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

Abstract

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 od2 mutant when its first 82 amino acids were deleted. The p ⌬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 ⌬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 p p were always found to multiply to a lesser extent in the presence of the protein expressed by p ⌬n. These data suggest that two distinct domains of the Ref(2)P protein are involved in the control of sigma virus multiplication.

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

“…Moreover, ubiquitin exhibited cytoplasmic staining ( Fig. 3, E -F ( Wyers et al, 1995 ). The ref (2)P od2 loss-of-function allele results in a protein lacking the PB1 domain and has a molecular mass of 85 kD, compared with the 100 kD mass of the wild-type protein ( Fig.…”

“…Importantly, both the PB1 and the UBA domains of Ref(2)P are necessary for the formation of protein aggregates, suggesting that both the abilities of Ref (2)P to multimerize and bind ubiquitinated proteins are necessary functions required during the in vivo formation of protein aggregates. (2)P od3 fl ies are described in Wyers et al, (1995) . For the induction of clones in the adult brain, larvae were heat shocked on days 4 and 5 (L2 and L3 of larvae development) for 1 h and 30 min at 37 ° C in a circulating water bath.…”

“…The brains were then incubated overnight at 4 ° C in blocking solution containing the primary antibodies. The primary antibodies used in the present study were the following: mouse anti -mono-and polyubiquitinated proteins (Clone FK2; BIOMOL International, L.P.) used at a concentration of 1:500; rabbit polyclonal antibody against D. melanogaster Ref(2)P protein ( Wyers et al, 1995 ) used at a dilution of 1:1,000; and mouse anti-tau1 (Millipore) used at a dilution of 1:1,000. After incubation with the respective primary antibody, brains were washed four times with blocking solution.…”

The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy

“…To examine whether the reduced MDC-positive structures in some cells of dying larval salivary glands from sigmar S are associated with a decrease in autophagic flux, we examined salivary gland cells using the Drosophila Ref(2)P antibody ( Nezis et al, 2008 ; Wyers et al, 1995 ) at early (16 hours APF) and late (25–26 hours APF) stages. Ref(2)P, a Drosophila homolog of the mammalian p62 protein, localizes to age-induced protein aggregates as well as to aggregates resulting from reduced autophagic or proteasomal activity ( Nezis et al, 2008 ).…”

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

“…Although Ref(2)P was originally identified as a factor involved in male fertility and sigma virus replication, 41 it contains all the structural hallmarks of a p62 ortholog, including the PB1, ZZ and UBA domains. 17,42 Interestingly, Δvps15 Drosophila larvae accumulated numerous Ref(2)P-positive structures, indicative of impaired metabolism of protein aggregates. Consistent with this, the Δvps15 mutants also accumulated ubiquitin-positive structures.…”