Sigma Rhabdovirus Research Articles

RNA editing by the host ADAR system affects the molecular evolution of the Zika virus.

Zika virus (ZIKV) is a mosquito‐transmitted flavivirus, linked to microcephaly and fetal death in humans. Here, we investigate whether host‐mediated RNA editing of adenosines (ADAR) plays a role in the molecular evolution of ZIKV. Using complete coding sequences for the ZIKV polyprotein, we show that potential ADAR substitutions are underrepresented at the ADAR‐resistant GA dinucleotides of both the positive and negative strands, that these changes are spatially and temporally clustered (as expected of ADAR editing) for three branches of the viral phylogeny, and that ADAR mutagenesis can be linked to its codon usage. Furthermore, resistant GA dinucleotides are enriched on the positive (but not negative) strand, indicating that the former is under stronger purifying selection than the latter. ADAR editing also affects the evolution of the rhabdovirus sigma. Our study now documents that host ADAR editing is a mutation and evolutionary force of positive‐ as well as negative‐strand RNA viruses.

Detection of rhabdovirus viral RNA in oropharyngeal swabs and ectoparasites of Spanish bats

Rhabdoviruses infect a variety of hosts, including mammals, birds, reptiles, fish, insects and plants. As bats are the natural host for most members of the genus Lyssavirus, the specificity of the amplification methods used for active surveillance is usually restricted to lyssaviruses. However, the presence of other rhabdoviruses in bats has also been reported. In order to broaden the scope of such methods, a new RT-PCR, able to detect a diverse range of rhabdoviruses, was designed. The method detected 81 of 86 different rhabdoviruses. In total, 1488 oropharyngeal bat swabs and 38 nycteribiid samples were analysed, and 17 unique rhabdovirus-related sequences were detected. Phylogenetic analysis suggested that those sequences detected in bats did not constitute a monophyletic group, even when originating from the same bat species. However, all of the sequences detected in nycteribiids and one sequence obtained from a bat did constitute a monophyletic group with Drosophila melanogaster sigma rhabdovirus.

Cellular and Molecular Aspects of Rhabdovirus Interactions with Insect and Plant Hosts

The rhabdoviruses form a large family (Rhabdoviridae) whose host ranges include humans, other vertebrates, invertebrates, and plants. There are at least 90 plant-infecting rhabdoviruses, several of which are economically important pathogens of various crops. All definitive plant-infecting and many vertebrate-infecting rhabdoviruses are persistently transmitted by insect vectors, and a few putative plant rhabdoviruses are transmitted by mites. Plant rhabdoviruses replicate in their plant and arthropod hosts, and transmission by vectors is highly specific, with each virus species transmitted by one or a few related insect species, mainly aphids, leafhoppers, or planthoppers. Here, we provide an overview of plant rhabdovirus interactions with their insect hosts and of how these interactions compare with those of vertebrate-infecting viruses and with the Sigma rhabdovirus that infects Drosophila flies. We focus on cellular and molecular aspects of vector/host specificity, transmission barriers, and virus receptors in the vectors. In addition, we briefly discuss recent advances in understanding rhabdovirus-plant interactions.

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.

A Gain-of-Function Screen for Genes That Affect the Development of the Drosophila Adult External Sensory Organ.

IN natural populations of Drosophila melanogaster throughout the world, a minority of individuals are usually infected by the Rhabdovirus sigma. This virus does not integrate into the fly chromosomes and is only vertically transmitted through male and female gametes ([Fleuriet 1999][1]). Of the fly

Polymorphism of the Drosophila melanogaster — sigma virus system

AbstractIn natural populations of D. melanogaster, a minority of flies are usually infected by the sigma Rhabdovirus, which is not contagious but vertically transmitted. A few parameters have been identified that affect the evolution of the Drosophila — sigma system. Various sets of values of these parameters in males mutually adapted to one another have been established in different populations. This study analyses female characteristics that were previously reputed not to vary much between populations. The newly collected data show that these characteristics (mainly transovarial transmission here) may also be polymorphic in the wild, and may contribute to the diversity of equilibria established between the virus and its host. The observations presented here, obtained from various populations, confirm the complexity of this system of two coevolving organisms: as already noted there seem to be as many different sets of values of parameters as there are populations.

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.

Gene 2 of the Sigma Rhabdovirus Genome Encodes the P Protein, and Gene 3 Encodes a Protein Related to the Reverse Transcriptase of Retroelements

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.

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.

Sequences of the N and M Genes of the Sigma Virus of Drosophila and Evolutionary Comparison

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 hemology 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.

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.

Study of the ref(2)P locus of Drosophila melanogaster

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.

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.

Genome Organization of the Sigma Rhabdovirus: Six Genes and a Gene Overlap

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.

Polymorphism of the hereditary rhabdovirus sigma in wild populations of its host, Drosophila melanogaster

Summary — In natural populations of Drosophila melanogaster throughout the world a minority of flies are infected by a rhabdovirus, sigma, which is not contagious but is transmitted through gametes. Transmission of the virus by males is a cornerstone for its maintenance in populations. The experiments reported in this paper show that in the wild European populations examined the efficiency of transmission by males is determined mainly by viral genotype. In African populations, genetic coadaptation of both partners can lead to a very low transmission of the virus by males. Evidence is also given of the coexistence in populations of different genotypes of the virus. The situation reported is thus another example of the genetic polymorphism displayed by the sigma virus in the wild.

Polymorphism of the hereditary rhabdovirus sigma in wild populations of its host, Drosophila melanogaster

Polymorphism of the hereditary rhabdovirus sigma in wild populations of its host, Drosophila melanogaster

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.

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.

Influence of the population size on the frequency of flies infected by sigma virus in populations of Drosophila melanogaster

The frequency of flies infected by the hereditary, noncontagious rhabdovirus sigma has been followed in experimental populations bred from very few adults. The average frequency of infected flies decreased. These results suggest that overwinterning might be one of the factors responsible for the discrepancy between wild and laboratory populations; in France, previous experiments have shown that about 20% of the individuals in natural populations are infected by the sigma virus, while in laboratory populations set up from samples collected in the wild, virus is detectable in almost the whole population. The effect of winter on the frequency of infected flies might be twofold: first, it has been shown previously that infected flies seem to be more sensitive to overwintering than uninfected flies; second, the reduction of population size might reduce the ability of the population to make up for the decrease during the summer generations.

Characteristics of the CO 2 sensitivity syndrome in Drosophila melanogaster: Introduction of non-permissive alleles of two ref genes in a maternal line stabilized for sigma virus

Summary The CO 2 sensitivity syndrome produced in Drosophila melanogaster by the sigma rhabdovirus is a motor paralysis observed when the insects are placed in an atmosphere containing a concentration of CO 2 above a certain threshold. The introduction of non-permissive alleles of two ref genes in a maternal line stabilized for sigma virus enabled us to demonstrate a cephalic sensitivity site which is attacked before the thoracic site as well as repair phenomena which eliminate the changes provoked by the virus and exposure to CO 2 . Thus the different forms of the syndrome obtained with different rhabdoviruses (vesicular stomatitis virus Indiana and Chandipura) are all reproducible with sigma virus by using various different D. melanogaster genotypes.