Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene.

The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.

Evolution and phylogeny of insect endogenous retroviruses.

BACKGROUND: The genome of invertebrates is rich in retroelements which are structurally reminiscent of the retroviruses of vertebrates. Those containing three open reading frames (ORFs), including an env-like gene, may well be considered as endogenous retroviruses. Further support to this similarity has been provided by the ability of the env-like gene of DmeGypV (the Gypsy endogenous retrovirus of Drosophila melanogaster) to promote infection of Drosophila cells by a pseudotyped vertebrate retrovirus vector. RESULTS: To gain insights into their evolutionary story, a sample of thirteen insect endogenous retroviruses, which represents the largest sample analysed until now, was studied by computer-assisted comparison of the translated products of their gag, pol and env genes, as well as their LTR structural features. We found that the three phylogenetic trees based respectively on Gag, Pol and Env common motifs are congruent, which suggest a monophyletic origin for these elements. CONCLUSIONS: We showed that most of the insect endogenous retroviruses belong to a major clade group which can be further divided into two main subgroups which also differ by the sequence of their primer binding sites (PBS). We propose to name IERV-K and IERV-S these two major subgroups of Insect Endogenous Retro Viruses (or Insect ERrantiVirus, according to the ICTV nomenclature) which respectively use Lys and Ser tRNAs to prime reverse transcription.

Characterization of the flamenco region of the Drosophila melanogaster genome.

The flamenco gene, located at 20A1-3 in the beta-heterochromatin of the Drosophila X chromosome, is a major regulator of the gypsy/mdg4 endogenous retrovirus. As a first step to characterize this gene, approximately 100 kb of genomic DNA flanking a P-element-induced mutation of flamenco was isolated. This DNA is located in a sequencing gap of the Celera Genomics project, i.e., one of those parts of the genome in which the "shotgun" sequence could not be assembled, probably because it contains long stretches of repetitive DNA, especially on the proximal side of the P insertion point. Deficiency mapping indicated that sequences required for the normal flamenco function are located >130 kb proximal to the insertion site. The distal part of the cloned DNA does, nevertheless, contain several unique sequences, including at least four different transcription units. Dip1, the closest one to the P-element insertion point, might be a good candidate for a gypsy regulator, since it putatively encodes a nuclear protein containing two double-stranded RNA-binding domains. However, transgenes containing dip1 genomic DNA were not able to rescue flamenco mutant flies. The possible nature of the missing flamenco sequences is discussed.

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline.

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.

Potentially active copies of the gypsy retroelement are confined to the Y chromosome of some strains of Drosophila melanogaster possibly as the result of the female-specific effect of the flamenco gene.

Gypsy is an endogenous retrovirus present in the genome of Drosophila melanogaster. This element is mobilized only in the progeny of females which contain active gypsy elements and which are homozygous for permissive alleles of a host gene called flamenco (flam). Some data strongly suggest that gypsy elements bearing a diagnostic HindIII site in the central region of the retrovirus body represent a subfamily that appears to be much more active than elements devoid of this site. We have taken advantage of this structural difference to assess by the Southern blotting technique the genomic distribution of active gypsy elements. In some of the laboratory Drosophila stocks tested, active gypsy elements were found to be restricted to the Y chromosome. Further analyses of 14 strains tested for the permissive vs. restrictive status of their flamenco alleles suggest that the presence of permissive alleles of flam in a stock tends to be associated with the confinement of active gypsy elements to the Y chromosome. This might be the result of the female-specific effect of flamenco on gypsy activity.

Expression of the Drosophila retrovirus gypsy as ultrastructurally detectable particles in the ovaries of flies carrying a permissive flamenco allele.

