The Toxoneuron nigriceps polydnavirus ANK1 protein induces apoptosis in insect S2 cells and Heliothis virescens hemocytes by interacting with Alix protein

The Toxoneuron nigriceps polydnavirus ANK1 protein induces apoptosis in insect S2 cells and Heliothis virescens hemocytes by interacting with Alix protein Intoduction: Toxoneuron nigriceps is an endophagous parasitoid of larval stages of tobacco budworm, Heliothis virescens. The bracovirus associated with this wasp (TnBV) is currently being studied, and several genes expressed in parasitized host larvae have been isolated and their possible role partly elucidated. In this work, we studied the role of the viral gene TnBVank1 in the apoptosis. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IkB, inhibitor of the transcription nuclear factor kB. Methods: The role of TnBVank1 was investigated into H. virescens hemocytes by in vivo transient expression and, in vitro, in Drosophila Schneider's S2 cells which stably expressed TnBVank1. The success of in vivo and in vitro transfections was detected by RT-PCR and western blot. Apoptosis was detected by caspase-3 activity. ANK1 interactor proteins were reveled by coimmunoprecipitation experiments which indicated that ANK1 bound ALG-2- interacting protein X (Alix), an interactor of apoptosis-linked gene protein 2 (ALG-2). Alix was silenced by RNAi to study the function of ANK1-Alix interaction. Results/Conclusion: We show that when ANK1 was stably expressed in insect S2 cells, it stimulated caspase-3 activity. Furthermore, we get the same results into H. virescens hemocytes by in vivo transient expression of TnBVank1. Silencing Alix, ANK1 was no longer able to cause apoptosis in S2 cells and into H. virescens hemocytes. Collectively, these results indicate that ANK1 induces apoptosis by interacting with Alix, suggesting that this role could be relevant in the suppression of host immune response observed in tobacco budworm larvae parasitized by T. nigriceps.