Expression and functional studies of the Microplitis demolitor bracovirus IkB-like gene family
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Complex signaling pathways regulate the innate immune system of insects, with NF-kBtranscription factors playing a central role in the activation of antimicrobial peptides and otherimmune genes. Although numerous studies have characterized the immune responses of insectsto pathogens, comparatively little is known about the counterstrategies pathogens have evolvedto circumvent host defenses. Among the most potent immunosuppressive pathogens of insectsare polydnaviruses that are symbiotically associated with parasitoid wasps. Here, I report thatthe Microplitis demolitor bracovirus (MdBV) encodes a family of genes with homology toinhibitor kB (IkB) proteins from insects and mammals. Relative quantitative real-time PCRindicated that MdBV IkB-like genes were transcribed at different levels and exhibited tissuespecificpatterns of activity. Western blot analyses indicated that the recombinant IkB-like H4and N5 proteins remained in the cytoplasm and did not degrade upon stimulation. These viralproteins inhibited the signal-dependent cleavage of Rel/NF-kB as well as actively sequesteredRel/NF-kB complexes in the host cytoplasm. Functional analysis of two of these genes, H4 andN5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduced theexpression of drosomycin and attacin reporter constructs, which are under NF-kB regulationthrough the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 andN5 bound to the Rel proteins Dif and Relish, and N5 also weakly bound to Dorsal. H4 and N5also inhibited binding of Dif and Relish to kB sites in the promoters of the drosomycin andcecropin A1 genes. Collectively, these results indicate that H4 and N5 function as irreversibleIkBs and circumstantially suggest that other IkB-like gene family members are involved in thesuppression of the insect immune system.