The endogenous retrovirus gypsy is controlled by the Drosophila gene flamenco (flam). New insertions of gypsy occur in any individual Drosophila if its mother is homozygous for the flam1 permissive allele and contains functional gypsy proviruses. The ovaries of flam1 females also contain high amounts of gypsy RNAs. Unexpectedly however, gypsy derepression does not occur in the flam1 female germ-line proper but in the somatic follicular epithelium of the ovary. Since extracts from these females are able to efficiently infect the germ-line of a strain devoid of active gypsy proviruses, we assume that a similar kind of germ-line infection, which would occur inside the flam1 females themselves, could be required for gypsy insertions to occur in their progeny. This hypothesis was confirmed by electron microscopy observations showing that non-enveloped intracytoplasmic particles containing gypsy RNAs accumulate in the apical region of the flam1 follicle cells, close to specific membrane domains to which the gypsy envelope proteins are targeted, whereas both are absent in the flam+ controls. Low amounts of similar virus-like particles were also observed in flam1 oocytes, but it is not yet known whether they entered passively or as a result of membrane fusion. This is the first report of the beginning of a retrovirus cycle in invertebrates and these observations should be taken into account when explaining the maternal effect of the flamenco gene on the multiplication of gypsy proviruses.

About the origin of retroviruses and the co-evolution of the gypsy retrovirus with the Drosophila flamenco host gene.

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. According to phylogenetic data, gypsy belongs to the same group as the Ty3 class of LTR-retrotransposons, which suggests that retroviruses evolved from this kind of retroelements before the radiation of vertebrates. There are other invertebrate retroelements that are also likely to be endogenous retroviruses because they share with gypsy some structural and functional retroviral-like characteristics. Gypsy is controlled by a Drosophila gene called flamenco, the restrictive alleles of which maintain the retrovirus in a repressed state. In permissive strains, functional gypsy elements transpose at high frequency and produce infective particles. Defective gypsy proviruses located in pericentromeric heterochromatin of all strains seem to be very old components of the genome of Drosophila melanogaster, which indicates that gypsy invaded this species, or an ancestor, a long time ago. At that time, Drosophila melanogaster presumably contained permissive alleles of the flamenco gene. One can imagine that the species survived to the increase of genetic load caused by the retroviral invasion because restrictive alleles of flamenco were selected. The characterization of a retrovirus in Drosophila, one of the most advanced model organisms for molecular genetics, provides us with an exceptional clue to study how a species can resist a retroviral invasion.

Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene.

Gypsy displays striking similarities to vertebrate retroviruses, including the presence of a yet uncharacterized additional open reading frame (ORF3) and the recent evidence for infectivity. It is mobilized with high frequency in the germline of the progeny of females homozygous for the flamenco permissive mutation. We report the characterization of a gypsy subgenomic ORF3 RNA encoding typical retroviral envelope proteins. In females, env expression is strongly repressed by one copy of the non-permissive allele of flamenco. A less dramatic reduction in the accumulation of other transcripts and retrotranscripts is also observed. These effects correlate well with the inhibition of gypsy transposition in the progeny of these females, and are therefore likely to be responsible for this phenomenon. The effects of flamenco on gypsy expression are apparently restricted to the somatic follicle cells that surround the maternal germline. Moreover, permissive follicle cells display a typically polarized distribution of gypsy RNAs and envelope proteins, both being mainly accumulated at the apical pole, close to the oocyte. We propose a model suggesting that gypsy germinal transposition might occur only in individuals that have maternally inherited enveloped gypsy particles due to infection of the maternal germline by the soma.

Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster.

Retroviruses are commonly considered to be restricted to vertebrates. However, the genome of many eukaryotes contains mobile sequences known as retrotransposons with long terminal repeats (LTR retrotransposons) or viral retrotransposons, showing similarities with integrated proviruses of retroviruses, such as Ty elements in Saccharomyces cerevisiae, copia-like elements in Drosophila, and endogenous proviruses in vertebrates. The gypsy element of Drosophila melanogaster has LTRs and contains three open reading frames, one of which encodes potential products similar to gag-specific protease, reverse transcriptase, and endonuclease. It is more similar to typical retroviruses than to LTR retrotransposons. We report here experiments showing that gypsy can be transmitted by microinjecting egg plasma from embryos of a strain containing actively transposing gypsy elements into embryos of a strain originally devoid of transposing elements. Horizontal transfer is also observed when individuals of the "empty" stock are raised on medium containing ground pupae of the stock possessing transposing elements. These results suggest that gypsy is an infectious retrovirus and provide evidence that retroviruses also occur in invertebrates.

I factors in Drosophila melanogaster: transposition under control.

