At the beginning of the 1970s Picard and L'Héritier reported that crosses between particular strains of Drosophila melanogaster produce progeny exhibiting genetic abnormalities. In 1976 Picard reported that the factor responsible for IR hybrid dysgenesis is a transposable element, the first discovered in Drosophila , and named it the I factor. The determination of its sequence showed that it belongs to the class of non-long terminal repeat retrotransposons (NLRs) also known as long interspersed nucleotidic elements (LINEs). The I factor is one of the models used to study the mechanism of transposition of NLRs because it can be mobilized at high frequency by dysgenic crosses, giving the opportunity to study the molecular mechanism of transposition in vivo. The D. melanogaster species can be divided into two classes of strains according to the IR system of hybrid dysgenesis, inducer (or I) and reactive (or R). I strains contain several complete and functional I factors, R strains do not. The mechanism of transposition of the I factor is thought to be related to target-primed reverse transcription (TPRT) requiring a full-length RNA intermediate. The study of deletion derivatives indicates that the sequences comprised between nucleotides 41 and 100 might be involved in the inhibition of somatic expression. The study of deletion derivatives showed that more than one region of the protein is involved in DNA binding and that the cysteine-rich motifs are not essential for this, but are required for the formation of the high molecular weight structures.

According to the current model of non-LTR retrotransposon (NLR) mobilization, co-expression of the RNA transposition intermediate, and the proteins it encodes (ORF1p and ORF2p), is a requisite for the formation of cytoplasmic ribonucleoprotein complexes which contain necessary elements to complete a retrotransposition cycle later in the nucleus. To understand these early processes of NLR mobilization, here we analyzed in vivo the protein and RNA expression patterns of the I factor, a model NLR in Drosophila. We show that ORF1p and I factor RNA, specifically produced during transposition, are co-expressed and tightly co-localize with a specific pattern (Loc+) exclusively in the cytoplasm of germ cells permissive for retrotransposition. Using an ORF2 mutated I factor, we show that ORF2p plays no role in the Loc+ patterning. With deletion derivatives of an I factor we define an RNA localization signal required to display the Loc+ pattern. Finally, by complementation experiments we show that ORF1p is necessary for the efficient localization of I factor RNA. Our data suggest that ORF1p is involved in proper folding and stabilization of I factor RNA for efficient targeting, through Loc+ patterning, to the nuclear neighborhood where downstream steps of the retrotransposition process occur.

To study the expression of the I factor, a non-long-terminal-repeat retrotransposon responsible for I-R hybrid dysgenesis in Drosophila melanogaster, we have tagged the ORF1 protein (ORF1p) by inserting the HA epitope in its N-terminal region. In transgenic flies, this modification is compatible with a high rate of autonomous transposition and allows direct estimation of the transposition frequency. I factor transposes in the germline of females (SF) that are daughters from crosses between I strain males (which contain active copies of the I factor) and R strain females (which do not). We analyzed the expression pattern of ORF1p by indirect immunofluorescence. Its expression correlates with retrotransposition. During oogenesis ORF1p appears unexpectedly as a cytoplasmic product, which accumulates with a specific pattern into the oocyte. A comparison of the expression patterns under conditions that modify the transposing activity of the element clarifies some aspects of I-factor functioning in the transposition process.

I factors in Drosophila melanogaster are non-LTR retrotransposons similar to mammalian LINEs. They transpose at very high frequencies in the germ line of SF females resulting from crosses between reactive females, devoid of active I factors, and inducer males, containing active I factors. The vermilion marked IviP2 element was designed to allow easy phenotypical screening for retrotransposition events. It is deleted in ORF2 and therefore cannot produce reverse transcriptase. IviP2 can be mobilized at very low frequencies by actively transposing I factors in the germ line of SF females. This paper shows that IviP2 can be mobilized more efficiently in the germ line of strongly reactive females in the absence of active I factors, when it is trans-complemented by the product of ORF2 synthesized from the hsp70 heat-shock promoter. This represents a promising step toward the use of marked I elements to study retrotransposition and as tools for mutagenesis.

