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 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.

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.

[Concentration, purification, and isotopic labeling of sigma virus]. / Concentration, purification et marquage isotopique du virus sigma

Sigma virus has been concentrated by means of polyethylene-glycol and diafiltration; the latter method is more efficient in preserving infectivity. When purified on sucrose gradient, sigma banded in a sharp peak without any polydispersion. We are now able to label sigma genome with 3H-uridine in Drosophila cell cultures.

Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.

Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection.

Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.

Restricted expression of viral glycoprotein in vesicular stomatitis virus-infected Drosophila melanogaster cells.

Vesicular stomatitis virus (VSV) establishes a non-cytopathic persistent infection in Drosophila melanogaster cells. The synthesis of the viral glycoprotein G was specifically inhibited during a post-transcriptional step, whereas the synthesis and turnover of its mRNA were not modified compared with the other viral mRNAs. Another viral glycoprotein, migrating slightly faster than G protein on an SDS-polyacrylamide gel, was detected in infected Drosophila cells. This protein showed most of the characteristics of the intracellular Gs protein found in infected vertebrate cells. The amounts of G protein integrated into mature virions and of soluble Gs protein secreted into the culture medium were reduced greatly during VSV infection in Drosophila cells.

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.

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.