I factors are responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. They belong to the LINE class of mobile elements, which transpose via reverse transcription of a full-length RNA intermediate. I factors are active members of the I element family, which also contains defective I elements that are immobilized within peri-centromeric heterochromatin and represent very old components of the genome. Active I factors have recently invaded natural populations of Drosophila melanogaster, giving rise to inducer strains. Reactive strains, devoid of active I factors, derive from old laboratory stocks established before the invasion. Transposition of I factors is activated at very high frequencies in the germline of hybrid females issued from crosses between females from reactive strains and males from inducer strains. It results in the production of high rates of mutations and chromosomal rearrangements as well as in a particular syndrome of sterility. The frequency of transposition of I factors is dependent on the amount of full-length RNA that is synthesized from an internal promoter. This full-length RNA serves both as an intermediate of transposition and presumably as a messenger for protein synthesis. Regulators of transposition apparently affect transcription initiation from the internal promoter. The data presented here lead to the proposal of a tentative model for transposition.

The Doc transposable element in Drosophila melanogaster and Drosophila simulans: genomic distribution and transcription.

The mobile element Doc is similar in structure and coding potential to the LINE families found in various organisms. In this paper, we analyze the insertional and structural polymorphism of this element and show that it appears to have a long evolutionary history in the genome of D. melanogaster. Like the family of I elements, the Doc family seems to display three types of elements: full length elements, defective members that have recently transposed and long since immobilized members common to each D. melanogaster strain. These three classes of Doc elements seem to be present in D. simulans, a closely related species to D. melanogaster. Furthermore, we show that Doc is transcribed as a polyadenylated RNA of about 5 kb in length, presumed to be a full length RNA. This transcript is present in different tissues and at different stages of Drosophila development. These results are compared with previous records on the chromosomal distribution of LINEs or other transposable element families. Doc transcription is analyzed in an attempt to understand the link between Doc transcription and transposition.

Properties of transgenic strains of Drosophila melanogaster containing I transposable elements from Drosophila teissieri.

I factors are transposable elements of Drosophila melanogaster similar to mammalian LINEs, that transpose by reverse transcription of an RNA intermediate and are responsible for the I-R system of hybrid dysgenesis. There are two categories of strains in this species: inducer, that contain about 15 I elements at the various sites on chromosomal arms, and reactive, that lack active I factors. I elements occur in various Drosophila species. Potentially functional I factors from Drosophila teissieri can transpose when introduced by P-element-mediated transformation in a reactive strain of Drosophila melanogaster. We have studied the properties of Drosophila melanogaster strains into which such an I factor from Drosophila teissieri, named Itei, was introduced. Typical hybrid dysgenesis is produced when males carrying Itei are crossed with reactive females. However, more than one copy of the element seems necessary to produce dysgenic traits, whereas only one I factor of Drosophila melanogaster seems to be sufficient. The copy number of Itei in transformed lines maintained by endogamous crosses increases rapidly and stabilizes at values similar to those observed in inducer strains. As Drosophila teissieri contains much fewer copies than the Drosophila melanogaster strains, this suggests that the copy number of I elements is not simply regulated by sequences present in the element itself.

I elements and the Drosophila genome.

LINEs are a large class of transposable elements in eukaryotes. They transpose by reverse transcription of an RNA intermediate. I elements of Drosophila melanogaster belong to this class and are responsible for the I-R system of hybrid dysgenesis. Many results indicate that at the beginning of the century natural populations of this species were devoid of active I elements and that they were invaded by functional I elements in the last decades. Many Drosophila species contain both defective and active I elements. It seems that the latter were lost in Drosophila melanogaster before its spread throughout the world, and that the recent invasion results from the spread of functional elements originating either from another species by horizontal transfer or from an isolated population of the same species. These data are discussed, as well as their significance in evolutionary processes.

Evidence for retrotransposition of the I factor, a LINE element of Drosophila melanogaster.

LINEs are transposable elements found in various eukaryotes such as plants, protists, insects, and mammals. Their transposition is usually difficult to study, particularly in humans, where some diseases have been shown to result from LINE insertion mutations. This is due to the fact that most copies of any particular family of elements are defective and that their transposition frequency is low. By contrast, the I factor of Drosophila melanogaster transposes at high frequency during I-R hybrid dysgenesis and is a good model for studying the LINE element superfamily. LINEs encode putative polypeptides showing similarities with viral reverse transcriptases but, unlike viral retrotransposons, they do not have terminal repeats and their ability to transpose by reverse transcription has previously only been inferred from structural analysis. Here we present direct evidence for LINE retrotransposition. Transposition of an I factor marked by an intron resulted in accurate removal of the intron.

