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

Abstract

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.

Figure 1.—

D. melanogaster Ref(2)P proteins used in this study. The PB1 (PFAM00564), ZZ (zinc finger, ZZ type, PFAM00569), and UBA (PFAM00627) domains are indicated by shaded boxes, with their localizations on the amino acid sequence. Solid boxes represent deletions and thick lines represent single amino acid changes. (A) Typical permissive Ref(2)P^(+++)O1 and typical restrictive Ref(2)P^(dpp)Pa sequences. The modifications found along the restrictive protein, including the dpp′ mutations, are shown below the sequence. (B) Ref(2)P proteins expressed by recombinant transgenes (Dru et al. 1993; Wayne et al. 1996; Marchler-Bauer et al. 2003).

Figure 2.—

Dose of A3 or 23 sigma virus necessary to infect flies according to the genotype at the ref(2)P locus. The concentration of the efficient inoculum was determined by the end-point dilution method from a dilution series of each viral stock. Error bars are defined for the limits of the confidence interval at the 5% level of significance. The reference strain was ref(2)P⁻/ref(2)P⁻. The size of inoculum necessary to infect these flies was arbitrarily defined as 1.00. The y-axis values are the logarithm of the smallest inoculum able to infect flies (Contamine 1981); thus positive values on the histogram reflect an increase in the minimum quantity of virus necessary for infection while negative values reveal a decreased quantity of virus compared to the control [log(1) = 0]. k and k′ are the minimum inoculum size necessary to infect homozygous permissive flies with A3 and 23 sigma virus, respectively. Values expressed in the number of k or k′ on top of each bar represent the minimum inoculum size necessary to infect flies of the corresponding genotype. The ref(2)P⁻/ref(2)P⁻ are ref(2)P^od3/Df(2L)E55. When these control flies are compared to hemi-zygotes [ref(2)P⁽⁺⁺⁺⁾/ref(2)P⁻ or ref(2)P^(dpp′)/ref(2)P⁻], the results are the same regardless of whichever ref(2)P⁻ was used to build hemi-zygote flies [ref(2)P^od3 or Df(2L)E55]. The number of copies of ref(2)P^(dpp′) (dashed lines) and ref(2)P⁽⁺⁺⁺⁾ (solid lines) is indicated under the histogram.

Figure 3.—

Immunodetection of Ref(2)P in different transgenic fly lines. After immunoprecipitation of Ref(2)P from 100 or 25 flies, protein samples were loaded on a 10% SDS–PAGE gel and transferred before immunodetection with an anti-Ref(2)P antibody. Fly lines homozygous for ref(2)P^O1 transgenes bearing different combinations of dpp′ mutations in a ref(2)P⁻/ref(2)P⁻ loss-of-function background. Similar levels of protein were observed in each fly line. These levels were not distinguishable from what is observed in a wild-type permissive fly line (OM, resident).

Figure 4.—

Role of the p′ mutation in restrictivity for viral strain A3 (A) and 23 (B) and of the PB1 domain alone on restrictivity for the A3 strain (A). The concentration of the efficient inoculum necessary to infect adult flies and the error bars were determined as in Figure 2. The size of inoculum necessary to infect ref(2)P⁻/ref(2)P⁻ (i.e., in the absence of any transgene) was arbitrarily defined as 1.00. (A) Role of the p′ mutation and PB1 domain in sensitivity to the A3 virus. The transgenes used are indicated in the figure. (B) Effect of ref(2)P mRNA quantity on sensitivity to the viral strain 23: role of the p′ mutation. On the x-axis, mRNA quantity for one copy of the permissive allele is arbitrarily defined as 1. Additional copies of transgenes in Drosophila do not necessarily result in a proportional increase in mRNA. ▵, ref(2)P^(dp+)O1; ○, ref(2)P^(dpp′)O1.

Figure 5.—

Evolution of the Ref(2)P PB1 domain. This tree was built according to the principle of parsimony. The external reference is D. simulans Ref(2)P (Wayne et al. 1996). No synonymous mutation is observed in the PB1 coding sequence among the 14 fully sequenced haplotypes. The number of sequenced alleles corresponding to each category is in parentheses.