Polydnavirus Ank Proteins Bind NF-κB Homodimers and Inhibit Processing of Relish

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-kappa B (NF-kappa B) transcription factors, and inhibitor kappa B (I kappa B) proteins that complex with and regulate NF-kappa Bs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with I kappa Bs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as I kappa B mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IkBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the I kappa B Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and -N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the I kappa B domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-kappa B signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral I kappa B binding affinities, while also showing that viral mimics disable Relish processing.