Control of sigma virus multiplication by the ref(2)P gene of Drosophila melanogaster: an in vivo study of the PB1 domain of Ref(2)P.

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.

Genetic and molecular features of Su(P), a gene that interacts with ref(2)P in male fertility of Drosophila melanogaster.

The ref(2)P gene is involved in the control of sigma rhabdovirus multiplication in Drosophila melanogaster. ref(2)P activity is also necessary for male fertility. However, in one-third of laboratory strains tested, males that lacked ref(2)P activity were fertile. In all such strains studied, the male sterility phenotype was abolished due to the presence of a particular allele at the Su(P) locus, at 73B1-2. These spontaneous suppressor alleles were dominant. We were able to induce dominant suppressor alleles at the Su(P) locus by X-ray mutagenesis and hybrid dysgenesis, suggesting that null alleles of Su(P) confer the dominant suppressor phenotype. The Su(P) gene was cloned by P element tagging. The P element-tagged alleles identified a Su(P) transcript as a 1.4-kb mRNA produced in the soma of both males and females, which is also abundant in ovaries.

Transposable elements behavior following viral genomic stress in Drosophila melanogaster inbred line.

To analyze the behavior of endogenous transposable elements under genomic stress, a Drosophila melanogaster inbred line was submitted to three kinds of viral perturbations. First, a retroviral plasmid containing the avian Rous Associated Virus type 2 (RAV-2) previously deleted for the viral envelope coding gene (env) was introduced by P element transformation into the Drosophila genome. An insertion of this avian retroviral sequence was detected by in situ hybridization in site 53C on polytene chromosome arm 2R. Second, Drosophila embryos were injected with RAV-2 particles produced by cell culture after transfection with the retroviral plasmid. Third, the Drosophila melanogaster inbred line was stably infected by the sigma native virus. It appears that neither the offspring of the flies in which the viral DNA was found integrated nor those from the infected sigma flies showed copia or mdg1 element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdg1 element on the 2L chromosome, the copia element insertion pattern remaining stable. Thus, endogenous transposable elements show more instability in sublines injected with exogenous viral particles than in a transgenic subline containing a foreign viral insert, all transposable elements not being equally sensitive to such genomic stress.

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila.

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.

Localization of domains within the Drosophila Ref(2)P protein involved in the intracellular control of sigma rhabdovirus multiplication.

The ref(2)P gene of Drosophila melanogaster interferes with sigma rhabdovirus multiplication. This gene is highly variable, and the different alleles are considered permissive or restrictive according to their effects on virus replication. In all cases, the mechanisms involve intracellular interactions between the sigma virus and Ref(2)P proteins. We showed that the N-terminal domain of the Ref(2)P protein was required for its activity in vivo. The protein was inactive in the null p(od)2 mutant when its first 82 amino acids were deleted. The p delta n gene was constructed so that the first 91 amino acids coded for by the restrictive alleles could be expressed in vivo. It was active in a transformed line. This sequence was sufficient to impart a restrictive phenotype to an adult D. melanogaster fly after it was injected with the virus. However, the truncated protein expressed by p delta n did not have an effect on the hereditary transmission of the sigma virus to the offspring of the infected flies, even though it contained the restriction site. The native Ref(2)P protein has been previously shown to have conformation-dependent epitopes common with some of those of the viral N protein. We demonstrated the following. (i) These epitopes were found in a domain of the Ref(2)P protein distinct from the site involved in restriction. (ii) They were modified in the N protein of the haP7 sigma virus mutant selected as being adapted to the restrictive alleles of the ref(2)P gene; only one mutation in the N gene, leading to an amino acid substitution, distinguished the haP7 mutant from the original virus. (iii) The virus strains partially or totally adapted to the effects of the full restrictive protein expressed by pp were always found to multiply to a lesser extent in the presence of the protein expressed by p delta n. These data suggest that two distinct domains of the Ref(2)P protein are involved in the control of sigma virus multiplication.

Study of the ref(2)P locus of Drosophila melanogaster. II. Genetic studies of the 37DF region.

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P0 which permits sigma virus multiplication and ref(2)Pp which is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (= Df(2L)37D2-E1;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is more efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)Pnull flies are male sterile. These flies, like homozygous or hemizygous ref(2)P0 flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.

Immunological cross-reactions and interactions between the Drosophila melanogaster ref(2)P protein and sigma rhabdovirus proteins.

The ref(2)P gene is one of the Drosophila melanogaster genes involved in the inhibition of sigma rhabdovirus multiplication. The partial restriction of viral replication varies according to the ref(2)P alleles and virus strains and involves intracellular interactions between parasite and host products. We identified the protein encoded by the ref(2)P gene and produced polyclonal antibodies directed against the whole ref(2)P protein obtained from a recombinant baculovirus and against a part of the protein expressed as a fusion protein. These antibodies were used to study the interactions with sigma virus proteins by different immunoprecipitation techniques. We showed that the native ref(2)P protein shared conformation-dependent common epitopes with the viral structural genome-associated N protein. Furthermore, the cellular protein was found to be associated in complexes with the viral P protein required for RNA polymerase activity. The significance of these observations in the control of sigma virus multiplication by its host is discussed.