The nucleotide sequence of the genes 2 and 3 of theDrosophilarhabdovirus sigma was determined from cDNAs to viral genome and poly(A)+mRNAs. Gene 2 comprises 1032 nucleotides and contains a long ORF encoding a molecular weight 35,208 polypeptide present in infected cells and in virions which migrates in SDS–PAGE as a doublet ofMrabout 60 kDa. The distribution of acidic charges as well as the electrophoretic properties of the protein are characteristic of the rhabdovirus P proteins. Gene 3 comprises 923 nucleotides and contains a long ORF capable of coding a polypeptide of 298 amino acids of MW 33,790. The putative protein (PP3) is similar in size to a minor component of the virions. Computer analysis shows that the sequence of PP3 contains three motifs related to the conserved motifs of reverse transcriptases.

The genome of the sigma rhabdovirus of Drosophila melanogaster consists of six genes in the order 3' N-2-3-4-G-L 5'. The nucleotide sequences of the N and of the fourth genes were determined from cDNA clones. Each gene contained a single long open reading frame encoding polypeptides with predicted MW of 50 and 25 kDa, respectively. Evidence that these genes encode the nucleocapsid N and the matrix M proteins were obtained using antibodies raised against recombinant proteins derived from the cloned genes and expressed in Escherichia coli. The M and N predicted amino acid sequences were compared with those of other rhabdoviruses. The M protein of sigma virus shared a similar domain arrangement to the other M proteins, but it showed very little sequence conservation. The N protein of sigma virus showed no significant homology with its counterpart in SYNV or IHNV and VSHV; it did, however, show sequence homology with the N of four vesiculoviruses and two lyssaviruses. The extent of amino acid identity suggests that sigma virus occupies an intermediate evolutionary position between these two genera. Some conserved motifs in the M and N proteins were deduced from the comparisons.

The ref(2)P gene of Drosophila melanogaster was identified by the discovery of two alleles, Po and Pp, respectively, permissive and restrictive for sigma rhabdovirus multiplication. A surprising variability of this gene was first noticed by the observation of size differences between the transcripts of permissive and restrictive alleles. In this paper, another restrictive allele, Pn, clearly distinct from Pp, is described: it exhibits a weaker antiviral effect than Pp and differs from Pp by its molecular structure. Five types of alleles were distinguished on the basis of their molecular structure, as revealed by S1 nuclease analysis of 17 D. melanogaster strains; three alleles were permissive and two restrictive. Comparison of the sequences of four haplotypes revealed numerous point mutations, two deletions (21 and 24 bp) and a complex event involving a 3-bp deletion, all affected the coding region. The unusual variability of the ref(2)P locus was confirmed by the high ratio of amino acid replacements to synonymous mutations (7:1), as compared to that of other genes, such as the Adh (2:42). Nevertheless, nucleotide sequence comparison with the Drosophila erecta ref(2)P gene shows that selective pressures are exerted to maintain the existence of a functional protein. The effects of this high variability on the ref(2)P protein are discussed in relation to its specific antiviral properties and to its function in D. melanogaster, where it is required for male fertility.

The sequence of the sigma virus glycoprotein gene has been reported. We report here the cloning of the cDNAs to four other genes, their physical map on the genome, the sequencing of the intergenic regions, and the determination of the gene junction signals. This analysis reveals unusual traits for a rhabdovirus: four genes map upstream of the glycoprotein gene and the glycoprotein gene overlaps the upstream gene by 33 nucleotides. Northern analysis did not show bicistronic transcripts of the overlapping genes, whereas it revealed bicistronic transcripts of two genes which are separated by a stretch of six nucleotides. This leads us to reconsider the significance of rhabdovirus consensus gene-end sequences and to integrate these data into the models proposed for polymerase functioning.

