Directed expression of the HIV-1 accessory protein Vpu in Drosophila fat-body cells inhibits Toll-dependent immune responses

Abstract

Human immunodeficiency virus 1 (HIV-1) expresses several accessory proteins that manipulate various host-cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCF(beta-TrCP) complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non-phosphorylated form, Vpu2/6, in fat-body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial-peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor-kappa B (NF-kappa B) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF-kappa B-mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF-kappa B signalling in vivo.

Figure 1

Viral protein U is expressed and phosphorylated in fat-body cells of transgenic adult flies. Expression of viral protein U (Vpu) was detected in the fat-body of UAS–Vpu; yolk–GAL4 females by immunofluorescence (IF) using an anti-Vpu serum (A–C, visible light; B–D, ultraviolet light). (A,B) UAS–Vpu flies do not express Vpu in the absence of the yolk–GAL4 driver. A few fat-body cells are indicated with an arrow and have a normal form. (E) Protein extracts prepared from flies expressing Vpu–HA (haemagglutinin; lanes 1 and 2) and Vpu2/6–HA (lanes 3 and 4) were incubated in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of calf intestinal alkaline phosphatase (CIP) before separation by SDS–polyacrylamide gel electrophoresis and analysis by western blotting using an anti-HA antibody.

Figure 2

Directed expression of viral protein U makes flies highly susceptible to fungal infections. Directed expression of viral protein U (Vpu) but not Vpu2/6 makes flies susceptible to fungal infection (B–D) but does not affect resistance to Gram-negative bacterial infection (A). Female adults were pricked with a needle that was previously dipped into (A) a pellet of Erwinia carotovora, or concentrated solutions of spores of (C) Aspergillus fumigatus or (D) Neurospora crassa, or were naturally infected with Beauveria bassiana (B). The spatzle (spz) gene is required to resist fungal infections (B–D), whereas mutations in dredd make flies highly susceptible to Gram-negative bacterial infection (A).

Figure 3

Viral protein U interferes with the Drosophila Toll pathway but not with the Imd pathway. (A) Northern blot analysis of Drosomycin (Drs) and Metchnikowin (Mtk) expression in flies expressing Vpu or Vpu2/6 collected 48 h or 72 h after natural infection by the fungus Beauveria bassiana. Expression of Vpu, but not Vpu2/6, affects Drs and Mtk induction after fungal infection, as does the spatzle mutation spz^rm7. (B) Northern blot analysis of Diptericin (Dpt) expression in flies expressing Vpu collected 6 h and 24 h after injection of Gram-negative bacteria (Escherichia coli). Vpu expression did not affect Dpt expression, whereas the dredd mutation strongly affected its induction. (C) Northern blot analysis of Drs and Mtk expression in female adults expressing Vpu or Vpu2/6 in a wild-type (WT) or Toll^10B mutant background. Vpu expression blocks the Toll^10B-mediated constitutive expression of Drs and Mtk. C, control (uninfected flies); Rp49, Ribosomal protein 49.

Figure 4

Viral protein U blocks the degradation of Cactus. (A) Viral protein U (Vpu) affects Drosomycin (Drs) expression in S2 cells. Cells were co-transfected with 1 μg of an expression vector that contains a copper-inducible TollΔLRR construction (Tauszig et al., 2000), plus 1 μg of an empty expression vector (E), a vector that contains Vpu, or a vector that contains Vpu2/6, and 0.1 μg of a reporter plasmid that encodes luciferase under the control of the Drs promoter. Forty-eight hours after transfection, CuSO₄ (500 μM) was added to induce the expression of the TollΔLRR construct. After 60 or 120 min, the cells were harvested and luciferase activity was determined from cell extracts as described in the supplementary information online. C, control (no copper). (B) Vpu blocks Cactus degradation. S2 cells were stably transfected with a copper-inducible Torso–Pelle construct (Silverman et al., 2000). Cells were transfected with 1 μg of an empty expression vector (lanes 1 and 2) or with vectors that contain Vpu (lane 3) or Vpu2/6 (lane 4). Thirty-six hours after transfection, CuSO₄ was added to induce the expression of the Torso–Pelle construct. Cellular protein extracts were prepared and were analysed on a western blot using an anti-Cactus antibody. A nonspecific band (indicated with an asterisk) was used as a loading control.

Figure 5

Slimb is not involved in the activation of the Toll pathway after fungal infection at the adult stage. Northern blot analysis of Drosomycin (Drs) expression in wild-type (WT) flies or slimb^m flies collected 48 h and 72 h after infection with the fungus Beauveria bassiana (Bb). The slimb^m mutation does not affect Drs induction after fungal infection. The slimb^m mutant has been described elsewhere (Grima et al., 2002). C, control (uninfected flies); Rp49, Ribosomal protein 49.