Unusual variability of the Drosophila melanogaster ref(2)P protein which controls the multiplication of sigma rhabdovirus.

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.

Molecular analysis of ref(2)P, a Drosophila gene implicated in sigma rhabdovirus multiplication and necessary for male fertility.

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.

Genetic resistance to viral infection: the molecular cloning of a Drosophila gene that restricts infection by the rhabdovirus sigma.

The ref(2)P gene of Drosophila melanogaster has two common alleles, ref(2)Po which permits the infection of flies by the rhabdovirus sigma (sigma), and ref(2)Pp which is restrictive for sigma infection. This gene has been cloned by P element tagging and shown to code for two RNAs in adult flies. These RNAs are expressed in both males and females, but only the larger is expressed in ovaries. Both transcripts are shorter, by about 50 nucleotides, in flies carrying the ref(2)Pp allele than in those carrying ref(2)Po. The dominance relationships of these two alleles, and the fact that ref(2)Pnull alleles are permissive to sigma infection, suggest that the ref(2)Po product is antimorphic to that of the ref(2)Pp allele.

[Locus ref(2)P of Drosophila melanogaster. I. Cytogenetic localization of ref(2)P]. / Etude du locus ref(2)P de Drosophila melanogaster. I. Localisation cytogénétique de ref(2)P.

The ref(2)P gene is a Drosophila gene which acts on Sigma virus multiplication. From recombination experiments the ref(2)P gene was located between hk and pr. This location was confirmed by the study of eight chromosomic aberrations, without ref(2)P gene activity, obtained following X irradiation. From the cytological study of three of these chromosomes and from the study of the ref(2)P gene activity of chromosomic aberrations obtained by other workers in the same region of the second chromosome, the ref(2)P gene was cytologically located in 37E3-37F3.

The late functions of Drosophila sigma virus.

Several criteria can be used to characterize the temperature-sensitive mutants of sigma virus: the lability of infectious centers initiated by the efficient viral particles following the inoculation; the localization of the temperature-sensitive period; the hereditary transmission of infection in a maternal Drosophila line at high temperature; the evolution of the CO2 sensitivity symptom and of the viral titer per fly for hereditarily infected flies kept at a permissive temperature and shifted to a restrictive one. Experimental data concerning the last two criteria are shown and discussed here. From all the criteria, three classes of ts mutants can be defined: the first class (prototype ts4) are the mutants blocked in hereditary transmission at high temperature. These mutants are affected during the whole viral cycle but the viral maturation is not directly affected. The second class (ts9, haP27) are the mutants directly affected in viral maturation. For these latter, the hereditary transmission in a maternal line is efficient at high temperature, and only a late temperature sensitive period is observed. The third class (prototype haP7) are the mutants for which only an early temperature-sensitive period is observed. In fact, for these mutants, the viral information is strongly heat-sensitive at the beginning of viral cycle. This defect is rapidly corrected, and the hereditary transmission, late viral functions and viral maturation are as efficient as those of wild type virus, at high temperature. Using temperature-sensitive mutants similar to the wild type virus at permissive temperature, a formal description of the viral cycle at 20 degrees C in the standard flies can be obtained. The long period necessary to perform an entire viral cycle (about 60 hours) is a result of the restrictive action on late viral functions at 20 degrees C of the Oe allele of the ref(3)O host gene present in the standard flies.

Role of the Drosophila genome in Sigma virus multiplication. I. Role of the ret(2)P gene; selection of host-adapted mutants at the nonpermissive allele Pp.

The allele Pp of gene ref(2)P of Drosophila melanogaster is known to inhibit the multiplication of certain strains of Sigma virus. From a viral strain for which the allele PP of ref(2)P is nonpermissive, host adapted (haP) mutants were selected after mutagenesis in vivo. Mutagenized virus was extracted from infected flies fed with 5-fluorouracil. The mutagenized virus was used to inoculate restrictive flies to screen haP mutants. The surviving viruses were studied in permissive and restrictive hosts. In a first test the capability to induce CO2 symptom in permissive and in restrictive flies was studied for each surviving clone. In a second test the efficiency to initiate infection in permissive and in restrictive flies was compared to the wild-type efficiency for each putative adapted clone. From 111 surviving viruses, 23 haP mutants were thus isolated. The proportion of temperature-sensitive mutants was higher among haP mutants than among non-haP brother clones. Five ts-haP mutants appeared more temperature sensitive in the restrictive host than in the permissive one. In the five cases, the viral functions affected by the mutation were early, i.e., occurring within 24 hr of the infection of a cell at 20 degrees and related to the replication of the viral genome. These results suggest that viral proteins required for early viral functions are implicated in the interaction with the PP allele of the ref(2)P host gene.