citation ; Song et al. 1994Citation ; Chalvet et al. 1998, 1999Citation ; Teysset et al. 1998Citation ). Under some circumstances, they can be rendered infective, but if they persist in the genome of a given species, their dynamic of expansion does not generate deleterious effects in their hosts. Endogenous retroviruses can be subject to constraints in order to acquire a lifestyle that is compatible with their host (Boeke and Stoye 1997Citation ). Transposition and/or infectious properties of gypsy seem to be controlled by the flamenco (flam) host gene (Bucheton 1995Citation ; Prud'homme et al 1995Citation ; Pélisson et al. 1997Citation ; Song et al. 1997Citation ). gypsy is stable in most Drosophila strains, but it transposes at high frequency in the unstable (MG) strains (Kim, Belyaeva, and Aslanian 1990Citation ; Lyubomirskaya et al. 1990, 1993Citation , Prud'homme et al. 1995Citation ), where the number of copies is higher than in stable strains. On the other hand, gypsy is widely extended among species from Sophophora and Drosophila subgenera (Stacey et al. 1986Citation ; de Frutos, Peterson, and Kidwell 1992Citation ; Loreto et al. 1998Citation ; Biémont and Cizeron 1999Citation ). This widespread distribution indicates the existence of ancient gypsy elements in Drosophila. If the specific characteristics of the gypsy retrotransposon of D. melanogaster were already present in the ancestral elements, transpositionally burst and/or infective events could have occurred in their evolutiary history. In species other than D. melanogaster, gypsy-like retrotransposons seem to be mainly located at the chromocentric region, with a small number of copies on the chromosome arms (Junakovic et al. 1998Citation ; Biémont and Cizeron 1999Citation ; Vieira et al. 1999Citation ). No vestiges of uncontrolled expansions can be inferred from these data; on the contrary, a strict control seems to limit the number of gypsy elements in the genomes of Drosophila species.

The gypsy elements analyzed in this work belong to the obscura species group of Drosophila. Since the pioneering works of Sturtevant (1942)Citation and Buzzati-Traverso and Scossiroli (1955)Citation , the phylogenetic relationships among species of this group have been extensively analyzed (Powell [1997]Citation for a review). Although phylogenetic relationships within species of the obscura group are not completely resolved, a division of the group into Palearctic and Nearctic species has been classically proposed. The Nearctic group presents the pseudoobscura and affinis subgroups, and Palearctic species are divided into subosbcura and obscura subgroups. A fifth subgroup, microlabis, which encompasses African species, has been described (Cariou et al. 1988Citation ). The phylogenetic analysis carried out here extends over species of the Nearctic and Palearctic subgroups, as no microlabis species were available. gypsy elements from species of the obscura group seem to constitute a highly homogeneous monophyletic group in which elements from Nearctic and Palaearctic species are sharply differentiated. The high bootstrap values support the monophyly of the pseudoobscura clade and the divergence between obscura and subosbcura subgroups. In summary, phylogenetic relationships among gypsy elements of the obscura group nearly coincide with those of its hosts, and we can conclude that no evidence of horizontal transmission has been found. We find that the location of D. bifasciata in this phylogenetic tree is quite remarkable. As we discussed previously, D. bifasciata ambiguously clustered with different species of the obscura group in the various phylogenetic analyses carried out before (Powell 1997Citation ). In addition, P and bilbo transposable elements from D. bifasciata are fairly diverged with respect to those of the obscura group (Clark, Maddison, and Kidwell 1994Citation ; Hagemann, Miller, and Pinsker 1994Citation ; Hagemann, Haring, and Pinsker 1996Citation ; García-Planells et al. 1998Citation ; Blesa, Gandía, and Martínez-Sebastián, unpublished data).

In contrast to the evolutionary pattern of gypsy elements among the obscura group species, elements from D. hydei and D. virilis are closely related to obscura-like elements; they turn out to be nearly identical to gypsy elements from Neartic species. It had already been established that gypsy elements of D. subosbcura were much closer to the ones from D. virilis than to those of D. melanogaster (Alberola and de Frutos 1993, 1996Citation ). Horizontal transfer between D. virilis and D. subobscura was proposed to explain this striking similarity. Our present data agree with this hypothesis. Horizontal transfer has been invoked to explain the evolutionary patterns of several families of transposable elements. In addition to evolution of the P element, for which horizontal transfer events are strongly documented (Daniels et al. 1990Citation ; Houck et al. 1991Citation ; Kidwell 1993Citation ; Clark and Kidwell 1997Citation ), apparent cases of horizontal transfer have been reported in mariner from Drosophila (Maruyama and Hartl 1991Citation ; Robertson 1993Citation ), SURL elements from echinoids (Springer et al. 1995Citation ; Gonzalez and Lessios 1999Citation ), magellan from Zea species (Purugganan and Wessler 1994Citation ), gypsy/Ty3-like elements from the tomato (Su and Brown 1997Citation ), and diverse vertebrate species (Miller et al. 1999Citation ), among others. Recently, direct evidence for a recent horizontal transfer of the copia between D. melanogaster and D. willistoni has also been reported (Jordan, Matyunina, and McDonald 1999Citation ). However, horizontal transfer as a mechanism to explain these phylogenetic discrepancies must be proposed with caution. Although transference of gypsy obscura-like elements from affinis species to the virilis group could have occurred, the scenario seems to be more complex. Both D. hydei (X74538) and (X74539) gypsy elements are truncated and rather degraded copies, lack LTRs, and are located on the heterochromatic Y chromosome (Hochstenbach et al. 1994, 1996Citation ). To decisively establish the existence of horizontal transfer will necessitate additional data about the presence of functional env genes in both species, as well as the evolution rates of active and defective elements. In fact, chromosome stochastic losses, existence of ancestral polymorphism, and variable evolution rates have been considered as alternative hypotheses (Capy, Anxolabéhère, and Langin 1994Citation ; Capy et al. 1997Citation ), and we cannot disregard them with our data.

Among the Ty3/gypsy family, gypsy is considered a retrovirus because it has the env gene, which is necessary for infection. All gypsy elements analyzed in this work have an env domain. Most of the obscura-like elements maintain the structural characteristics of an enveloped protein (obviously, these characteristics refer to approximately 100 amino acids of the N-terminal region). It will be interesting to analyze the full env region of the obscura-like gypsy elements in order to ascertain if gypsy retroviral lineages other than the prototypic element of D. melanogaster exist.

Acknowledgements

This work was supported by grant PB96–0803 DGYCIT.

Footnotes

Pierre Capy, Reviewing Editor

1 Present address: Unidad de Genética y Diagnóstico Prenatal, Hospital Universitario La Fe, Avda, Campanar, Valencia, Spain. Back

2 Keywords: Drosophila, gypsy, retrotransposons endogenous retroviruses evolution of transposable elements Back

3 Address for correspondence and reprints: Rosa de Frutos, Departamento de Genética, Facultad de Ciencias Biológicas, Dr. Moliner 50, 46100 Burjasot, Valencia, Spain. E-mail: rosa.frutos{at}uv.es Back

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Accepted for publication March 31, 2000.