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. A permissive allele was cloned and sequenced. The structural gene (3.1 kbp) is divided into three exons. The mRNAs are heterogeneous in size. They differ only in the 5′ end of the first exon. The sequence upstream of the short mRNAs contains classical promoter elements. No TATA and CAAT boxes are appropriately positioned upstream of the initiation sites of the long mRNAs, but several repeats, palindromic sequences and inverted CAAT boxes are present. These observations, together with the tissue‐dependent distribution of short and long transcripts, support the hypothesis of the existence of at least two classes of genuine initiation sites. The long size of the untranslated leader RNA region suggests a control of gene expression at the translation level. The same translation product of 599 amino acids (76.3 kd) is predicted for all mRNAs, but the in vitro translation product migrates in SDS‐PAGE with a higher apparent mol. wt (115‐125 kd). The putative ref(2)P protein contains internal repeats, PEST regions which may be signals for protein degradation, and interesting structural motifs such as zinc finger and amphiphilic helices. These later motifs could be mitochondrial pre‐sequences. The degeneration of mitochondria is observed in the spermatids of sterile male flies homozygous for the loss‐of‐function alleles. The amino acid sequence of the ref(2)P product shows no homology with any known protein from the data banks.

Sigma virus, the hereditary agent of a CO2-induced paralysis of Drosophila, is classified as a rhabdovirus on a molecular basis. We have purified its genome which after 32P-labelling was used as a probe to detect mRNAs in infected cells. A cDNA copy of the entire coding region of the glycoprotein mRNA was cloned. Nucleotide and deduced amino acid sequences were determined and compared to previously known sequences of other rhabdovirus glycoproteins to determine the relatedness of Sigma virus to other viruses of this group.

SUMMARY Drosophila X virus described here appeared as a contaminant in Drosophila melanogaster. It is pathogenic for the inoculated flies, inducing anoxia sensitivity and death in these insects. An assay based on these symptoms in flies has been developed. Immunofluorescence has been used to study the characteristics of infected Drosophila cell cultures. A permanent infection can be established in these cultures. This virus is morphologically similar to several ungrouped vertebrate and invertebrate viruses like IPNV, IBDV and Tellina tenuis virus. Its possible origin is discussed.

SUMMARY Purified Drosophila X virus (DXV) particles have been analysed. They band at a density of 1.345 g/ml in CsCl. The virion proteins have been resolved into six major polypeptide species (mol. wt. 100000, 50000, 49000, 44000, 33000 and 27000) by polyacrylamide gel electrophoresis. The RNA sediments at 5S and 14S in sucrose gradients. The 5S RNA is sensitive to pancreatic RNase and the 14S RNA is resistant in its native form and sensitive after denaturation. The 14S RNA can be resolved into two equimolar fractions by polyacrylamide gel electrophoresis. The estimates of the mol. wt. of the two RNA species depends upon their structure. If they exist as double-stranded molecules their electrophoretic mobility compared to that of reovirus type 3 RNAs indicates for each species an average mol. wt. of 2.2 × 106.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.

Drosophila P virus was described by Plus & Duthoit (1969) as a small icosahedral virus, 25 nm. in diameter, endemic in many Drosophila populations. In naturally infected strains, this virus has little effect on flies. When injected, however, it causes female sterility and reduces the life span of both sexes by half (David & Plus, 1971). Just before death, injected flies become turgid with liquid, suggesting malfunction of the Malpighian tubules responsible for water exchanges in insects. An electron microscopic study was undertaken in order to characterize the virus and the response of the virus to certain chemicals was observed. The P virus exhibited properties consistent with those of the picornaviruses. Heterozygous Drosophila melanogaster vg, free from known Drosophila viruses: P (Plus & Duthoit, 1969), σ (L'Héritier, 1970) and Iota (Jousset, 1970), were used as standard flies for injections.