Molecular characteristics of the heterochromatic I elements from a reactive strain of Drosophila melanogaster.

There are two categories of strains in Drosophila melanogaster with respect to the I-R system of hybrid dysgenesis. The inducer strains contain particular transposable elements named I factors. They are not present in the strains of the other category called reactive (R) strains. Defective I elements are present in the pericentromeric regions of both categories of strains. This last subfamily of I sequences has not yet been described in detail and little is known about its origin. In this paper, we report that the defective I elements display an average of 94% of sequence identity with each other and with the transposable I factor. The results suggest that they cannot be the progenitors of the present day I factors, but that each of these two subfamilies started to evolve independently several million years ago. Furthermore, the sequence comparison of these I elements with an active I factor from Drosophila teissieri provides useful information about when the deleted I elements became immobilized.

A long interspersed repetitive element--the I factor of Drosophila teissieri--is able to transpose in different Drosophila species.

Long interspersed repetitive elements (LINEs) are transposable elements present in many species. In mammals they are difficult to study because most of them are defective and their transposition frequency is low. The I factor of Drosophila melanogaster is a LINE element that is particularly interesting because its transposition occurs at high frequency during I-R hybrid dysgenesis. This phenomenon occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several complete 5.4-kilobase I factors at various sites on the chromosomal arms. Reactive strains are devoid of complete I factors. Many results indicate that active I factors have invaded the D. melanogaster genome recently. To study the evolutionary history of I elements, we have cloned and sequenced a potentially active I factor from Drosophila teissieri. It is flanked by a target-site duplication and terminates at the 3' end by tandem repeats of the sequence TAA. When introduced into the germ line of a reactive strain of D. melanogaster by P element-mediated transformation, it is able to transpose and induces hybrid dysgenesis. This strengthens the hypothesis of a recent reinvasion of the D. melanogaster genome by active I factors giving rise to the inducer strains. They could have originated by horizontal transfer from another species. Such events also could occur for other LINE elements and might explain the spread of new variants in mammalian genomes. Moreover, the results give a further insight into I factor functional organization.

Characterization of 5' truncated transposed copies of the I factor in Drosophila melanogaster.

I factors in Drosophila melanogaster are transposable elements structurally related to Mammalian LINEs. Their transposition is activated at high frequencies during I-R hybrid dysgenesis and is associated with the production of mutations of various sorts. Very few of these mutations have been studied at the molecular level; those reported so far result either from chromosomal rearrangements or from insertions of complete I factors. We have analysed three I-R induced yellow mutations and have found that one of them is due to the insertion of an I element very similar to the complete I factor, whereas the other two are due to insertions of I elements that are truncated at their 5' ends; one of them exhibits an unusual 3' end. We discuss possible mechanisms of production of such modified I elements.

The beta heterochromatic sequences flanking the I elements are themselves defective transposable elements.

Phylogenetic studies suggest that mobile element families are unstable components of the Drosophila genome. Two examples of immobilization of a transposable element family are presented here: as judged by their constant genomic organization among unrelated strains, the F and I element families have been respectively immobilized for a long time in D. simulans and in the reactive D. melanogaster strains (these are the laboratory strains which escaped the recent I invasion of D. melanogaster natural populations). All the elements of these defective families are located in the beta heterochromatic portion of the genome. Moreover, most if not all of the beta heterochromatic sequences into which the defective I elements are embedded are themselves non-mobile members of various nomadic families such as mdg 4, 297, 1731, F and Doc. These results are discussed with special emphasis on the possible nomadic origin of beta heterochromatin components and on the mechanisms of evolutionary turnover of the transposable element families.

I elements of Drosophila melanogaster generate specific chromosomal rearrangements during transposition.

We report a detailed molecular analysis of three chromosomal rearrangements, which have been produced during I-R hybrid dysgenesis in Drosophila melanogaster. They all disrupt the yellow gene. One of them is a deletion; the other two are inversions, which may be interpreted as the results of recombination events between I elements inserted at their break points. These events appear to occur at the time of transposition and involve integrating rather than resident I elements. They are produced by a mechanism very similar to homologous ectopic recombination.